Full text of "كتب في علم الميكروبيولوجي"

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K Alexander

Dennis Strete

ISBN: 0-07-248744-5

Publication Date: March 2003

Overview

A modern general microbiology laboratory manual that combines the procedural details of a
laboratory manual with the photographic support of a laboratory atlas. The 46 class-tested
laboratory experiments are divided into 9 specialty areas, and the extensive four-color illustration
program includes 220 photos and micrographs plus 150 line drawings.

Features

• An extensive full-color art program integrated into the laboratory exercises allows students to not only conduct a
variety of laboratory exercises but also to interpret and confirm their results with the help of the large collection of color
photographs.

• Unique exercise! ! Simulation of Infectious Disease Transmission (Lab Exercise 44). Developed in conjunction with
the pioneering program "The Biology Project" at the University of Arizona, this exercise allows class members to trade
simulated "body fluids" in a random pattern coordinated by the lab instructor. ELISA testing makes it clear to students
how easily the mock pathogen has passed through intermediaries to individuals in distant locations (across the lab).

• Emphasis on modern lab safety issues. Besides the usual safety advisories, this manual includes a table ranking the
Biosafety Level of every bacteria used in the lab exercises, specific guidelines for working with bacteria in each
Biosafety Level, and prominent icons throughout the lab exercises advising students of the Biosafety Level of the
bacteria in use. Safety Stops throughout the manual also remind students of particular hazards in each exercise. No
other lab manual on the market provides the Biosafety Level cautions and identification.

Alexander-Strete-Niles:

Front Matter

Preface

©The McGraw-Hill

Lab Exercises in
Organismal and Molecular
Microbiology

Companies, 2003

Preface

When students move from the lecture hall to the micro-
biology laboratory, they need help bridging the
gap between the theory and the practice of what they are
learning. The equipment is unfamiliar, the procedures
are unfamiliar, and many of the materials they are han-
dling are unfamiliar. Linking the information from their
classroom lectures to the laboratory procedures is nec-
essary for their ultimate success. Our goal for this
laboratory manual is to provide the bridge that helps
students integrate their classroom lectures with their
laboratory experiences. This integrated approach is
the only way to ensure understanding and mastery in
microbiology.

Features

Class-tested experiments have been vetted in our
own courses and provide a thoughtful progression
of opportunities — from basic lab techniques, such
as Exercises 9-15 on various staining techniques,
to more challenging exercises, such as the simu-
lated epidemic in Exercise 44: "Enzyme- linked
Immunosorbent Assay (ELISA)." This building-
block approach allows students to develop
comfort and confidence in their laboratory skills.

Exceptional full-color art program includes over
250 of our own photographs created specifically
for these laboratory exercises, plus 150 line
drawings of equipment, procedures, and results.
Students can easily confirm their results and
procedures by referring to the illustrations.

Exceptional attention to safety issues is given
throughout the manual. A basic lab safety section
beginning on page xi includes a table identifying
the biosafety level of every organism used in the
experiments. The BSL 2 icon @ appears where
appropriate to remind students of the needed safety
precautions when working with pathogens. Caution
symbols ^ appear throughout the lab manual to
provide students with additional safety warnings
as needed.

Organization

Our 46 exercises are organized into the following nine
sections:

Section I
Section II
Section III
Section IV
Section V
Section VI

Survey of Microscopic Organisms

Staining Techniques

Bacterial Cultivation

Bacterial Identification

Medical Microbiology

Controlling the Risk and Spread
of B acterial Infections

Section VII Bacterial Genetics
Section VIII Viruses

Section IX

Hematology and Serology

The standard presentation of each section makes it easy
for both students and lab managers to prepare for an
exercise. Each exercise:

1 . Opens with a short background that conveys only
information relevant to the exercise.

2. Lists all needed materials, by category.

3. Presents procedures for the exercise in easy-to-
follow steps and includes special notes, hints, and
instructions to ensure success.

4. Integrates all photographs and line drawings into
the text of the exercise where they will provide
the student with the most support.

5. Includes a tear-out laboratory report conveniently
located at the end of the exercise.

Instructor Support Material

An Instructor Image Bank provides digital files in the
easy-to-use JPEG format for all of the photos and line
art included in this lab manual. They are organized by
section and placed in PowerPoint sets for easy access.
These may prove useful for lab preparation packets,
testing, or discussion sessions. Ask your McGraw-Hill
representative for further details.

Alexander-Strete-Niles:

Front Matter

Preface

©The McGraw-Hill

Lab Exercises in
Organismal and Molecular
Microbiology

Companies, 2003

The Instructor's Manual for this set of labora-
tory exercises may be found online at:

www.mhhe.com/biosci/ap/labcentral/

It provides answers to lab report questions, tips for lab
exercise success, and other useful information.

Acknowledgments

In the end, our hope is that we have put together a man-
ual that will serve as a valuable teaching tool for the
microbiology laboratory. Our efforts were greatly aided
by the following reviewers, whom we gratefully
acknowledge:

Daniel R. Brown, Sante Fe Community College
Kathy Buhrer, Tidewater Community College
Linda E. Fisher, University of Michigan, Dearborn
Georgia Ineichen, Hinds Community College
Hubert Ling, County College of Morris
Rita Moyes, Texas A&M University
Richard C. Renner, Laredo Community College
Ken Slater, Utah Valley State College

Kristin M. Snow, Fox Valley Technical College
Carole Rehkugler, Cornell University
Paul E. Wanda, Southern Illinois University,
Edwardsville

Our gratitude is also extended to our publishing team at
McGraw-Hill:

Colin Wheatley, Publisher/Sponsoring Editor

Jean Sims Fornango, Senior Developmental Editor

Tami Petsche, Marketing Manager

Gloria Schiesl, Project Manager

Sandy Ludovissy, Production Supervisor

Wayne Harms, Designer

Carrie Burger, Photo Editor

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

Front Matter

Safety Guidelines for the
Microbiology Laboratory

General Guidelines for Every Lab Session

1 . Wear appropriate clothing and shoes to the laboratory. Shoes must completely cover the feet to
provide protection from broken glass and spills.

2. Place all books, backpacks, purses, etc., in an area designated by your laboratory instructor. Carry to
your work area only the items you will use in the lab.

3. Wash your hands thoroughly with antibacterial soap before beginning the lab session.

4. Wipe your work area with disinfectant, and allow to air-dry before beginning the lab session.

5. Do not perform activities in the lab until you are given instructions by your laboratory instructor.

6. Do not eat, drink, smoke, or apply makeup while working in the laboratory.

7. If you cut or burn yourself while working, report this immediately to your laboratory instructor.

8. Broken glassware should be immediately brought to the attention of your laboratory instructor. Bro-
ken glass should be placed in a special sharps container for disposal and not in the

trash container.

9. If using a Bunsen burner, tie back long hair. Do not lean over the countertop. When in use, always be
aware of the flame. Keep flammable items away from the flame. Turn off the burner when not in use.

10. Before leaving the lab, make sure all items have been returned to their appropriate location.

1 1 . After your work area is clear, wipe down your countertop with disinfectant before leaving.

12. Wash your hands thoroughly with antibacterial soap before leaving the lab.

13. Do not remove any item from the lab unless you have been directed to do so by the laboratory
instructor.

Guidelines for Working with Biosafety Level (BSL) 1 Bacteria

Handling live bacteria in the laboratory, even those considered nonpathogenic, requires special guidelines
beyond the general guidelines already mentioned. All bacteria are potentially pathogenic, especially if
they gain entry into the human body. So observe the following guidelines when handling the biosafety
level (BSL) 1 bacteria listed in the summary table.

1. Do not put anything into your mouth when working with cultures. Do not pipette by mouth; use a
pipette aid instead. Keep your hands, pencil, pen, etc., away from your mouth, eyes, and nose.

2. When inoculating cultures, sterilize the loop or needle before placing it on the counter.

3. Always keep tubes in test tube racks when working with liquid media. Do not stand them up or lay
them down on the countertop where they may spill.

4. If you accidentally spill a culture, cover the spill with a paper towel, flood it with disinfectant, and
notify your laboratory instructor.

5. Place all used culture media, paper towels, gloves, etc., into the waste container designated by your
laboratory instructor. A separate waste container for sharps (slides, pipettes, swabs, broken glass,
etc.) will also be provided. All this waste will be autoclaved before disposal or reuse. Do not throw
any of these items into the trash container.

6. If you have a burn or wound on one of your hands, cover it with a plastic strip and wear disposable
gloves for added protection.

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

Front Matter

Safety Guidelines for the
Microbiology Laboratory

Guidelines for Working with Biosafety Level (BSL) 2 Bacteria

Handling pathogenic bacteria in the laboratory requires special guidelines beyond the general guidelines and
those for BSL 1 bacteria. The following additional guidelines apply when working with the biosafety level
(BSL) 2 bacteria listed in the summary table.

1 . When handling pathogens, access to the laboratory must be restricted to only those working in
the lab.

2. Disposable gloves and a lab coat must be worn. The gloves should be disposed of in a container des-
ignated by the instructor. The lab coat must be removed before leaving and kept in a designated area
of the lab.

3. Avoid creating aerosols when working with pathogens. If there is a chance of creating tiny airborne
droplets, work under a safety hood.

Summary of Biosafety

Levels for Infectious Agents

Biosafety level (BSL)

Description of infectious agents

Examples from this lab manual

Agents that typically do not cause

Alcaligenes denitrificans

disease in healthy adults; they

Ale ali genes faecalis

generally do not pose a disease

Bacillus cereus

risk to humans.

Bacillus subtilis

Corynebacterium pseudodiphtheriticum

Enterobacter aerogenes

Enterococcus faecalis

Escherichia coli

Micrococcus luteus

Neisseria sicca

Proteus vulgaris

Pseudomonas aeruginosa

Serratia marcescens

Staphylococcus epidermidis

Staphylococcus saprophyticus

Agents that can cause disease in

Klebsiella pneumoniae

healthy adults; they pose

Mycobacterium phlei

moderate disease risk to

Salmonella typhimurium

humans.

Shigella flexneri
Staphylococcus aureus
Streptococcus pneumoniae
Streptococcus pyogenes

Agents that can cause disease in

None; these agents are not used in

healthy adults; they are airborne

this lab manual.

and pose a more serious disease

risk to humans.

Agents that can cause disease in

None; these agents are not used in

healthy adults; they pose a

this lab manual.

lethal disease risk to humans;

no vaccines or therapy

available.

Xll

Alexander-Strete-Niles:

Front Matter

Safety Guidelines for the

Lab Exercises in Microbiology Laboratory Companies, 2003

Organismal and Molecular
Microbiology

Universal Precautions

All human blood and certain other body fluids are treated as if they are infectious for blood-borne pathogens,
such as human immunodeficiency virus (HIV), hepatitis B virus (HBV), and hepatitis C virus (HCV).
Such precautions are the rule among nurses, doctors, phlebotomists, and clinical laboratory personnel,
and are a critical component of infection control.

1 . Wear gloves.

2. Change gloves when they are soiled or torn.

3. Remove gloves when you are finished handling a specimen, and before you touch other objects such
as drawer handles, door knobs, refrigerator handles, pens/pencils, and paper.

4. Wash hands thoroughly with soap and water after removing gloves.

5. Dispose of gloves and blood-contaminated materials in a biohazard receptacle.

Additional precautions that may not apply to this laboratory exercise:

6. Wear a lab coat when soiling with blood or body fluids is possible.

7. Wear a mask, goggles, or glasses with side shields if splashing of the face is possible.

Safety Commitment

I have read and understand the safety guidelines described above. I declare my commitment to safety in
the microbiology laboratory and promise to follow each rule during the course of the semester.

Name

Date

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

I. Survey of Microscopic
Organisms

1. Structure, Function, and
Use of the Microscope

Structure, Function,

and Use of the Microscope

The study of microscopic organisms is greatly aided by
the use of microscopes. The light microscope (LM) mag-
nifies objects up to 1,000 times (l,000x) and can be used
to study cell size, shape, and arrangement. However, the
LM gives little information about internal cell structures.
The internal details of a cell are studied using a trans-
mission electron microscope (TEM), since useful mag-
nifications of up to 100,000x are possible. The infection
of a cell by viruses or bacteria can also be studied using
a TEM. In addition, a three-dimensional view of cells in
their natural environment is possible with a scanning
electron microscope (SEM). Useful magnifications of up
to 20,000x are obtained with a SEM.

This exercise is designed to familiarize you with the
structure, function, and use of the light microscope. In
addition, TEM and SEM views of cells will be provided
for comparison.

Prepared slides (2)
Blood (human)
Budding yeast

Equipment
Microscope

Miscellaneous supplies
Immersion oil
Lens paper

1 . Familiarize yourself with the structure and
function of the light microscope by reviewing
the following: (a) the microscope in figure 1.1;
(b) the parts of the microscope and their
functions in table 1.1; and (c) the magnifications
obtained using different objectives in table 1.2.
Complete step 1 of the laboratory report.

Table 1 A Functions of the Parts of

the Light Microscope*

Part

Function

Ocular (eyepiece)

Magnifies image, usually lOx

Thumb wheel

Adjusts distance between
oculars to match your eyes

Lock screw

Secures head after rotation

Head

Holds oculars

Arm

Holds head and stage

Revolving

Rotates objective lenses

nosepiece

into viewing position

Objective

Magnifies image, usually low
(4x), medium (lOx), high dry
(40x), and oil-immersion
(100X)

Slide holder

Fixed and movable parts
secure slide on stage

Mechanical

Includes slide holder and is

stage

used to locate specimen

10.

Stage

Holds slide

11.

Stage aperture

Admits light

12.

Condenser

Focuses light on specimen
and fills lens with light

13.

Diaphragm lever

Controls amount of light
entering stage aperture

14.

Substage-
adjustment knob

Raises and lowers condenser

15.

Mechanical-

Moves slide back and forth

stage control

on stage

16.

Light source

Illuminates specimen

17.

Coarse-

Rapidly brings specimen into

adjustment knob

focus

18.

Fine-adjustment

Slowly brings specimen into

knob

sharp focus

19.

Base

Supports microscope

*Parts are listed in order from top to bottom, and their numbers
correspond to those in figure 1.1.

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

I. Survey of Microscopic
Organisms

1. Structure, Function, and
Use of the Microscope

Structure, Function, and Use of the Microscope EXERCISE 1 3

(3) Lock screw

(5) Arm

(7) Objective

(10) Stage

(15) Mechanical-
stage control

(17) Coarse-
adjustment knob

(18) Fine-
adjustment knob J

(14) Substage-

adjustment knob

(19) Base

(1) Ocular

(2) Thumb
wheel

(4) Head

(6) Revolving
nosepiece

(9) Mechanical
stage

(8) Slide holder

(11) Stage aperture
near center

(12) Condenser

(13) Diaphragm
lever

(16) Light source

Figure 1.1 The parts of the microscope.

Table 1 .2 Total Magnification Possible

with Different Objective Lenses
of the Light Microscope

Power

Low
Medium
High dry
Oil-

lmmersion

Objective Ocular Total

lens lens magnification

lOx

40x

lOOx

lOx
lOx
lOx
lOx

40x

lOOx

400x

1 ,000x

2. Table 1.3 lists the steps for using the light
microscope. Follow these steps carefully as you
examine two slides: human blood and budding
yeast. Using figure 1.2 as a guide, identify as
many of the cell types and structures as you can
For each slide, record in the laboratory report
what you see at 40x, lOOx, 400x, and l,000x.

3 . Examine the photographs of the TEM
(figure 1.3) and the SEM (figure 1.4).
Also examine the images of cells that these
microscopes provide (figures 1.5-1.8).
How do these views of cells differ from
those provided by the light microscope?

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

I. Survey of Microscopic
Organisms

1. Structure, Function, and
Use of the Microscope

4 SECTION I Survey of Microscopic Organisms

Carry the microscope upright with two hands (figure 1.9, p. 10). Place the microscope on the countertop, plug it in,
and turn on the light. Follow these steps as you examine the human blood and budding yeast slides:

1 . Clip the slide into place on the stage using the slide holder.

2. Use the mechanical- stage control to move the slide so that the specimen is centered over the condenser.

3. Rotate the nosepiece to position the 4x objective (figure 1.10a, p. 11). When this objective is in place over the
specimen, move the coarse-adjustment knob until the stage and objective are as close together as possible.

4. While looking through the oculars, move the coarse-adjustment knob to slowly increase the distance between the
stage and the objective. Stop when the specimen comes into focus.

5. Adjust the distance of the ocular lens by moving the thumb wheel until two images become one.

6. Close your left eye, and focus for the right eye using the fine-adjustment knob. Close your right eye, and focus for
the left eye using the focusing ring on the left ocular lens. Open both eyes and move the fine-adjustment knob
until a sharp image is obtained. You are now ready to make your observations at 40x total magnification.

7. Center the specimen, and then rotate the nosepiece to position the lOx objective (figure 1.10&, p. 11). Since most
microscopes are parfocal, the only adjustment that should be necessary is the fine adjustment. When the image is
sharp, make your observations at lOOx total magnification.

8. Rotate the nosepiece to position the 40x objective (figure 1.10c, p. 11). Move the fine-adjustment knob, and make
your observations at 400x total magnification.

9. Move the 40x objective out of the way, and place a drop of immersion oil on top of the specimen. Position the
lOOx oil-immersion objective (figure lAOd, p. 11). Move only the fine-adjustment knob. You may need to open
the iris diaphragm with the diaphragm lever to allow more light to enter the objective lens. Make your
observations at 1 ,000x total magnification.

10. When observations are complete, position the 4x objective lens and wipe the oil off the oil-immersion objective
with a piece of lens paper. Remove the slide from the stage, and wipe off the oil if the specimen is covered by a
coverslip. If not, let the oil drain off by placing the slide upright in a slide box.

11. When finished, turn off the light, unplug the cord, and wrap it around the base. Return the microscope to the
storage cabinet.

Lobed nucleus

Nuclei

Parent cell

Red blood cells

Neutrophils

(a)

Buds

Lymphocytes

Yeast cells

(b)

Figure 1.2 (a) Formed elements of human blood (l,000x); (b) Yeast cells (l,000x)

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

I. Survey of Microscopic
Organisms

1. Structure, Function, and
Use of the Microscope

Structure, Function, and Use of the Microscope EXERCISE 1 5

Figure 1.3 Transmission electron microscope (TEM)

Figure 1.4 Scanning electron microscope (SEM).

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

I. Survey of Microscopic
Organisms

1. Structure, Function, and
Use of the Microscope

6 SECTION I Survey of Microscopic Organisms

Figure 1.5 TEM view of white blood cells showing the internal structures characteristic of eucaryotic cells (12,000x).

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

I. Survey of Microscopic
Organisms

1. Structure, Function, and
Use of the Microscope

Structure, Function, and Use of the Microscope EXERCISE 1 7

,.*&- . '.V ■ *. .... -.

•-Sjia .-;• ......

■■*"•"

v,c- : -.v..

.-■■ ■ ' > -■

Figure 1.6 TEM view of a virus-infected cell. Viruses are the circular particles with dark centers (20,000x).

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

I. Survey of Microscopic
Organisms

1. Structure, Function, and
Use of the Microscope

8 SECTION I Survey of Microscopic Organisms

Figure 1.7 TEM view of a C 'hlamydia-ini "ected cell. Chlamydia bacteria are the numerous dark circles (3,OOOx).

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

I. Survey of Microscopic
Organisms

1. Structure, Function, and
Use of the Microscope

Structure, Function, and Use of the Microscope EXERCISE 1 9

Figure 1.8 SEM view of fungal hyphae on the surface of a potato leaf (5,OOOx)

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

I. Survey of Microscopic
Organisms

1. Structure, Function, and
Use of the Microscope

10 SECTION I Survey of Microscopic Organisms

Figure 1.9 Method used to carry the light microscope.

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

I. Survey of Microscopic
Organisms

1. Structure, Function, and
Use of the Microscope

Structure, Function, and Use of the Microscope EXERCISE 1

(a) 4x objective

(b) lOx objective

(d) lOOx oil-immersion objective

Figure 1.10 Positions of light microscope objectives when viewing the specimen.

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

I. Survey of Microscopic
Organisms

1. Structure, Function, and
Use of the Microscope

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

I. Survey of Microscopic
Organisms

1. Structure, Function, and
Use of the Microscope

Name

Lab Section

EXERCISE

Laboratory Report

Date

Structure, Function, and Use of the Microscope

1 . Identify the parts (a-f ) of the microscope below, and fill in their functions

Part

Function

Part

d. _

Function

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

I. Survey of Microscopic
Organisms

1. Structure, Function, and
Use of the Microscope

14 SECTION I Survey of Microscopic Organisms

2. Depict the morphology of a few representative cells at each total magnification. Try to draw the cells
at the size scale you observed.

a. Human blood

Draw and label the
cell types you find.

40x

lOOx

400x

l,OOOx

b. Budding yeast

Draw and label parent
cells and buds you find.

40x

lOOx

400x

1 ,OOOx

Alexander-Strete-Niles:

I. Survey of Microscopic

1. Structure, Function, and

Lab Exercises in Organisms Use of the Microscope Companies, 2003

Organismal and Molecular
Microbiology

Structure, Function, and Use of the Microscope EXERCISE 1 15

3. Which microscope (LM, TEM, or SEM) would be most useful to study the following?
a. Size of cells

b. Whether or not a cell has a nucleus (i.e., is procaryotic or eucaryotic)

c. Whether or not a cell is infected with viruses

d. A three-dimensional view of cells attached to a surface

e. Cell shapes and arrangements

f. Cells infected with Chlamydia

4. Answer the following questions in the space provided.

a. (1) Give the general formula used to calculate the total magnification:

x = total magnification

(2) What is the total magnification when using the lOOx oil-immersion objective lens?
b. In general, should the condenser be kept close to or far from the stage? Explain.

c. When increasing magnification from high dry to oil-immersion, should the iris diaphragm be
open or closed? How is this done? Does this adjustment increase or decrease the light reaching
the objective lens?

d. Explain why oil must be used with the oil-immersion lens.

e. Based on your observations of blood cells and yeast cells, which total magnification would you
recommend for best viewing? Explain.

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

I. Survey of Microscopic
Organisms

2. Micro. Comparisons of
Microorganisms, Multi.
Parasites & Micro. Invert.

Microscopic Comparisons of Microorganisms,

Multicellular Parasites, and Microscopic
nvertebrates

Microorganisms (bacteria, cyanobacteria, fungi, pro-
tozoans, and algae) and small animals (multicellular
parasites and microscopic invertebrates) display a vari-
ety of shapes and sizes (table 2.1). Figure 2.1 depicts

Kingdom Plantae Kingdom Fungi Kingdom Animalia

(Multicellular eucaryotes)

Nonphotosynthetic
(absorb food)

Photosynthetic

Kingdom Protista

Kingdom Monera

Nonphotosynthetic
(ingest food)

Protozoans and algae
(unicellular eucaryotes)

Bacteria and cyanobacteria
(unicellular procaryotes)

(a) Whittaker system

Eucaryotes

Fungi

Animals

Eubacteria

Other bacteria

Cyanobacteria

Plants

Protozoans

Archaebacteria

Extreme thermophiles,
halophiles, and methanogens

(b) Woese system

Figure 2.1 Two classification systems recognized by
biologists and microbiologists: (a) the five-kingdom
classification system of R. H. Whittaker; (b) the
three-domain system of C. Woese.

two widely accepted classification systems for these
organisms. The Whittaker system, which consists of five
kingdoms, emphasizes differences in cellular traits and
nutrition, while the Woese system, which consists of
three domains, emphasizes differences in biochemical
traits. Neither system includes the viruses, due to their
unique makeup and method of replication.

In this exercise, you will use the microscope to
make comparisons of the microscopic organisms exam-
ined in Section I. You will learn to make size measure-
ments, and will measure a variety of microscopic
organisms. After you measure, be sure to note the mor-
phology of the microorganisms, multicellular parasites,
and microscopic invertebrates.

Prepared slides (8)

Select one slide from each category in
table 2. 1 .

Equipment

Light microscope

Miscellaneous supplies
Immersion oil
Lens paper
Ocular micrometer
Stage micrometer slide

1 . Clip the stage micrometer slide into position
on the stage, and position the scale over the
condenser (figure 2.2a, b). Focus on the scale
using the 4x objective lens.

2. Align the ocular micrometer and stage
micrometer scales as depicted in figure 2.2c.
Now follow figure 2. 2d to calibrate the ocular
micrometer for the 4x objective lens.

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

I. Survey of Microscopic
Organisms

2. Micro. Comparisons of
Microorganisms, Multi.
Parasites & Micro. Invert.

18 SECTION I Survey of Microscopic Organisms

Table 2.1 Typical Sizes of Selected

Microscopic Organisms

Microscopic

Size (in

Organism

microns, |x)

Bacteria

Bacillus

Escherichia coli

2-3

Spirillum

Staphylococcus

Treponema pallidum

Cyanobacteria

Oscillatoria (filament)

400

Yeasts (fungi)

Saccharomyces (with bud)

Molds (fungi)

Aspergillus (conidiophore)

1,200

Rhizopus (zygospore)

400

Protozoans

Amoeba proteus

300

Paramecium caudatum

200

Algae

Diatoms (centric)

100

Diatoms (pennate)

Dinoflagellates

100

Spirogyra (filament)

2,500

Volvox (colony)

200

Multicellular parasites

Clonorchis sinensis

7,500

(liver fluke)

Dipylidium caninum

2,500

(tapeworm proglottid)

Microscopic invertebrates

Cyclops

500

Daphnia

500

Nauplius larvae

600

Tick

2,500

4x objective

Ocular micrometer

(b)

Stage micrometer

V V

(c)

Sample calculation from (c):

Stage micrometer Ocular micrometer

40x:

(1) 0.5 mm 20 ocular units (ou's)

(2) 1 .0 mm 40 ocular units (ou's)

(d)

Calibration

0.025 mm/ou
0.025 mm/ou
Average = 25 u/ou

Figure 2.2 Calibration of the ocular micrometer.

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

I. Survey of Microscopic
Organisms

2. Micro. Comparisons of
Microorganisms, Multi.
Parasites & Micro. Invert.

Microscopic Comparisons of Microorganisms, Multicellular Parasites, and Microscopic Invertebrates EXERCISE 2 19

Table 2.2 Calculations in the Calibration of the Ocular Micrometer

Stage micrometer

Ocular micrometer

Calibration

a. 40x
1. .

b. lOOx
1. _

c. 400x
1. _

d. l,OOOx

Calibration at lOOx/10

Average

3. Repeat the calibration steps for the lOx and 40x
objectives. To calculate the calibration for the
lOOx objective, take the calibration for the lOx
objective and divide by 10. Record your ocular
calibration results in table 2.2 and in the
laboratory report.

4. Select one slide from each category in table 2.1
(eight total). Using your ocular calibration
results, calculate and record in the laboratory

report the size of each organism at the
appropriate magnification. When comparing your
results to those in table 2.1, do not expect results
for every organism to be exactly like those
shown, since the size of individual cells and
cell groupings may vary.

5. Also be sure to depict the morphology of
each organism in the circles provided in the
laboratory report.

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

I. Survey of Microscopic
Organisms

2. Micro. Comparisons of
Microorganisms, Multi.
Parasites & Micro. Invert.

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

I. Survey of Microscopic
Organisms

2. Micro. Comparisons of
Microorganisms, Multi.
Parasites & Micro. Invert.

Name

Lab Section

EXERCISE

Laboratory Report

Date

Microscopic Comparisons of Microorganisms, Multicellular
Parasites, and Microscopic Invertebrates

1. Record your ocular calibration results from table 2.2.

40x:

lOOx:
400x:

|Li/ocular unit (ou)

u/ou

l,OOOx:

u/ou

2. Determine the size of each of the eight selected organisms by multiplying the length you measured in
ocular units by the appropriate ocular calibration result recorded in question 1 . Also sketch each

organism in the circle provided.
Bacteria

Cyanobacteria

Organism

Magnification

Length (in ou's)

Organism

Magnification

Length (in ou's)

Size(

ou's X

u/ou) =

Size(

ou's X

u/ou) =

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

I. Survey of Microscopic
Organisms

2. Micro. Comparisons of
Microorganisms, Multi.
Parasites & Micro. Invert.

22 SECTION I Survey of Microscopic Organisms

Yeasts (fungi)

Molds (fungi)

Organism

Magnification

Length (in ou's)

Size(

ou's X

|u/ou) =

Organism

Magnification

Length (in ou's)

Size(

ou's X

u/ou) =

Protozoans

Algae

Organism

Magnification

Length (in ou's)

Organism

Magnification

Length (in ou's)

Size(

ou's X

|Ll/0U) =

Size(

ou's X

|Ll/0U) =

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

I. Survey of Microscopic
Organisms

2. Micro. Comparisons of
Microorganisms, Multi.
Parasites & Micro. Invert.

Microscopic Comparisons of Microorganisms, Multicellular Parasites, and Microscopic Invertebrates EXERCISE 2 23

Multicellular parasites

Microscopic invertebrates

Organism

Magnification

Length (in ou's)

Organism

Magnification

Length (in ou's)

Size(

ou's X

|u/ou) =

Size(

ou's X

|u/ou) =

3. List the eight organisms based on size, from smallest (1) to largest (8). Also list the magnification used to

view each organism.

Organism

Size (ji)

Magnification used for viewing

2.
3.
4.
5.
6.
7.
8.

4. Answer the following questions in the space provided.

a. Based on your measurements and morphological observations, describe how the following
microorganisms are different from one another.

How are cyanobacteria different from bacteria?

How are yeasts different from bacteria?

How are molds different from bacteria and yeasts?

How are protozoans different from bacteria?

How are protozoans different from algae?

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

I. Survey of Microscopic
Organisms

2. Micro. Comparisons of
Microorganisms, Multi.
Parasites & Micro. Invert.

24 SECTION I Survey of Microscopic Organisms

b. Based on the Whittaker system, to which kingdom do the following organisms belong?

Kingdom

Organism

Protozoans

Yeasts

B ac teri a

Microscopic invertebrates

Algae

Molds

Multicellular parasites

Cyanobacteria

5. Identify each of the following photos as bacteria, cyanobacteria, yeasts, molds, protozoans, algae,
a multicellular parasite, or a microscopic invertebrate.

**i -■* y k

r/-*i< r £&T ,iH

F '-t v- »-**

*-*v~t.'Y<

*■>

/*j:

-■v

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

I. Survey of Microscopic
Organisms

3. Microbial

Procaryotes:Bacteria and
Cyanobacteria

Microbial Procaryotes: Bacteria
and Cyanobacteria

Bacteria and cyanobacteria are both procaryotic

microorganisms that belong to the Kingdom Monera
in the Whittaker classification scheme. All pathogenic
bacteria and most environmental bacteria are het-
erotrophic, lacking the light-absorbing pigments nec-
essary to carry out photosynthesis. In contrast,

cyanobacteria are autotrophic, containing the neces-
sary light- absorbing pigments to carry out photosyn-
thesis. Cyanobacteria and algae are responsible for the
majority of the organic production that occurs in aquatic
environments and wet soils.

Bacteria come in a variety of cell shapes, includ-
ing rod, club, spirillum, spirochete, vibrio, and coc-
cus (figure 3.1(2). When bacteria grow (one cell dividing

Short rod

Long rod i

CZZ)CZ)

Diplobacilli

( X )( )( ) Streptobacilli

X_Z>

cz>

Cords

Club

V-shapes

Spirillum

Spirochete J\/f\/\/\/\/

Vibrio

Coccus

Diplococci

Tetrads

QGGQQO

Streptococci

Staphylococci

(a) Cell shapes

(b) Cell arrangements
(after cell division)

Figure 3.1 (a) Cell shapes and (b) cell arrangements in bacteria.

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

I. Survey of Microscopic
Organisms

3. Microbial

Procaryotes:Bacteria and
Cyanobacteria

26 SECTION I Survey of Microscopic Organisms

Colonies

Chains

Filaments

Figure 3.2 Cell arrangements in cyanobacteria.

to become two, the two cells dividing to become four,
the four cells dividing to become eight, and so on), cells
may separate or remain together. If cells remain
together, a number of cell arrangements are possible,
such as diplobacilli, streptobacilli, cords, V-shapes,
diplococci, tetrads, streptococci, and staphylococci
(figure 3.1b). Cell shape and arrangement are important
characteristics used to identify bacteria.

Cyanobacteria come in a variety of shapes and
arrangements as well. Their cells may be spherical or
cubical, and arranged in a colony, chain, or filament
(figure 3.2).

In this exercise, you will examine the variety of
cell shapes and arrangements seen in bacteria and
cyanobacteria.

Materials

Prepared slides
Bacteria (11)

Bacillus (large rods and streptobacilli)
Coryne bacterium diphtheriae (club and

V- shapes); causes diphtheria
Escherichia coli (short rods)
Micrococcus luteus (cocci and tetrads)
Mycobacterium tuberculosis (rods and

cords); causes tuberculosis
Neisseria gonorrhoeae (cocci and

diplococci); causes gonorrhea
Spirillum volutans (spirillum)
Staphylococcus epidermidis (cocci and

staphylococci)
Streptococcus pyogenes (cocci and

streptococci); causes strep throat
Treponema pallidum (spirochete); causes

syphilis
Vibrio cholerae (vibrio); causes cholera

Cyanobacteria (4)
Anabaena (chains)
Gleocapsa (colony)
Nostoc (chains)
Oscillatoria (filaments)

Equipment

Light microscope

Miscellaneous supplies
Immersion oil

Lens paper

Procedure

1 . Examine each of the prepared slides of bacteria
using the oil-immersion lens. Note the variety
of cell shapes and arrangements displayed

by bacteria.

2. Examine the prepared slides of cyanobacteria
using the lOx or 40x objective lens. Note the
variety of forms displayed by cyanobacteria.

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

I. Survey of Microscopic
Organisms

3. Microbial

Procaryotes:Bacteria and
Cyanobacteria

Name

Lab Section

EXERCISE

Laboratory Report

Date

Microbial Procaryotes: Bacteria and Cyanobacteria

1 . Draw the bacteria and cyanobacteria you observed. Depict cell size, shape, and arrangement as
accurately as possible.

a. Bacteria

Bacillus
Magnification

Cell shape

Corynebacterium diphtheriae

Magnification

Cell arrangement

Cell shape

Cell arrangement

Escherichia coli

Magnification

Cell shape

Micrococcus luteus

Magnification

Cell shape

Cell arrangement

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

I. Survey of Microscopic
Organisms

3. Microbial

Procaryotes:Bacteria and
Cyanobacteria

28 SECTION I Survey of Microscopic Organisms

Mycobacterium tuberculosis

Magnification

Cell shape

Cell arrangement

Neisseria gonorrhoeae

Magnification

Cell shape

Cell arrangement

Spirillum volutans

Magnification

Cell shape

Staphylococcus epidermidis

Magnification

Cell shape

Cell arrangement

Streptococcus pyogenes

Magnification

Cell shape

Treponema pallidum

Magnification

Cell shape

Cell arrangement

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

I. Survey of Microscopic
Organisms

3. Microbial

Procaryotes:Bacteria and
Cyanobacteria

Microbial Procaryotes: Bacteria and Cyanobacteria EXERCISE 3 29

Vibrio cholerae

Magnification

Cell shape

b. Cyanobacteria

Anabaena
Magnification

Cell shape

Cell arrangement

Gleocapsa
Magnification

Cell shape

Cell arrangement

Nostoc

Magnification

Cell shape

Oscillatoria
Magnification

Cell shape

Cell arrangement

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

I. Survey of Microscopic
Organisms

3. Microbial

Procaryotes:Bacteria and
Cyanobacteria

30 SECTION I Survey of Microscopic Organisms

2. Answer the following questions in the space provided,
a. How are bacteria and cyanobacteria similar? Dissimilar?

b. Why is "cyanobacteria" a more appropriate term than "blue-green algae"?

c. Can cell shape and arrangement be useful in bacterial identification? If so, give three specific
examples based on your observations.

3. Identify the cell shape and arrangement depicted in the following photographs of bacteria and
cyanobacteria. Also give an example of a genus with these traits.

J7-- 7 — t 1 — j _ i# . , 1 . W I — I

"*■ J.

a. Cell shape
Genus

b. Cell shape

Cell arrangement
Genus

c. Cell shape

Cell arrangement
Genus

?*»* r 5

d. Cell shape

Cell arrangement

e. Cell shape
Genus

f. Cell shape

Cell arrangement

Genus

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

I. Survey of Microscopic
Organisms

3. Microbial

Procaryotes:Bacteria and
Cyanobacteria

Microbial Procaryotes: Bacteria and Cyanobacteria EXERCISE 3 31

••

■

•

(

■

> r

■

r i

^ -*

>»

g. Cell shape
Genus

h. Cell shape

Cell arrangement

i. Cell shape

Cell arrangement

Genus

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

I. Survey of Microscopic
Organisms

4. Microbial Eucaryotes:
Fungi

Microbial Eucaryotes: Fungi

Fungi exhibit a diversity of growth forms, such as
yeasts, molds, mushrooms, cup fungi, and lichens

(figure 4.1a). These organisms reproduce in a variety of
ways: (1) formation of a bud from a parent yeast cell;

(2) addition of new cells to chains of cells called
hyphae; and (3) production of asexual and sexual
spores (figure 4.1b). The type of sexual spore produced,
whether zygospore, ascospore, or basidiospore, is
used to classify fungi into groups.

Bud

Vegetative cells
of hyphae

Cap
Gills

Stipe

Yeast

Mold

Mushroom

Cup fungus

Lichen (fruticose)

(a) Growth forms

Sporangiospores

Asexual

Sporangium

Sporangiophore

Conidia

Conidiophore

(b) Spore types

Figure 4.1 (a) Growth forms and (b) spore types in fungi

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

I. Survey of Microscopic
Organisms

4. Microbial Eucaryotes:
Fungi

34 SECTION I Survey of Microscopic Organisms

This exercise will introduce you to the variety
of growth forms in fungi and their methods of
reproduction.

Fungal cultures on Sabouraud dextrose agar (4)
Aspergillus (mold)
Penicillium (mold)
Rhizopus (mold)
Saccharomyces (yeast)

Prepared slides of fungi (6)

Candida albicans (pathogenic yeast);

causes candidiasis
Coprinus (mushroom with basidiospores

on gills)
Peziza (cup fungus with ascospores)
Physcia (lichen with fungi and algae

symbiosis)
Rhizopus (bread mold with zygospores)
Saccharomyces (brewing and baking yeast

with buds)

Dry specimens of fungi obtained locally (2)
Lichens (on a tree branch)
Mushrooms (from a field or market)

Equipment

Dissecting microscope
Light microscope

Miscellaneous supplies
Clear tape
Glass slides
Immersion oil

Lactophenol cotton blue (for staining molds)
Lens paper

1 . a. Examine the colonies of the four fungal

cultures. The use of a dissecting microscope
may aid your examination.

b. After examining the colonies, make a pressure
tape preparation of the three mold cultures

using the steps outlined in figure 4.2. Examine
this preparation using the light microscope.
Note the structures you see, including hyphae
and asexual spores.

2. Examine the six prepared slides of fungi using
the light microscope. Note the distinctive
structure of each fungus examined, including
hyphae, buds, conidia, zygospores, ascospores,
and basidiospores.

3. Examine and record your observations of the dry
specimens of a mushroom and lichens on a tree
branch.

(a) Using a pipette, place a
drop of lactophenol
cotton blue on the
center of the slide.

(b) Hold a piece of
clear tape in a
U-shape, sticky side
down.

Sticky side

Colony of mold

(d) Place tape sticky side
down in a drop of
lactophenol cotton
blue.

(e) Fold extra length of
tape around edges of
slide. Examine
microscopically.

Figure 4.2 Pressure-tape preparation of fungi.

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

I. Survey of Microscopic
Organisms

4. Microbial Eucaryotes:
Fungi

Name

Lab Section

EXERCISE

Laboratory Report

Date

Microbial Eucaryotes: Fungi

1 . Record your results from the examination of fungal cultures

Fungal culture

Colony description

Aspergillus (mold)

Penicillium (mold)

Rhizopus (mold)

Saccharomyces (yeast)

2. Draw from the microscopic examination of pressure-tape preparations of the three mold cultures

Aspergillus
Magnification

Penicillium
Magnification

Rhizopus
Magnification

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

I. Survey of Microscopic
Organisms

4. Microbial Eucaryotes:
Fungi

36 SECTION I Survey of Microscopic Organisms

3. Draw the organisms you observed in the prepared slides of fungi

Candida albicans, pathogenic
yeast

Magnification

Coprinus, mushroom
(gill with basidiospores)

Magnification

Peziza, cup fungus
(ascospores)

Magnification

Physcia, lichen

(fungal filaments and algae)

Magnification

Rhizopus, bread mold
(zygospores)

Saccharomyces, yeast
(cells with buds)

Magnification

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

I. Survey of Microscopic
Organisms

4. Microbial Eucaryotes:
Fungi

Microbial Eucaryotes: Fungi EXERCISE 4 37

4. a. Draw a mushroom, and label the following parts: stipe, cap, and gills.

b. Draw a lichen on a tree branch. What two components form a lichen?

5. Answer the following questions in the space provided,
a. Describe two differences between molds and yeasts

b. Name two characteristics that are used to distinguish one fungus from another.

c. Aspergillus fumigatus causes an infection of the lungs called aspergillosis. How do you think this
disease is acquired?

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

I. Survey of Microscopic
Organisms

4. Microbial Eucaryotes:
Fungi

38 SECTION I Survey of Microscopic Organisms

6. Identify the following photos.

• '

■

1 ■

■■

•

_ - .

■ ';•■■>
■ . • '-

. i > j

•' u il

V ^1

,^r

J-

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

I. Survey of Microscopic
Organisms

5. Microbial Eucaryotes:
Protozoans and Algae

Microbial Eucaryotes: Protozoans and Algae

In the Whittaker classification scheme, protozoans and
algae are members of the Kingdom Protista. Proto-
zoans are unicellular, nonphotosynthetic protists that are
widespread in aquatic environments and wet soils. In

this group, the type of organelle for motility is an impor-
tant trait in classification. Protozoans have pseudo-
podia, cilia, or flagella, with the exception of the
members of one group, the sporozoans, which do not
have any of these structures (figure 5.1).

Sarcodina

(amebas)

Pseudopodia

Amoeba

Ciliophora

(ciliates)

Cilia

,,„„33?

Paramecium

Mastigophora

(flagellates)

Flagella

Trichomonas

Apicomplexa

(sporozoans)

Ring stage
Red blood cell

Plasmodium

Figure 5.1 Representative protozoans, listed by phylum name.

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

I. Survey of Microscopic
Organisms

5. Microbial Eucaryotes:
Protozoans and Algae

40 SECTION I Survey of Microscopic Organisms

Table 5.1 Distinguishing Traits of the Algae

Algal group (division)

Distinguishing traits

Diatoms (Chrysophyta)

Dinoflagellates (Pyrrophyta)
Euglenoids (Euglenophyta)
Green algae (Chlorophyta)

Brown algae (Phaeophyta)
Red algae (Rhodophyta)

Unicellular or chains of cells; silica cell walls consisting of overlapping halves;
freshwater and marine

Unicellular; armor of cellulose plates; 2 flagella; marine

Unicellular; red eyespot; 1 or 2 flagella; 2 to many chloroplasts; freshwater

Unicellular, colonial, and filamentous micro-algae; multicellular macro-algae;
dominant pigment chlorophyll (green); freshwater and marine

Multicellular macro-algae; dominant pigment fucoxanthin (brown); marine

Multicellular macro-algae; dominant pigment phycobilins (red); marine

Algae are photosynthetic protists that inhabit
aquatic environments, where they are the primary
agents responsible for the synthesis of organic mole-
cules. They occur in a variety of forms, including uni-
cellular, colonial, and filamentous micro-algae, and
large, multicellular macro-algae. Several traits, such
as morphology and photosynthetic pigments, are used
to classify algae into the six groups shown in figure
5.2 and listed in table 5.1.

In this exercise, you will experience the diversity of
the Kingdom Protista by examining a variety of pro-
tozoans and algae.

Materials

Prepared slides or live cultures
Free-living protozoans (2)
Amoeba (ameba)
Paramecium (ciliate)

Prepared slides

Pathogenic protozoans (2)

Plasmodium (sporozoan); causes malaria
Trichomonas vaginalis (flagellate);
causes trichomoniasis

Micro-algae (6)

Cladophora (filamentous green algae)
Diatoms (unicellular and chain-forming

chrysophytes)
Dinoflagellates (unicellular pyrrophytes)
Euglena (unicellular euglenoids)
Spirogyra (filamentous green algae)
Volvox (colonial green algae)

Preserved whole specimens of macro-algae (3)
Padina (brown algae)
Sargassum (brown algae)
Ulva (green algae)

Pond water sample

Equipment

Light microscope

Miscellaneous supplies
Coverslips
Glass slides
Immersion oil
Lens paper
Pasteur pipette with bulb

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

I. Survey of Microscopic
Organisms

5. Microbial Eucaryotes:
Protozoans and Algae

Microbial Eucaryotes: Protozoans and Algae EXERCISE 5 41

Chrysophyta

(diatoms)

Pyrrophyta

(dinoflagellates)

Euglenophyta

(euglenoids)

Chlorophyta

(green algae)

Phaeophyta

(brown algae)

Rhodophyta

(red algae)

Centric

Pennate

r* n

Ceratium

Euglena

Spirogyra

Volvox

Sargassum

Polysiphonia

Figure 5.2 Representative algae, listed by division name

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

I. Survey of Microscopic
Organisms

5. Microbial Eucaryotes:
Protozoans and Algae

42 SECTION I Survey of Microscopic Organisms

1 . Examine microscopically the prepared slides or
live specimens of free-living protozoans and the
prepared slides of pathogenic protozoans. Live
specimens of free-living protozoans can be
examined using the wet mount preparation steps
depicted in figure 5.3. For all protozoans, note
the presence of pseudopodia, cilia, or flagella.

2. Examine microscopically the prepared slides of
micro- algae. During your examination, note
features such as cell morphology, cell
arrangement, shape of chloroplasts, and
unique structures.

3. Visually examine the preserved whole specimens
of macro- algae, noting morphology, color,

and unique structures.

4. Prepare several wet mounts of pond water using
the procedure in figure 5.3. The pond water
sample should contain both protozoans and
micro- algae in the bottom sediment, so make sure
you get some of this material with your pipette.
Draw a few of the representative organisms you
see under the microscope.

(a) Obtain water from the bottom of
a live specimen container or pond
water sample, and place on a
glass slide.

(b) Using a pair of forceps, position a
coverslip over the sample.

Figure 5.3 Wet mount preparation for viewing live specimens in pond water.

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

I. Survey of Microscopic
Organisms

5. Microbial Eucaryotes:
Protozoans and Algae

Name

Lab Section

EXERCISE

Laboratory Report

Date

Microbial Eucaryotes: Protozoans and Algae

1 . Draw your microscopic observations of free-living and pathogenic protozoans
a. Free-living protozoans

Amoeba (ameba)

Magnification

Pseudopodia, cilia, or

flagella?

Paramecium (ciliate)

Magnification

Pseudopodia, cilia, or

flagella?

b. Pathogenic protozoans

Plasmodium (sporozoan)

Magnification

Pseudopodia, cilia, or
flagella?

Trichomonas vaginalis (flagellate)

Magnification

Pseudopodia, cilia, or
flagella?

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

I. Survey of Microscopic
Organisms

5. Microbial Eucaryotes:
Protozoans and Algae

44 SECTION I Survey of Microscopic Organisms

2. Sketch your microscopic observations of micro-algae

Cladophora (green algae)

Magnification

Diatoms (chrysophytes)

Magnification

Dinoflagellates (pyrrophytes)

Magnification

Euglena (euglenoid)

Magnification

Spirogyra (green algae)

Volvox (green algae)

Magnification

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

I. Survey of Microscopic
Organisms

5. Microbial Eucaryotes:
Protozoans and Algae

Microbial Eucaryotes: Protozoans and Algae EXERCISE 5 45

3. Draw the general morphology of the preserved whole specimens of macro-algae.

Padina (brown algae)

Sargassum (brown algae)

Ulva (green algae)

4. Sketch several representative forms of protozoans and micro-algae you observed in the pond water,
a. Protozoans

Magnification

b. Micro-algae

Magnification

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

I. Survey of Microscopic
Organisms

5. Microbial Eucaryotes:
Protozoans and Algae

46 SECTION I Survey of Microscopic Organisms

5. Answer the following questions in the space provided.

a. On what basis are protozoan groups differentiated from one another?

b. On what basis are algal groups differentiated from one another?

c. How are protozoans and algae similar? Dissimilar?

6. Identify the following photos.

/* •

a. Genus

b. Genus

c. Genus

Algal group

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

I. Survey of Microscopic
Organisms

6. Flatworms and
Roundworms

Flatworms and Roundworms

Multicellular parasites include flatworms (flukes and
tapeworms) of the Phylum Platyhelminthes, and round-
worms of the Phylum Nematoda. While flatworms are
flattened in cross section, roundworms are round in
cross section.

Flukes are flatworms with oral suckers and ven-
tral suckers. Flukes, such as the Chinese liver fluke,
Clonorchis sinensis, and the blood fluke, Schistosoma
mansoni, infect humans, causing clonorchiasis and
schistosomiasis, respectively. Clonorchiasis is acquired
by ingesting raw or undercooked fish, while schisto-
somiasis is acquired when larvae penetrate human skin
(figure 6.1).

Tapeworms are flatworms that have an anterior
scolex for intestinal attachment and produce reproduc-
tive segments called proglottids. The tapeworms of the
genus Taenia infect humans, causing taeniasis. This
infection is contracted by ingesting undercooked beef
or pork (figure 6.2).

Roundworms also infect humans, including the
roundworm Ascaris lumbricoides, the cause of ascari-
asis; Enterobius vermicularis, the cause of enterobiasis;
and Trichinella spiralis, the cause of trichinosis. Ascari-
asis and enterobiasis are contracted by ingesting food
or water contaminated with roundworm eggs, while
trichinosis is contracted by consuming undercooked
pork (figure 6.3).

In this exercise, you will examine basic structural
characteristics and life cycle aspects of these multicel-
lular parasites.

Preserved specimen (1)

Ascaris (roundworms, male and female)

Prepared slides
Flukes (5)

Clonorchis (adult)

Clonorchis (eggs)
Schistosoma (adult)
Schistosoma (eggs)
Schistosoma (cercaria)

Tapeworms (4)
Taenia (scolex)

Taenia (mature or gravid proglottid)
Taenia (eggs)
Taenia (cysticercus)

Roundworms (5)
Ascaris (eggs)
Enterobius (adult)
Enterobius (eggs)
Trichinella (adult)
Trichinella (larvae)

Equipment
Microscope

Miscellaneous supplies
Immersion oil
Lens paper

1 . Examine the prepared slides of flukes,
noting unique structures, such as oral and
ventral suckers.

2. Examine the prepared slides of tapeworms,
noting unique structures, such as the scolex
and reproductive proglottids.

3. a. Examine the prepared slides of the

roundworms, noting unique structures.

b. Examine the preserved specimens of Ascaris
lumbricoides. Note the morphological
differences between the male and female.

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

I. Survey of Microscopic
Organisms

6. Flatworms and
Roundworms

48 SECTION I Survey of Microscopic Organisms

Clonorchis

Metacercaria
develops in
fish muscle.

Adult flukes
(in humans)

Schistosoma adult (male)

Eggs in stool

Cercaria
penetrates
fish muscle

{Clonorchis)

Cercaria

penetrates

human skin

{Schistosoma)

Eggs develop into

swimming miracidium

in water.

Swimming cercaria
escapes.

Miracidium penetrates
freshwater snail host.

Schistosoma cercariae

Figure 6.1 Life cycles of two flukes, Clonorchis sinensis and Schistosoma mansoni.

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

I. Survey of Microscopic
Organisms

6. Flatworms and
Roundworms

Flatworms and Roundworms EXERCISE 6

Taenia saginata
scolex

Taenia solium
scolex

Adult tapeworms
(in human intestine)

Scolex

attaches to

intestine.

Gravid

proglottids

in stool

Cysticercus
excysts in
intestine.

Eggs from
proglottids

Ingestion of

undercooked

beef or pork

by humans

Eggs develop

into cysticercus

in muscle.

Figure 6.2 Life cycle of the tapeworm, Taenia.

Ingestion by
cows or pigs

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

I. Survey of Microscopic
Organisms

6. Flatworms and
Roundworms

50 SECTION I Survey of Microscopic Organisms

Enterobius
adult

Ascaris
adults

Trichinella
adults

Ingestion of

undercooked

pork

Larvae in

pig muscle

{Trichinella)

Adult roundworms
(in human intestine)

Adults produce

larvae in muscle

(Trichinella).

Eggs deposited on

inanimate objects;

picked up on hands by

others and ingested

Eggs deposited by
female in perianal
region; picked up

on fingers by
scratching

(Enterobius)

Eggs in stool
(Ascaris)

Ingestion of food or

water contaminated

by eggs

Figure 6.3 Life cycle of three roundworms

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

I. Survey of Microscopic
Organisms

6. Flatworms and
Roundworms

Name

Lab Section

EXERCISE

Laboratory Report

Date

Flatworms and Roundworms

1 . Sketch the specimens you examined
a. Flatworms: Flukes

Clonorchis (adult)

Magnification

Clonorchis (eggs)

Magnification

Schistosoma (adult)

Magnification

Schistosoma (eggs)

Magnification

Schistosoma (cercaria)

Magnification

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

I. Survey of Microscopic
Organisms

6. Flatworms and
Roundworms

52 SECTION I Survey of Microscopic Organisms

b. Flatworms: Tapeworms

Taenia (scolex)

Magnification

Taenia (proglottid, mature or gravid)

Magnification

Taenia (eggs)
Magnification

Taenia (cysticercus)

Magnification

c. Roundworms

Ascaris (eggs)

Enterobius (adult)

Magnification

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

I. Survey of Microscopic
Organisms

6. Flatworms and
Roundworms

Flatworms and Roundworms EXERCISE 6 53

Enterobius (eggs)

Magnification

Trichinella (adult)

Magnification

Trichinella (larvae)

Magnification

Ascaris (male)

Ascaris (female)

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

I. Survey of Microscopic
Organisms

6. Flatworms and
Roundworms

54 SECTION I Survey of Microscopic Organisms

2. Check the morphological features that apply.

Multicellular
parasite

Body
flattened in
cross section

Body

round in

cross section

Oral/ventral
suckers

Scolex with
suckers/hooks

Reproductive

segments
(proglottids)

Flukes

Tapeworms

Roundworms

3. Fill in this table.

Multicellular parasite

How Contracted?

Name of disease

Flukes

Clonorchis

Schistosoma

Tapeworms
Taenia

Roundworms
Ascaris

Enterobius

Trichinella

4. Identify the following photos.

Alexander-Strete-Niles:

1. Survey of Microscopic

7. Zooplankton

©The McGraw-Hill

Lab Exercises in
Organismal and Molecular
Microbiology

Organisms

Companies, 2003

Zooplankton

A variety of microscopic invertebrates make up the
small animal plankton, or zooplankton, of aquatic envi-
ronments. Zooplankton feed on microscopic plant
plankton, or phytoplankton, and in turn are fed upon
by the higher-trophic-level consumers in aquatic food
chains (figure 7.1). Zooplankton, therefore, are a criti-
cal food chain link in aquatic environments.

Zooplankton are subdivided into two categories,
holoplankton and meroplankton. Holoplankton are the
permanent members of the zooplankton, and include
copepods, cladocerans, rotifers, and ostracods. Mero-
plankton are the temporary members of the zooplank-
ton, and include the larval stages of benthic marine
animals such as polychaetes, gastropods, barnacles,
crabs, and starfish. Larvae change into adult animals
and settle to the bottom to take on the benthic lifestyle.

In this exercise, you will examine prepared slides
of representative zooplankton from freshwater and
marine environments. Marine zooplankton will include
examples of both holoplankton and meroplankton. You
will also examine preserved samples of plankton, if
these are available.

Prepared slides

Freshwater zooplankton (3)
Cyclops (copepod)
Daphnia (cladoceran)
Rotifers

Marine zooplankton (10)

Bipinnaria (early starfish larvae)

Brachiolaria (late starfish

Calanus (copepod)

Megalops (late crab larvae)

Nauplius (early barnacle larvae)

Ostracod

Planula (early jellyfish larvae)

Trochophore (early polychaete larvae)

Veliger (gastropod larvae)

Zoea (early crab larvae)

Preserved plankton samples (2)
Freshwater
Marine

Equipment

Light microscope
Dissecting microscope

Miscellaneous supplies
Immersion oil
Lens paper

Pasteur pipette with bulb
Sample dish

1 . Examine the prepared slides of freshwater
zooplankton.

2. Examine the prepared slides of marine
zooplankton, including both holoplankton and
meroplankton.

3. Examine plankton samples, if available. Transfer
some of the sample to a dish with a Pasteur
pipette. Examine the contents in the dish with a
dissecting microscope, and note the types of
zooplankton you see.

Light

Water

Carbon dioxide
Nutrients

Phytoplankton
1° producers

Zooplankton
1° consumers

Plankton feeders

2° consumers

Higher-level consumers
374° consumers

Figure 7.1 Food chain diagram of aquatic ecosystems.

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

I. Survey of Microscopic
Organisms

7. Zooplankton

Alexander-Strete-Niles:

1. Survey of Microscopic

7. Zooplankton

©The McGraw-Hill

Lab Exercises in
Organismal and Molecular
Microbiology

Organisms

Companies, 2003

Name

Lab Section

EXERCISE

Laboratory Report

Date

Zooplankton

1 . Draw the freshwater zooplankton you observed in the prepared slides

Cyclops (copepod)

Magnification

Daphnia (cladoceran)

Magnification

Rotifers

Magnification

2. Draw the marine zooplankton you observed in the prepared slides

Bipinnaria (starfish larva)
Magnification

Brachiolaria (starfish larva)
Magnification

Alexander-Strete-Niles:

1. Survey of Microscopic

7. Zooplankton

©The McGraw-Hill

Lab Exercises in
Organismal and Molecular
Microbiology

Organisms

Companies, 2003

58 SECTION I Survey of Microscopic Organisms

Calanus (copepod)

Magnification

Megalops (crab larva)

Magnification

Nauplius (barnacle larva)

Magnification

Ostracod
Magnification

Planula (jellyfish larva)

Trochophore (polychaete larva)

Magnification

Alexander-Strete-Niles:

1. Survey of Microscopic

7. Zooplankton

©The McGraw-Hill

Lab Exercises in
Organismal and Molecular
Microbiology

Organisms

Companies, 2003

Zooplankton EXERCISE 7 59

Veliger (gastropod larva)

Magnification

Zoea (crab larva)

Magnification

3. Draw several of the common zooplankton organisms you observed in the plankton samples

Freshwater plankton
4. Answer the following questions in the space provided.

Marine plankton

a. Depict an aquatic food chain showing the position of the zooplankton examined in this exercise

b. Explain the difference between holoplankton and meroplankton.

Alexander-Strete-Niles:

1. Survey of Microscopic

7. Zooplankton

©The McGraw-Hill

Lab Exercises in
Organismal and Molecular
Microbiology

Organisms

Companies, 2003

60 SECTION I Survey of Microscopic Organisms

5. Identify the following members of the zooplankton

J-

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

I. Survey of Microscopic
Organisms

8. Disease Vectors

Disease Vectors

Disease-causing microorganisms can be transmitted in a
variety of ways, including through air, food and water, and
sexual contact. Vectors also transmit disease-causing
microorganisms.

Most disease vectors are arachnids or insects that
belong to the Phylum Arthropoda. Arthropod vectors
include ticks, lice, mosquitoes, and fleas (figure 8.1).
These organisms bite humans and in the process trans-
mit pathogens.

In this exercise, you will examine the arthropod
vectors of human diseases.

Chrysops (deer fly)
Culex (mosquito)
Dermacentor (tick)
Glossina (tsetse fly)
Ixodes (tick)
Ornithodorus (tick)
Pediculus (human louse)
Xenopsylla (rat flea)

Equipment

Light microscope
Dissecting microscope

Examine the prepared slides of arthropod vectors, noting
their size, distinguishing structures, and unique features.

Prepared slides (10)
Aedes (mosquito)
Anopheles (mosquito)

Infected individual

Pediculus (human louse)

Dermacentor (tick)

Culex (mosquito)

Xenopsylla (rat flea)

Uninfected individual

Figure 8.1 Selected examples of arthropod disease vectors.

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

I. Survey of Microscopic
Organisms

8. Disease Vectors

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

I. Survey of Microscopic
Organisms

8. Disease Vectors

Name

Lab Section

EXERCISE

Laboratory Report

Date

Disease Vectors

1 . Draw the organisms you observed in the prepared slides

Aedes (mosquito)
Disease transmitted

Anopheles (mosquito)
Disease transmitted _

Chrysops (deer fly)
Disease transmitted

Culex (mosquito)
Disease transmitted

Dermacentor (tick)
Disease transmitted

Glossina (tsetse fly)
Disease transmitted

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

I. Survey of Microscopic
Organisms

8. Disease Vectors

64 SECTION I Survey of Microscopic Organisms

Ixodes (tick)
Disease transmitted

Ornithodorus (tick)
Disease transmitted

Pediculus (human louse)
Disease transmitted

Xenopsylla (rat flea)
Disease transmitted .

2. Answer the following questions in the space provided
a. Explain how these organisms transmit diseases.

b. Explain why certain diseases transmitted by vectors, such as Lyme disease, occur more frequently in

certain areas.

c. Ixodes, the tick vector of Lyme disease, can be found attached to the skin after a walk in the woods.
What would you recommend to a person going to the woods? What would you recommend to a
person returning from the woods?

Alexander-Strete-Niles:

II. Staining Techniques

9. Negative Stain

©The McGraw-Hill

Lab Exercises in
Organismal and Molecular
Microbiology

Companies, 2003

Negative Stain

Morphological stains color either bacterial cells them-
selves or their backgrounds to allow a clear microscopic
view of cells. Such clear views provide information
about cell size, shape, and arrangement.

Stains that color bacterial cells themselves carry a
positive charge and are called basic stains. Basic stains
color bacterial cells because they are attracted to the
negatively charged cell surface. Basic stains include
crystal violet, methylene blue, and safranin.

Stains that color the background surrounding bac-
terial cells carry a negative charge and are called acidic
stains. Acidic stains are repelled by the negatively
charged bacterial cell surface and, hence, color only the

background (figure 9.1). However, this still provides a
clear microscopic view, because the bacterial cells are
seen in outline. Acidic stains include congo red,
nigrosin, and india ink.

A single acidic stain used to color the background
around cells is called a negative stain. There are two
advantages of a negative stain: (1) it allows more accu-
rate determination of cell size and shape, since the pro-
cedure requires no heating or staining of cells (which
can cause cell shrinkage); and (2) it facilitates the
microscopic observation of cells that are difficult to
stain, such as spirilli and spirochetes.

In this exercise, you will use a single acidic stain
to determine the cell morphology of several bacterial
cultures.

Rod

Coccus

Cells and background
are colorless.

Acidic stain colors
the background; cells
remain colorless.

* -

■r

Bacillus cere us

Figure 9.1 The negative stain.

Staphylococcus epidermidis

Alexander-Strete-Niles:

II. Staining Techniques

9. Negative Stain

©The McGraw-Hill

Lab Exercises in
Organismal and Molecular
Microbiology

Companies, 2003

Negative Stain EXERCISE 9 67

Cultures (24-48-hour broth)
Bacillus cere us (rod)
Staphylococcus epidermidis (coccus)

Stains

Nigrosin, india ink, or congo red

Equipment

Light microscope

Miscellaneous supplies
Bunsen burner and striker
Disposable gloves (optional)
Glass slides
Immersion oil
Inoculating loop
Lens paper
Wax pencil

Procedure

1 . Place a drop of nigrosin, india ink, or congo red
near the edge of a clean glass slide.

2. Aseptically obtain a loopful of a broth culture
of Bacillus cereus by following the steps in
figure 9.2.

3 . Transfer the loopful of culture to the drop of stain
on the slide, and mix the culture into the drop, as
shown in figure 93a, b. Always flame your loop
before setting it down!

4. Follow steps c-e in figure 9.3 to complete your
preparation of a negative stain of Bacillus cereus.

5. Repeat steps 1-4 to prepare a negative stain of a
broth culture of Staphylococcus epidermidis.

6. After you have completed both negative stains,
examine them using the oil-immersion objective.

(a) Shake the culture tube from
side to side to suspend the
organisms.

(b) Heat the loop and wire to red
hot.

(.'

(d) After allowing the loop to
cool for 5 seconds, remove a
loopful of organisms. Avoid
touching the sides of the tube

(e) Flame the mouth of the tube
again.

(f ) Return the cap to the tube, and
place the tube in a test tube rack.
Transfer the loopful of organisms

Figure 9.2 Aseptic procedure for removing an organism from a broth culture

Alexander-Strete-Niles:

II. Staining Techniques

9. Negative Stain

©The McGraw-Hill

Lab Exercises in
Organismal and Molecular
Microbiology

Companies, 2003

68 SECTION II Staining Techniques

Drop of stain

Clean glass slide
(a)

Add loopful of culture, and mix.

(b)

(c)

Direction of
movement

(d)

Second slide

(45° angle with first slide)

Bacteria-stain suspension spreads
along back edge of slide.

Spread suspension

Allow to air-dry.

Stained material forms a thin film,
(e)

Figure 9.3 Negative staining procedure

Alexander-Strete-Niles:

II. Staining Techniques

9. Negative Stain

©The McGraw-Hill

Lab Exercises in
Organismal and Molecular
Microbiology

Companies, 2003

Name

Lab Section

EXERCISE

Laboratory Report

Date

Negative Stain

1 . Draw the cell shapes and arrangements you observed

Bacillus cereus

Magnification

Staphylococcus epidermidis

Magnification

Cell shape

Cell arrangement

2. Answer the following questions in the space provided.

Cell shape

Cell arrangement

a. Explain why nigrosin, india ink, and congo red do not stain bacterial cells

b. What are the advantages of negative stains?

Alexander-Strete-Niles:

II. Staining Techniques

10. Smear Preparation and

©The McGraw-Hill

Lab Exercises in
Organismal and Molecular
Microbiology

the Simple Stain

Companies, 2003

Smear Preparation
and the Simple Stain

The use of a single basic stain to color bacterial cells
is called a simple stain. Basic stains employed for this
purpose include safranin, crystal violet, and methyl-
ene blue. These stains color the bacterial cells so that
they are clearly visible with the microscope (figure

10.1). Since this procedure requires the heat-fixation of
a smear prior to stain application, it does result in some
cell shrinkage.

In this exercise, you will use a single basic stain
to color the cells of several bacterial cultures to reveal
their morphological characteristics.

Rod

Coccus

Rod and coccus mix

Cells are transparent
prior to staining.

Basic stain is added
so that cells are colored

Safranin

€fr®>

Crystal violet

Methylene blue

^L*

Pseudomonas aeruginosa

Figure 10.1 The simple stain.

y/%frfc?

Staphylococcus epidermidis

Pseudomonas aeruginosa
and Staphylococcus
epidermidis

Alexander-Strete-Niles:

II. Staining Techniques

10. Smear Preparation and

©The McGraw-Hill

Lab Exercises in
Organismal and Molecular
Microbiology

the Simple Stain

Companies, 2003

72 SECTION II Staining Techniques

Materials

Cultures (24-48 hour broth or agar)
Pseudomonas aeruginosa (rod)
Staphylococcus epidermidis (coccus)

Stains

Crystal violet, methylene blue, or safranin

Equipment

Light microscope

Miscellaneous supplies
Bibulous paper
Bunsen burner and striker
Clothespin

Disposable gloves (optional)
Glass slides
Immersion oil
Inoculating loop or needle

Lens paper

Staining tray

Water bottle with tap water

Wax pencil

Procedure

Smear Preparation

1 . Aseptically obtain a loopful of a broth culture of
Pseudomonas aeruginosa by following the steps
described in Exercise 9 (figure 9.2). If an agar
culture is used instead of broth, follow the steps
depicted in figure 10.2. Note: A loop or needle
can be used to transfer from an agar culture;
in either case, transfer only a pinhead amount
of growth.

Figure 10.2 Bacterial smear preparation and the simple stain procedure

(a) Flame the loop (or needle) to
red-hot to sterilize.

(b) Touch the loop (or needle) to
an isolated colony to pick up a
pinhead amount of growth.

(c) Transfer the growth to a drop of
water on a slide and thoroughly
mix to obtain a slightly milky
color. This mixture must be
air-dried and heat-fixed
before staining.

(d) Cover the heat-fixed smear with
stain and allow to sit for 60 seconds.

(e) After 60 seconds, wash off the
stain with a water rinse. After drying,
the stained smear is ready to observe.

Alexander-Strete-Niles:

II. Staining Techniques

10. Smear Preparation and

©The McGraw-Hill

Lab Exercises in
Organismal and Molecular
Microbiology

the Simple Stain

Companies, 2003

Smear Preparation and the Simple Stain EXERCISE 10 73

2. Transfer the loopful of broth culture to a glass
slide, and spread it out into a circle. If an agar
culture is used instead of broth, mix the pinhead
amount of culture into a drop of water on a glass
slide as shown in figure 10.2c. Prepare a mixture
that is only slightly milky in color. Do not
prepare a heavy suspension!

3 . Allow the slide to air-dry before heat-fixation.
Heat gently by passing the slide over the flame
several times. After heat-fixation, the slide is
ready to stain.

4. Repeat steps 1-3 to prepare a smear of
Staphylococcus epidermidis and to prepare a
smear of a mixture of Pseudomonas aeruginosa
and Staphylococcus epidermidis.

Simple Stain

1 . Apply crystal violet, methylene blue, or safranin
to the three smears, and let stand for 60 seconds
(figure 10.2J). Cover the entire smear with
stain!

2. After 60 seconds, gently wash off the stain with
tap water (figure 10.2c). Blot the slide with
bibulous paper, and examine using the oil-
immersion objective.

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

II. Staining Techniques

10. Smear Preparation and
the Simple Stain

Alexander-Strete-Niles:

II. Staining Techniques

10. Smear Preparation and

©The McGraw-Hill

Lab Exercises in
Organismal and Molecular
Microbiology

the Simple Stain

Companies, 2003

Name

Lab Section

EXERCISE

Laboratory Report

Date

Smear Preparation and the Simple Stain

1. Draw your results from the simple stains.

Pseudomonas aeruginosa

Magnification

Cell shape
Stain used

Color of cells

Pseudomonas aeruginosa and
Staphylococcus epidermidis mix

Magnification

Staphylococcus epidermidis

Magnification

Cell shape

Cell arrangement
Stain used

Color of cells

Cell shapes

Cell arrangement
Stain used

Color of cells

Alexander-Strete-Niles:

II. Staining Techniques

10. Smear Preparation and

©The McGraw-Hill

Lab Exercises in
Organismal and Molecular
Microbiology

the Simple Stain

Companies, 2003

76 SECTION II Staining Techniques

2. Answer the following questions in the space provided.

a. What is the purpose of heat- fixation? What happens if you heat-fix too much?

b. Was your smear too thick when you viewed it (i.e., cells clumped too close together)? Why is a thick
smear undesirable?

c. Explain why methylene blue, crystal violet, and safranin stain differently from nigrosin, india ink, and
congo red.

d. What are the advantages and disadvantages of a simple stain?

e. Although crystal violet and safranin can be used as simple stains, can you think of any reason it would
be preferable to use methylene blue?

Alexander-Strete-Niles:

II. Staining Techniques

11. Gram Stain

©The McGraw-Hill

Lab Exercises in
Organismal and Molecular
Microbiology

Companies, 2003

Gram Stain

Most bacteria possess a cell wall that contains either a
thick peptidoglycan layer or a thin peptidoglycan layer
with an additional outer membrane composed of
lipopolysaccharide (figure 11.1). This chemical dif-
ference in bacterial cell walls is identified with the
Gram stain. The Gram stain is the stain most frequently
used to identify unknown bacterial cultures, because
it yields information on Gram reaction, cell size, cell
shape, and cell arrangement.

During the Gram- staining procedure, all bacteria
are stained purple by crystal violet, the primary stain.
Bacterial cells that have a thick peptidoglycan layer

retain the crystal violet during subsequent decoloriza-
tion and counterstain steps. These bacteria appear pur-
ple when viewed with the microscope and are referred
to as Gram-positive (figure 11.2). Bacterial cells that
have a thin peptidoglycan layer and an added outer
lipopolysaccharide layer lose the crystal violet during
the decolorization step and take up the counterstain
safranin. These bacteria appear red when viewed with
the microscope and are referred to as Gram-negative
(figure 11.2).

In this exercise, you will use the Gram stain on
selected 18-24-hour bacterial cultures, as well as on a
sample from your teeth or from yogurt.

Thick peptidoglycan layer

Cytoplasmic Periplasm
membrane

(a) Gram-positive

Outer layer of
lipopolysaccharide

Thin

peptidoglycan

layer

Cytoplasmic
membrane

(b) Gram-negative

Periplasm

ism^W

Figure 11.1 Differences in bacterial cell walls, (a) Gram-positive.

(h) Gram-necrarive.

(b) Gram-negative.

Alexander-Strete-Niles:

II. Staining Techniques

11. Gram Stain

©The McGraw-Hill

Lab Exercises in
Organismal and Molecular
Microbiology

Companies, 2003

78 SECTION II Staining Techniques

Materials

Cultures to select from (18-24-hour broth
or agar)

Bacillus cereus (Gram-positive rod)
Enterobacter aerogenes (Gram-negative rod)

Enterococcus faecalis (Gram-positive coccus)
Escherichia coli (Gram-negative rod)
Neisseria sicca (Gram-negative coccus)
Proteus vulgaris (Gram-negative rod)
Pseudomonas aeruginosa (Gram- negative rod)
Staphylococcus epidermidis (Gram-positive
coccus)

Gram-positive
Rod Coccus

Gram-negative
Rod Coccus

Gram-negative rod and
Gram-positive coccus mix

Cells are transparent prior to staining.

Cells are colored purple by primary
stain crystal violet and mordant
Gram's iodine.

The decolorizing agent, ethyl alcohol,
removes purple from Gram-negative
cells; Gram-positive cells retain stain.

Gram-negative cells take up the
counterstain, safranin, and are colored
red; Gram-positive cells remain purple.

Bacillus cereus

Staphylococcus epidermidis

Enterococcus faecalis

Escherichia coli

77 " *^~ I

m. mm

Pseudomonas aeruginosa

E. coli and S. epidermidis

:.•.

N *

m i

Neisseria sicca

Proteus vulgaris

Enterobacter aerogenes

Figure 11.2 Gram stain results for Gram-positive and Gram-negative cells.

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11. Gram Stain

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Lab Exercises in
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Gram Stain EXERCISE 11

Stains

Crystal violet
Gram's iodine
Ethanol (95%)
Safranin

Equipment

Light microscope

Miscellaneous supplies
Bibulous paper
Bunsen burner and striker
Clothespin

Disposable gloves (optional)
Glass slides
Immersion oil
Inoculating loop or needle
Lens paper
Staining tray
Toothpick

Water bottle with tap water
Wax pencil
Yogurt

Select four bacterial cultures from the materials list.
Although a variety of bacteria can be used, one from
each of the following categories is recommended:
Gram-positive rod, Gram-positive coccus, Gram-negative
rod, Gram-negative coccus, and a mixture of a Gram-
positive coccus and a Gram-negative rod.

Smear Preparation

1. Following the steps outlined in Exercise 10
(figure 10.2), prepare smears of the four selected
bacterial cultures and a smear of a mixture.

Wash your hands before proceeding.

2. Obtain a clean toothpick from the container, and
use it to prepare a smear of scrapings from your
teeth, or from yogurt. To prepare a smear of teeth
scrapings, use the end of a toothpick to pick
material from between your teeth, and transfer it
to a drop of water on a glass slide. Break up and
mix the material into the drop as much as
possible using the end of the toothpick. When
finished, place the toothpick in a container of
disinfectant. Allow this mixture to air-dry, and
then heat-fix. To prepare a smear of yogurt, dip a

toothpick in a container of yogurt, and transfer a
small amount to a drop of water on a glass slide.
Mix the yogurt into the drop using the toothpick.
Allow this to air-dry before heat-fixation.

Gram Staining

1. Using the steps outlined in figure 11.3, Gram-
stain all prepared smears. Follow these steps
exactly as outlined. Do not over-decolorize! Tilt
the slide, and drip alcohol onto the smear until it
runs off clear. Stop decolorization at this point!

2. After Gram staining, examine all slides using the
oil-immersion objective. Note: Avoid viewing
areas of the slide where cells are clumped
together. Only view areas where individual cells
can be seen.

(a) Apply crystal violet for 1
minute.

(b) Rinse for 5 seconds with
water.

■•

(d) Rinse for 5 seconds with
water.

(e) Decolorize with 95%

ethanol for 15-30 seconds

(f ) Rinse for 5 seconds with
water.

(g) Counterstain with safranin
for 1 minute.

(h) Rinse for 5 seconds with
water.

(i) Blot dry with bibulous
paper.

Figure 11.3 Gram- stain procedure

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11. Gram Stain

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Lab Exercises in
Organismal and Molecular
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Alexander-Strete-Niles:

II. Staining Techniques

11. Gram Stain

©The McGraw-Hill

Lab Exercises in
Organismal and Molecular
Microbiology

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EXERCISE

Laboratory Report

Name

Date

Lab Section

Gram Stain

1 . Draw the results of your Gram stains

Gram-positive rod

Organism

Magnification

Cell shape

Cell arrangement
Cell color

Gram-positive coccus

Organism

Magnification

Cell shape

Cell arrangement
Cell color

Gram reaction

Gram-negative rod

Organism

Magnification

Cell shape

Cell arrangement
Cell color

Gram-negative coccus

Organism

Magnification

Cell shape

Cell arrangement
Cell color

Gram reaction

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11. Gram Stain

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82 SECTION II Staining Techniques

Gram-negative rod

and Gram-positive coccus mix

Organisms

Magnification

Cell shapes

Cell arrangements
Cell colors

Gram reactions

Teeth scrapings or yogurt

Sample

Magnification

Cell shapes

Cell arrangements
Cell colors

Gram reactions

2. Answer the following questions in the space provided.

a. Why are contrasting colors important in the Gram stain?

b. Explain why the alcohol decolorization step is so critical in the Gram stain

c. Explain how a Gram stain differs from a
(1) negative stain

(2) simple stain

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11. Gram Stain

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Lab Exercises in
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Microbiology

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Gram Stain EXERCISE 11

d. Why is an 1 8-24-hour culture necessary for a Gram stain?

e. Name several pathogenic Gram-positive and Gram-negative bacteria and the diseases they cause.

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12. Acid-fast Stain

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Lab Exercises in
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Acid-fast Stain

The acid-fast stain is used to distinguish certain bacteria
that contain a high content of the lipid mycolic acid in
their cell wall. This component makes the cell wall resis-
tant to most stains, but heated carbolfuchsin will penetrate
the cell wall, imparting a red color to cells that is not
removed when the decolorizing agent, acid-alcohol, is
added. Bacteria with this characteristic are referred to as
acid-fast (figure 12.1). The majority of bacteria do not
have as high a lipid content in their cell wall, so their cells
lose the red color when acid-alcohol is added. They then

take up the counterstain methylene blue. These bacteria
are referred to as non-acid-fast (figure 12.1).

Several pathogenic bacteria can be distinguished
by the acid-fast stain, including two species of mycobac-
teria — Mycobacterium tuberculosis, the causative agent
of tuberculosis, and Mycobacterium leprae, the causative
agent of leprosy. An acid-fast stain of sputum is impor-
tant in the diagnosis of tuberculosis. In addition, cer-
tain pathogenic species of the actinomycete genus
Nocardia are acid-fast, including Nocardia asteroides,
a causative agent of nocardiosis. The oocysts of the
sporozoan parasite Cryptosporidium are also acid-fast.

Cells prior to staining are colorless.

Cells are colored red by hot carbolfuchsin.

Acid-fast rod

Non-acid-fast rod

The decolorizing agent, acid-alcohol,
removes the red from non-acid-fast cells;
acid-fast cells retain the stain.

Non-acid-fast cells take up the counterstain,
methylene blue, and are colored blue;
acid-fast cells remain red.

Mycobacterium phlei

Pseudomonas aeruginosa

Figure 12.1 Acid-fast staining procedure.

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12. Acid-fast Stain

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Lab Exercises in
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86 SECTION II Staining Techniques

In this exercise, you will use the Ziehl-Neelsen
acid- fast stain method to demonstrate the acid- fast trait
in Mycobacterium phlei.

Cultures (5-7-day agar)

Mycobacterium phlei (acid-fast rod)

Pseudomonas aeruginosa (non-acid-fast rod)

All agents in red are BSL2 bacteria.

Stains

Carbolfuchsin
Acid- alcohol
Methylene blue

Equipment

Hot plate (optional, for heating carbolfuchsin)
Light microscope

Miscellaneous supplies
Bibulous paper
Bunsen burner and striker
Clothespin

Disposable gloves (optional)
Egg albumin solution
Glass slides
Immersion oil
Inoculating loop or needle
Lens paper
Staining tray

Water bottle with tap water
Wax pencil

Smear Preparation

1 . Prepare a smear of Mycobacterium phlei, an acid-
fast rod, and a smear of Pseudomonas
aeruginosa, a non-acid-fast rod. Follow the steps
outlined in Exercise 10 (see figure 10.2), with
one exception: Mix the culture into a drop of egg
albumin solution, instead of water. This solution
will help acid-fast cells adhere to the glass slide.
Note: If you have trouble transferring cells with a
needle, use a loop instead. Since mycobacteria
tend to clump together, use your inoculating
needle or loop to break up cell clumps as much
as possible into the drop.

Acid-fast Stain

1 . After smear preparation, follow the steps of the
Ziehl-Neelsen acid-fast staining procedure in
figure 12.2. Note: The carbolfuchsin can be
heated using either a hot plate (as depicted) or a
Bunsen burner flame. In either case, gently
steam only; do not boil. As the paper dries out,
add more carbolfuchsin to keep the paper moist.
After 5 minutes, remove the paper and continue
the steps as outlined.

2. After staining, examine both slides using the oil-
immersion objective.

(a) Apply carbolfuchsin to
saturate paper, and heat
for 5 minutes in an
exhaust hood.

(b) Remove paper, cool, and
rinse with water for 30
seconds.

\L>

(d) Rinse with water for 5
seconds.

(e) Counterstain with

methylene blue for about
2 minutes.

(f ) Rinse with water for 30
seconds.

T-

(g) Blot dry with bibulous
paper.

Figure 12.2 Ziehl-Neelsen acid-fast staining procedure

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12. Acid-fast Stain

©The McGraw-Hill

Lab Exercises in
Organismal and Molecular
Microbiology

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Name

Lab Section

EXERCISE

Laboratory Report

Date

Acid-fast Stain

1. Draw the results from your acid- fast stains.

Mycobacterium phlei

Magnification

Cell shape
Cell color

Acid- fast?

Pseudomonas aeruginosa

Magnification

Cell shape
Cell color

Acid-fast?

2. Answer the following questions in the space provided.

a. Explain these terms. Which one applies to species of Mycobacterium and Nocardia?
(1) acid- fast

(2) non-acid-fast

b. Name several pathogenic acid-fast bacteria and the diseases they cause

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13. Spore Stain

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Lab Exercises in
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Microbiology

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Spore Stain

Some bacteria produce an internal structure known as
an endospore during their life cycle (figure 13.1). This
structure is produced by the vegetative cell by a process
called sporogenesis and is released upon the death of
the cell. The resulting free spore is a dormant structure
that contains little water and carries out few chemical
reactions. Its highly resistant nature is due to two fac-
tors: (1) a multilayered outer covering containing pepti-
doglycan; and (2) the presence of a protein-stabilizing
molecule called dipicolinic acid. These components
allow spores to survive adverse conditions that no other
living thing could survive. When favorable conditions
return, the bacterial spore undergoes germination to
yield a vegetative cell (figure 13.1).

The detection of endospores is a useful character-
istic in the identification of some bacteria, including
species of Bacillus and Clostridium. Several species
of these genera are pathogenic: Bacillus anthracis
causes anthrax; Clostridium botulinum causes botulism;
Clostridium tetani causes tetanus; and Clostridium per -
fringens causes gas gangrene.

When spores are detected in bacteria, their size,
shape, and location are useful in identification. For

Endospore

Vegetative cell

Sporogenesis

Cell dies

Vegetative
cell

Free
spore

Germination

example, Bacillus cereus and Bacillus anthracis produce
an oval- shaped spore located in the center of the cell
(central) (figure 13. 2a). The spores of Bacillus anthracis
are small enough that, when inhaled, they can enter the
alveoli of the lungs, causing a disease known as inhala-
tion anthrax. Clostridium botulinum produces oval-
shaped spores located between the center and the end
of the cell (subterminal) (figure 13. 2b). Clostridium
tetani produces spherical endospores at the end of the cell
(terminal) (figure 13.2c). Terminal spores give this organ-
ism its characteristic "drumstick" appearance.

Due to their unique physical and chemical charac-
teristics, endospores do not readily stain using ordinary
staining procedures. However, basic stains easily color
the vegetative cells that produce endospores. As a result,
endospores can be seen in outline against the back-
ground of stained vegetative cells. For best observa-
tion and verification of their presence, spores should be
stained using a special procedure called the spore stain.

In a spore stain, the cells are heated in the pres-
ence of malachite green. Heating drives the malachite
green into the spore, where it is retained even during the
water rinse step. When viewed with the microscope,
spores are readily visible as green, oval- shaped or
spherical objects within or outside of vegetative cells
(figure 13.3). Water removes the malachite green from
vegetative cells, allowing them to pick up the counter-
stain safranin and appear red. Non- spore- forming bac-
teria appear as red rods with no green, oval-shaped or
spherical objects (figure 13.3).

Spore growth

(a) Central

(b) Subterminal

Figure 13.1 The life cycle of endospore-forming bacteria.

Figure 13.2 Location of an endospore.

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13. Spore Stain

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90 SECTION II Staining Techniques

Cells and spores are colorless prior to staining

Cells and spores are colored green with hot
malachite green.

The decolorizing agent, water, washes the
malachite green from cells; spores retain the
stain.

Cells are colored red with the counterstain,
safranin.

Figure 13.3 The spore stain.

Spore-forming rod

Non-spore-forming rod

O" Free spore
Central endospore

Bacillus cereus

Escherichia coli

In this exercise, you will use the Schaeffer-Fulton
endospore stain method to demonstrate the presence
of spores in Bacillus cereus.

Materials

Cultures (4-5 days on nutrient agar)
Bacillus cereus (spore-forming rod)
Escherichia coli (non- spore-forming rod)

Stains

Malachite green
Safranin

Equipment

Hot plate (optional, to heat malachite green)
Light microscope

Miscellaneous supplies
Bibulous paper
Bunsen burner and striker
Clothespin

Disposable gloves (optional)
Glass slides
Immersion oil
Inoculating loop or needle
Lens paper
Staining tray

Water bath (optional, to heat malachite green)
Water bottle with tap water
Wax pencil

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13. Spore Stain

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Lab Exercises in
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Spore Stain EXERCISE 13 91

Smear Preparation

1 . Prepare a smear of Bacillus cereus, a spore-
forming rod, and a smear of Escherichia coli, a
non- spore-forming rod, following the steps
outlined in Exercise 10 (see figure 10.2).

Spore Stain

1 . After smear preparation, follow the steps of the
Schaeffer-Fulton endospore stain method depicted
in figure 13.4. Note: Heating the malachite green
can be done using a water bath (as depicted), a hot
plate, or a Bunsen burner flame. In either case,
gently steam only; do not boil. As the paper
dries out, add more malachite green to keep the
paper moist. After 5 minutes, remove the paper
and continue the steps as outlined.

2. After staining, examine both slides using the
oil-immersion objective.

(a) Apply malachite green to saturate paper, and steam for 5
minutes.

(b) Remove paper, cool, and
rinse with water for 30
seconds.

m>\,

(d) Rinse with water for 30
seconds.

(e) Blot dry with bibulous
paper.

Figure 13.4 Schaeffer-Fulton endospore staining
procedure.

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13. Spore Stain

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Lab Exercises in
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Alexander-Strete-Niles:

II. Staining Techniques

13. Spore Stain

©The McGraw-Hill

Lab Exercises in
Organismal and Molecular
Microbiology

Companies, 2003

Name

Lab Section

EXERCISE

Laboratory Report

Date

Spore Stain

1 . Draw your results of the spore stains

Bacillus cereus

Magnification

Cell shape

Vegetative cell color

Endospores?

Color

Location in cell

Free spores?

Color

Escherichia coli

Magnification

Cell shape

Vegetative cell color

Endospores?

Free spores?

2. Answer the following questions in the space provided
a. Define these terms:
(1) endo spore

(2) sporogenesis

(3) germination

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94 SECTION II Staining Techniques

b. Explain why sporogenesis is not a form of bacterial reproduction

c. How do bacterial endospores differ from mold asexual spores (conidia)?

d. Why is a 4-5-day culture of Bacillus cereus required for this exercise instead of a 1-2-day culture?

e. Why is heat applied to the malachite green in the spore stain? What function does water serve in
this method?

f. Name several spore- forming pathogens and the diseases they cause.

3. Answer the following questions based on these photographs:

•

-»

^-f

J .

*vs

* \

s « 4

h^*

•

•^

a. What are the clear ovals?

Why are they not stained?

b. What is your tentative identification of this
spore-former?

What is stained?

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II. Staining Techniques

14. Capsule and Flagella

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Lab Exercises in
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Microbiology

Stains

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Capsule and Flagella Stains

Some bacteria have cell structures external to the cell
wall that are visible with the light microscope after spe-
cial staining. One of these structures is a capsule, an
extracellular layer surrounding the cell wall that is com-
posed of polysaccharides and polypeptides. Although
a capsule is resistant to staining, it can be revealed by
using a combination of acidic and basic stains. The
acidic stain colors the background, while the basic stain
colors the cell. The capsule appears as a clear halo

around the cell. Non-capsule-forming bacteria do not
have a halo around the cell (figure 14.1).

Several clinically important bacteria form capsules,
including Klebsiella pneumoniae and Streptococcus
pneumoniae, both causes of bacterial pneumonia. In
these and other bacteria, the capsule is considered a vir-
ulence factor, since it protects the cell from phagocy-
tosis by white blood cells.

A second external cell structure that is visible
after staining is the flagellum, a long, whiplike struc-
ture composed of protein and used by bacteria for

Capsule-forming rod

Non-capsule-forming rod

Cells and capsules are colorless
prior to staining.

Acidic stain colors the background.

Basic stain colors the cell; capsule
appears as clear halo between back-
ground and cell.

Alcaligenes denitrificans

Enterobacter aerogenes

Figure 14.1 Capsule stain.

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14. Capsule and Flagella

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Lab Exercises in
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Stains

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96 SECTION II Staining Techniques

motility. Bacteria that possess a flagellum (one) or fla-
gella (two or more) are referred to as motile, while bac-
teria that lack this structure are referred to as
nonmotile. Although motility test agar can determine
if bacteria are motile or nonmotile (see Exercise 18),
it does not provide information about the number or
arrangement of flagella. Only a flagella stain can pro-
vide this information. In a flagella stain, the stain
clumps around the surface of the flagella, widening
their diameter so that they can be seen with a light
microscope. Microscopic observation reveals several
flagella arrangements in bacteria: monotrichous,
amphitrichous, lophotrichous, and peritrichous
(figure 14.2). These arrangements are useful in bacte-
rial identification.

In this exercise, you will prepare a capsule stain
of an encapsulated and nonencapsulated culture. You
will not prepare flagella stains, since flagella are very
fragile and easily break off bacterial cells; but you will
examine several prepared slides of flagella stains.

Cultures ( 1 8-24-hour broth)

Enterobacter aerogenes (encapsulated rod)
Alcali genes denitrificans (nonencapsulated rod)

Stains

Acidic: india ink
Basic: crystal violet

Monotrichous

{Pseudomonas
aeruginosa)

Amphitrichous

{Spirillum volutans)

Lophotrichous

{Pseudomonas
marginalis)

Peritrichous

{Proteus vulgaris)

Figure 14.2 Flagella arrangements in bacteria.

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14. Capsule and Flagella

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Lab Exercises in
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Microbiology

Stains

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Capsule and Flagella Stains EXERCISE 14 97

Prepared slides

Proteus vulgaris (peritrichous flagella)
Spirillum volutans (amphitrichous flagella)

Equipment

Light microscope

Miscellaneous supplies
Bibulous paper
Bunsen burner and striker
Clothespin

Disposable gloves (optional)
Glass slides
Immersion oil
Inoculating loop
Lens paper
Staining tray

Water bottle with tap water
Wax pencil

Capsule Stain

1 . Study the steps for preparing a capsule stain
shown in figure 14.3. Then carefully follow this
procedure as you prepare a capsule stain of
two cultures: Enterobacter aerogenes, an
encapsulated rod, and Alcaligenes denitrificans •,
a nonencapsulated rod. Gently heat only;
gently rinse with water.

2. When finished staining, examine both slides
using the oil-immersion objective.

Flagella Stain

Examine the prepared slides of flagella stains using the
oil-immersion objective. Note the number and arrange-
ment of flagella.

(a) Two loopfuls of the organism are
mixed in a small drop of india ink

(b) The ink suspension of bacteria is
spread over the slide and air-dried

(d) Smear is stained with crystal violet for
1 minute.

(e) Crystal violet is gently washed off
with water.

(f ) Slide is blotted dry with bibulous
paper, and examined with oil-
immersion objective.

Figure 14.3 Procedure for demonstration of capsule.

Alexander-Strete-Niles:
Lab Exercises in
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Microbiology

II. Staining Techniques

14. Capsule and Flagella
Stains

Alexander-Strete-Niles:

II. Staining Techniques

14. Capsule and Flagella

©The McGraw-Hill

Lab Exercises in
Organismal and Molecular
Microbiology

Stains

Companies, 2003

Name

Lab Section

EXERCISE

Laboratory Report

Date

Capsule and Flagella Stains

1 . Draw the results of your capsule stains

Enterobacter aerogenes

Magnification

Color of background
Color of cells

Cell shape

Capsule?

Color

2. Draw the prepared slides of flagella you examined

Spirillum volutans

Magnification

Cell shape

Flagella present?
Arrangement _

Alcaligenes denitrificans

Magnification

Color of background
Color of cells

Cell shape

Capsule?

Proteus vulgaris

Magnification

Cell shape

Flagella present?
Arrangement _

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14. Capsule and Flagella

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Lab Exercises in
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Stains

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100 SECTION II Staining Techniques

3. Answer the following questions in the space provided.

a. What are bacterial capsules? How do capsules play a role in the establishment of disease?

b. Why must a combination of basic and acidic stains be used to reveal a capsule?

c. Name several capsule-forming bacteria and the diseases they cause.

d. Why are bacterial flagella visible with a light microscope only after a flagella stain?

e. Motility provided by bacterial flagella can be observed in wet mount preparations. What additional
information does a flagella stain provide? How can this information be useful?

4. Answer the following questions based on these photographs:

Is this organism encapsulated?

Identify (a)

Identify (b)
Identify (c)

"I > *

_ _

1, "^ •*

■ * *

* t »

- 4? >

• , r

v r*

, * ,

■t , • ■

" A ' *

Is this organism flagellated?

If yes, what is the arrangement?

Name one bacterium with this type of
arrangement?

Alexander-Strete-Niles:

II. Staining Techniques

15. The Staining

©The McGraw-Hill

Lab Exercises in
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Microbiology

Characterization of a
Bacterial Unknown

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The Staining Characterization
of a Bacterial Unknown

This exercise will allow you to apply what you have
learned in previous exercises to the identification of a
bacterial unknown. First, you will select or have
assigned to you one of the bacterial cultures from the
materials list, but you will not know which one. You will

then use the information provided in figure 15.1 and
table 15.1 to guide you through the characterization of
your unknown. Perform only those staining procedures
required as you work your way through the scheme.
When you are finished, you should be able to correctly
identify your bacterial unknown based on its staining
characteristics.

Gram stain (Exercise 11)

Gram-negative

Morphology
(Exercises 9, 10, 11)

Gram-positive

Morphology
(Exercises 9, 10, 11)

Coccus

Capsule stain
(Exercise 14)

Neisseria
sicca

Acid-fast Arrangement

stain (Exercises 10, 11)

(Exercise 12)

Alcaligenes
denitrificans

Enterobacter
aero genes

Spore stain
(Exercise 13)

Coryne bacterium
pseudodiphtheriticum

Bacillus
cereus*

Clusters

Short chains

Staphylococcus
epidermidis

Enterococcus
faecalis

Mycobacterium
phlei*

4-5 -day cultures will yield some Gram-negative cells.

Figure 15.1 Identification scheme for eight bacterial unknowns. You can use this
scheme when identifying your staining unknown.

101

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15. The Staining

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Characterization of a
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102 SECTION II Staining Techniques

Table 15.1 Cell Size,

Shape, and Arrangement of Bacterial Staining Unknowns

Bacterial culture

Cell size (|x)

Cell shape

Cell arrangement

Alcaligenes

0.5 x 1-2

Rod

Single cells or pairs

denitrificans

Bacillus cereus

1 x 3-5

Rod

Streptobacilli

Corynebacterium

0.5 x 1-2

Rod

Single cells or V- shapes

pseudodiphtheriticum

Enterobacter

0.5 x 1-2

Rod

Single cells or pairs

aero genes

Enterococcus faecalis

0.5-1

Coccus

Single cells, pairs, or short streptococci

Mycobacterium phlei

0.2 x 1-2

Rod

Single cells or cords

Neisseria sicca

0.5-1

Coccus

Single cells or diplococci

Staphylococcus

0.5-1.5

Coccus

Single cells, pairs, or staphylococci

epidermidis

Cultures ( 1 8-24-hour agar or broth)
Alcaligenes denitrificans
Bacillus cereus

Corynebacterium pseudodiphtheriticum
Enterobacter aerogenes
Enterococcus faecalis
Mycobacterium phlei
Neisseria sicca
Staphylococcus epidermidis

All agents in red are BSL2 bacteria.

Stains

Gram stain
Crystal violet
Gram's iodine
Ethanol (95%)
Safranin

Acid-fast stain
Carbolfuchsin
Acid-alcohol
Methylene blue

Spore stain

Malachite green
Safranin

Capsule stain

Acidic stain: india ink
Basic stain: crystal violet

Equipment

Hot plate (optional)
Light microscope

Miscellaneous supplies
Bibulous paper
Bunsen burner and striker
Clothespin

Disposable gloves (optional)
Egg albumin solution
Glass slides
Immersion oil
Inoculating loop or needle
Lens paper
Ocular micrometer
Stage micrometer slide
Staining tray

Wash bottle with tap water
Wax pencil

Alexander-Strete-Niles:

II. Staining Techniques

15. The Staining

©The McGraw-Hill

Lab Exercises in
Organismal and Molecular
Microbiology

Characterization of a
Bacterial Unknown

Companies, 2003

The Staining Characterization of a Bacterial Unknown EXERCISE 15 103

1 . You will select or have assigned to you an unknown
from the materials list. Record your unknown
number in the laboratory report.

2. Examine the information provided in figure 15.1
and table 15.1. Notice that the Gram stain must
be done first to determine Gram reaction and cell
morphology (figure 15.1). A negative stain or
simple stain may be used in conjunction with a

Gram stain to verify cell size, shape, and
arrangement (table 15.1). Notice that Gram-
positive and Gram-negative cocci will require no
additional staining, but Gram-positive and Gram-
negative rods will require one or more additional
stains for identification. So, whatever your Gram
stain results, continue to follow the identification
scheme downward until you identify your
unknown based on staining characteristics.

3. As you perform whatever stains are required,
record your results in the laboratory report.

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

II. Staining Techniques

15. The Staining
Characterization of a
Bacterial Unknown

Alexander-Strete-Niles:

II. Staining Techniques

15. The Staining

©The McGraw-Hill

Lab Exercises in
Organismal and Molecular
Microbiology

Characterization of a
Bacterial Unknown

Companies, 2003

Name

Lab Section

EXERCISE

Laboratory Report

Date

The Staining Characterization of a Bacterial Unknown

Unknown no.

1. Follow the information provided in figure 15.1 and table 15.1 to identify your staining unknown. Perform
only the stains required to identify your unknown.

2. Required staining results

Gram stain

(See Exercise 11.)

Magnification
Cell size

Cell shape

Cell arrangement
Cell color

Acid-fast stain

(See Exercise 12.)

Magnification

Cell shape

Cell arrangement
Cell color

Gram reaction

Acid-fast?

105

Alexander-Strete-Niles:

II. Staining Techniques

15. The Staining

©The McGraw-Hill

Lab Exercises in
Organismal and Molecular
Microbiology

Characterization of a
Bacterial Unknown

Companies, 2003

106 SECTION II Staining Techniques

Spore stain

(See Exercise 13.)

Magnification

Cell shape

Cell arrangement

Vegetative cell color

Endospores?

If yes, color?

If yes, location in cell

Free spores?

If yes, spore color?

3. Summary of the staining characteristics of my unknown

Capsule stain

(See Exercise 14.)

Magnification

Color of background
Color of cells

Cell shape

Capsule?

If yes, capsule color?

Unknown no.

Cell shape

Cell arrangement

Gram reaction

Acid-fast?

Spores?

Capsules?

4. After examining the information provided in figure 15.1 and table 15.1 and recording the results of

required stains, I conclude that my staining unknown is

Alexander-Strete-Niles:

II. Staining Techniques

15. The Staining

©The McGraw-Hill

Lab Exercises in
Organismal and Molecular
Microbiology

Characterization of a
Bacterial Unknown

Companies, 2003

The Staining Characterization of a Bacterial Unknown EXERCISE 15 107

5. Based on the information provided in figure 15.1 and table 15.1, fill in the following table for the eight
unknown cultures.

Unknown
culture

Cell
shape

Cell
arrangement

Gram
reaction

Acid-
fast

Spores

Capsules

Alcali genes
denitrificans

Bacillus cereus

Cory neb acterium
pseudodiphtheriticum

Enterobacter
aero genes

Enterococcus faecalis

Mycobacterium phlei

Neisseria sicca

Staphylococcus
epidermidis

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

Bacterial Cultivation

16. Bacteria & Fungi in the
Lab. Environ: The Necessity
of Aseptic Technique

Bacteria and Fungi in the Laboratory
Environment: The Necessity of

Aseptic Technique

Bacteria and Fungi in the Laboratory
Environment

Bacteria and fungi occur widely in the natural environ-
ment in association with air, water, soil, plants, and
animals. These microorganisms find their way into
our homes, offices, and buildings in a variety of ways:
(1) through open doors and windows; (2) on the bot-

toms of shoes; (3) on the surfaces of plants, pets, and
food; and (4) on the surfaces of our hands and clothes.

Bacteria and fungi also find their way into our lab-
oratory environment, where they can be found in the air
and on countertops (figure 16.1). We must be aware of
these microorganisms in the laboratory environment
when working with laboratory cultures.

To demonstrate their presence, you will use two
types of media to culture bacteria and fungi from the
laboratory environment: nutrient agar and Sabouraud

Fungi

Bacteria

(a) Bacteria and fungi from laboratory air.

(b) Fungi from laboratory air.

(d) Fungi from laboratory countertop

Figure 16.1 Bacteria and fungi from the laboratory environment.

110

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

Bacterial Cultivation

16. Bacteria & Fungi in the
Lab. Environ: The Necessity
of Aseptic Technique

Bacteria and Fungi in the Laboratory Environment: The Necessity of Aseptic Technique EXERCISE 16 111

Table 16.1 Components of Nutrient Agar

and Sabouraud Dextrose Agar

Nutrient agar*
(bacteria)

Sabouraud dextrose
agar (fungi)

Peptone

Beef extract 3 g

Agar

15 g

Peptone
Dextrose

Agar

10 g
40 g

15 g

Distilled water 1 ,000 ml Distilled water 1 ,000 ml

Final pH = 6.8

Final pH = 5.6

Source: The Difco Manual. Eleventh Edition. Difco Laboratories.
*Nutrient broth has the same formula, but does not contain agar.

Inhibiting organism
(chemical producer)

Inhibited
organism

Zone of
inhibition

Figure 16.2 Evidence of inhibition of one microbe by
another. The inhibiting organism is producing a chemical
that is active against the other organism.

dextrose agar (table 16.1). Nutrient agar contains
organic compounds, which support the growth of a wide
variety of bacteria, and agar as a solidifying agent. The
final pH of the medium is 6.8. Sabouraud dextrose agar
also contains organic compounds and agar, but the high
dextrose content (4%) and low pH (5.6) favor the
growth of fungi over bacteria. The medium can be made
even more selective for fungi through the addition of an
antibiotic, such as chloramphenicol. Together, these two
media will demonstrate the number and variety of bac-
teria and fungi in our laboratory environment.

Excluding Environmental
Contaminants from
Laboratory Cultures

Once your examination of culture media has revealed
the existence of bacteria and fungi in our laboratory
environment, you will be asked to consider how this
relates to working with pure cultures in the laboratory.
For example, can these environmental bacteria and fungi
contaminate our laboratory cultures? Can their entry
be prevented by using certain techniques designed to
exclude them? If such techniques exist, what are they?

Searching for Examples of Antibiosis

The primary focus of this exercise is to demonstrate
bacteria and fungi in the laboratory environment and
to consider techniques to exclude them from cultures.
A secondary focus is to find an example of antibiosis
on the media you inoculate with samples from the lab-
oratory environment. What is antibiosis? When envi-

ronmental microorganisms grow in close proximity
to one another, as occurs naturally in soil or unnaturally
in a culture medium, one microbe may produce a
chemical substance that inhibits the growth of another
microbe nearby. This phenomenon is called antibiosis.
Antibiosis is identified in culture media by a zone
of inhibition around the chemical-producing organ-
ism (figure 16.2). Examples of antibiosis in culture
media are not common, since the odds are low that you
will inoculate in close proximity an organism that pro-
duces a chemical substance inhibitory to another. You
and other laboratory students may collectively find only
one example on all your plates, but finding that one
example is the objective. When you see this example,
you will understand what Alexander Fleming saw in
1928 when he examined a plate in his laboratory. He
found that the growth of Staphylococcus aureus was
inhibited by a mold. The mold was identified as Peni-
cillium, and the chemical substance it produced was
later isolated and named penicillin. Penicillin proved to
be effective in treating infections caused by Staphylo-
coccus aureus in the human body. It became the first
antibiotic, an antimicrobial chemical agent of micro-
bial origin put into the human body to treat disease.
Since the introduction of penicillin, many other antibi-
otics of microbial origin have been discovered.

Streptomyces and Bacillus, Examples
of Antibiotic-Producers

Streptomyces is a genus of bacteria that is common in
soil. These bacteria are Gram-positive and produce rods
in branching filaments similar to those of fungi, but

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

Bacterial Cultivation

16. Bacteria & Fungi in the
Lab. Environ: The Necessity
of Aseptic Technique

112 Section III Bacterial Cultivation

Media

Streptomyces colonies

Figure 16.3 Swab results from the laboratory floor.
A number of white, powdery colonies of Streptomyces
are present.

the filaments have a much smaller diameter than those
of fungi. Streptomyces produces a small, white, pow-
dery colony on culture media (figure 16.3) and gives off
an "earthy," soil-like odor.

Bacillus is a genus of bacteria also common in soil.
This organism is a Gram-positive, endospore-forming
rod. Bacillus generally produces a large, flat colony that
is typically white or cream-colored.

Streptomyces and Bacillus are both sources of use-
ful antibiotics. Species of Streptomyces are the source
of more than half of all antibiotics effective against bac-
teria, including streptomycin, tetracycline, and chlo-
ramphenicol. They are also the source of the polyenes,
such as amphotericin B and nystatin, effective against
fungi. Species of Bacillus are the source of antibiotics
such as bacitracin, effective against Gram-positive bac-
teria, and polymyxin B, effective against Gram-nega-
tive bacteria.

A third focus of this exercise is to find one or both
of these common soil bacteria in the laboratory envi-
ronment. One of these bacteria may provide the exam-
ple of antibiosis in your culture media.

4 nutrient agar plates

4 Sabouraud dextrose agar plates

3 nutrient broth (or water) tubes, sterile

Stains

Gram stain
Crystal violet
Gram's iodine
Ethanol (95%)
Safranin

Equipment

Dissecting microscope
Incubator (set at 35 °C)
Light microscope

Miscellaneous supplies
Bibulous paper
Bunsen burner and striker
Clothespin

Cotton-tipped swabs, sterile (3)
Disposable gloves (optional)
Glass slides
Immersion oil
Inoculating needle
Lens paper
Staining tray

Wash bottle with tap water
Wax pencil

First Session: Inoculation of
Nutrient Agar and Sabouraud
Dextrose Agar Plates

1 . Remove the lids from a nutrient agar plate and
a Sabouraud dextrose agar plate. Leave these
two plates open to the laboratory air for
30-60 minutes.

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

Bacterial Cultivation

16. Bacteria & Fungi in the
Lab. Environ: The Necessity
of Aseptic Technique

Bacteria and Fungi in the Laboratory Environment: The Necessity of Aseptic Technique EXERCISE 16 113

Line 1

Swab

Sample
surface

Line 1

(end)

(a) A moistened swab is first rubbed
back and forth across the sample
surface to pick up microorganisms.

(b)

Microorganisms are then transferred to an
agar plate by rubbing the swab back and
forth along lines 1 and 2.

Figure 16.4 Swab inoculation of an agar plate.

2. Dip the cotton- tipped end of a sterile swab into a
tube of sterile water or nutrient broth. Blot the
excess liquid against the test tube wall. Use

the wetted end to rub back and forth across
the countertop of your work area; then inoculate
a nutrient agar plate (figure 16.4). Repeat
this process to inoculate a Sabouraud dextrose
agar plate.

3 . Wet a second sterile, cotton-tipped swab with
sterile water or broth, and use it to rub back and
forth across the floor below your work area.
Inoculate a second nutrient agar plate as before.
Repeat to inoculate a second Sabouraud dextrose
agar plate.

4. Wet a third sterile, cotton-tipped swab with
sterile water or broth, and use it to rub back and
forth across the skin on the inside of your left
hand. Inoculate a third nutrient agar plate as
before, and repeat to inoculate a third Sabouraud
dextrose agar plate.

5. Label the plates, and incubate the air, countertop,
and floor plates at room temperature (22°C).
Place the skin plates in an incubator set at

35 °C. Incubate all plates at least 3-4 days
before examining.

Second Session: Examination of
Nutrient Agar and Sabouraud Dextrose
Agar Plates

Number and Variety of Bacteria and Fungi
in the Laboratory Environment

1 . After incubation, examine all plates for
growth. Sketch a typical nutrient agar plate
and Sabouraud dextrose agar plate in the
laboratory report.

2. Record the number and variety of bacteria and
fungi on your plates in the table of your
laboratory report. Note: When determining
number, count only bacterial colonies on nutrient
agar plates and fungal colonies on Sabouraud
dextrose agar plates. Bacterial colonies are
smooth and round, while fungal colonies are
large and cottony in appearance (see figure

16. la). Note: When determining variety, look for
bacterial and fungal colonies that are different in
appearance. Colonies that look different (based
on size, shape, margin, texture, elevation,
pigmentation, etc.) represent different types. The
use of a dissecting microscope may help you
count and differentiate colonies.

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

Bacterial Cultivation

16. Bacteria & Fungi in the
Lab. Environ: The Necessity
of Aseptic Technique

114 Section III Bacterial Cultivation

Examples of Antibiosis on Plates

1 . After completing the drawings and table and
considering the implications of these results, go
back through your plates searching for examples
of antibiosis. Refer to figure 16.2 to determine if
a zone of inhibition is present on any of your
plates. If you find none, examine the plates of
other students. Generally, at least one example
can be found.

2. In the laboratory report, draw the example of
antibiosis you see. If time permits, Gram-stain
the two organisms in order to get an idea of what
antibiotic may be involved.

Presence of Streptomyces and Bacillus

1 . Examine your plates for signs of Streptomyces
and Bacillus. Their colony characteristics were
described previously in the "Background" section
of this exercise.

2. If you find suspect colonies, do a Gram stain to
verify your identification. Also, do a spore stain
on the suspect Bacillus colony if it turns out to be
a Gram-positive rod. If you have isolated species
of one or both of these bacteria, you have isolated
important antibiotic-producers. Did one of these
bacteria provide your example of antibiosis?

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

Bacterial Cultivation

16. Bacteria & Fungi in the
Lab. Environ: The Necessity
of Aseptic Technique

Name

Lab Section

EXERCISE

Laboratory Report

Date

Bacteria and Fungi in the Laboratory Environment: The Necessity
of Aseptic Technique

1. a. Draw a typical nutrient agar plate and Sabouraud dextrose agar plate inoculated with a
laboratory sample.

Nutrient agar plate (bacteria)

Sample

Sabouraud dextrose agar plate (fungi)

S ample

Total bacterial colonies

Total colony types

Total fungal colonies

Total colony types

b. Record your results for all plates in the following table.

Laboratory
sample

Nutrient agar (bacteria)
Total colonies Colony types

Sabouraud dextrose agar (fungi)
Total colonies Colony types

Air

Countertop

Floor

Skin

Which sample had the highest number of bacteria?

.Why'}

c. Based on your results, does the laboratory environment contain a large number and variety of bacteria

and fungi?

115

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

Bacterial Cultivation

16. Bacteria & Fungi in the
Lab. Environ: The Necessity
of Aseptic Technique

116 Section III Bacterial Cultivation

d. Describe several techniques you might use to keep these environmental bacteria and fungi from
contaminating your laboratory cultures.

2. a. Did you see any zones of inhibition on your plates? (yes or no)
Any zones on other students' plates? (yes or no)

b. If yes, draw a representative result indicating a zone of inhibition. Draw only the region of the
interacting organisms. Label the chemical-producing organism, the zone of inhibition, and the
inhibited organism.

c. Explain how your result is similar to that observed by Alexander Fleming in 1928.

d. Why was Fleming's observation historically important?

e. Gram-stain results: Antibiotic-producer in the drawing in (b)

Gram reaction

Cell shape

Bacteria or fungi?

Inhibited organism in the drawing in (b)

Gram reaction

Cell shape

Bacteria or fungi?

Do these results give you any clues as to what antibiotic is being produced? If so, describe
the possibilities.

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

Bacterial Cultivation

16. Bacteria & Fungi in the
Lab. Environ: The Necessity
of Aseptic Technique

Bacteria and Fungi in the Laboratory Environment: The Necessity of Aseptic Technique EXERCISE 16 117

3. a. Did you or another student have an isolate from the countertop or floor with the
following characteristics:

Small, white, powdery colony? (yes or no)

Gram-positive rods in branching filaments? (yes or no)

If you answered yes on both lines, you may have isolated Streptomyces, a common bacterium in soil
Can you explain how this organism gets into the lab?

What is the medical significance of this organism?

b. Did you or another student have an isolate from the countertop or floor with the
following characteristics:

Large, flat colony, white or cream-colored? (yes or no)

Gram-positive endospore-forming rod? (yes or no)

If you answered yes on both lines, you may have isolated Bacillus, a common bacterium in soil
Can you explain how this organism gets into the lab?

What is the medical significance of this organism?

4. Answer the following questions based on these photographs

(1)

(2)

a. Bacteria or fungi?

(1)

b. Type of medium?

How do you know?

c. Bacteria from air or skin?

(2)

How do you know?

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

Bacterial Cultivation

16. Bacteria & Fungi in the
Lab. Environ: The Necessity
of Aseptic Technique

118

Section III Bacterial Cultivation

Gram-negative rod

Fungi

Gram-positive,
endospore-forming rod

(1)

Gram-positive rods
in branching filaments

d. Name area of no growth (1)

Given these results, what group of antibiotics may
be indicated?

e. Name area of no growth (1)

Given these results, what group of antibiotics may
be indicated?

f. These two photographs indicate

what type of bacterium?

Where does this organism occur naturally?

What is the medical significance

of this organism?

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

Bacterial Cultivation

17. Preparation and
Inoculation of Growth
Media

Preparation and Inoculation
of Growth Media

Media Preparation

The cultivation of bacteria (i.e., their growth on a nutri-
ent medium) is necessary for subsequent isolation and
identification. A complex medium, one that contains
an array of organic nutrients, can grow a variety of bac-
teria. Media of this type include tryptic soy broth and
tryptic soy agar (table 17.1). In this exercise, you will
cultivate bacteria using different forms of these media,
including broth tubes, agar slants, agar deeps, and
agar plates (figure 17.1).

Media Sterilization

Before use, media must be sterilized in an autoclave
(figure 17.2). Sterilization is a process that destroys all

microbes in the medium. If autoclaving is not done,
these microbes will contaminate the culture you intro-
duce into the medium.

Media Inoculation

After media preparation and sterilization, a culture is
inoculated (introduced) into each medium. Media inoc-
ulation can be done using a variety of sterile instru-
ments, such as a loop, needle, swab, or pipette (figure
17.3). In all cases, care must be taken to avoid intro-
ducing environmental bacteria and fungi into the
medium with the culture. To prevent contamination of
your media, examine and carefully follow the aseptic
techniques outlined in table 17.2. These procedures
must become a standard part of your laboratory tech-
nique when working with pure cultures.

Table 17.1

Composition of Tryptic Soy
Broth and Tryptic Soy Agar

Tryptic soy I

broth

Tryptic soy agar*

Tryptone

17 g

Tryptone 15 g

Soy tone

Soy tone 5 g

Dextrose

2.5 g

Sodium 5 g
chloride

Sodium
chloride

Agar 15 g

Dipotassium
phosphate

2.5 g

Distilled water 1 ,000 ml

Final pH =

7.3

Final pH = 7.3

Source: The Difco Manual. Eleventh Edition. Difco Laboratories.
The addition of 5% sheep blood makes blood agar, a medium used
to cultivate fastidious bacteria and to determine hemolytic reactions.

Figure 17.1 Different forms of culture media. From left
to right: broth tube, agar slant (front view), agar slant (side
view), agar deep, and agar plate (top and side views).

119

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

Bacterial Cultivation

17. Preparation and
Inoculation of Growth
Media

120

Section III Bacterial Cultivation

Table i 7.2 Basic Aseptic Techniques to

Keep Unwanted Bacteria
and Fungi out of Your Cultures

Prior to culture transfer:

1 . Close doors. This reduces air currents that
suspend particles.

2. Wash hands. This removes bacteria from
your hands.

3. Disinfect countertops. This kills bacteria and
mold spores on your work area.

During culture transfer:

1. Sterilize loop or needle. Hold the loop or needle
in a Bunsen burner flame until red-hot.

2. Hold test tubes at an upward angle, and flame
the opening. This reduces the likelihood that
suspended particles will enter.

3. Keep lids on plates when not in use. This reduces
the chances of contamination.

4. Keep movements in your work area to a
minimum. Needless movements create air
currents that suspend particles.

Figure 17.2 An autoclave, used to sterilize media.

i-Trfv-f" *- ~— >■

_ — '

-*rr-

^=Sr

-_■

Figure 17.3 Instruments used to inoculate media. From
bottom to top: standard loop, calibrated loop (disposable),
needle, cotton-tipped swab, and pipette with safety bulb.

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

Bacterial Cultivation

17. Preparation and
Inoculation of Growth
Media

Preparation and Inoculation of Growth Media EXERCISE 17 121

Media Incubation and Examination

Inoculated media must be incubated to allow time for
bacterial growth. After incubation, the microbial growth
in tubes and plates is visible and can be examined. Dur-
ing examination, look for contamination by unwanted
bacteria and fungi. The presence of more than an occa-
sional contaminant suggests the need for better asep-
tic technique.

Materials

Culture (24-hour broth)
Escherichia coli

Media

Tryptic soy agar
Tryptic soy broth

Equipment
Autoclave
B alance
Hot plate
Incubator (set at 35 °C)

Miscellaneous supplies
Aluminum foil
Bunsen burner and striker
Distilled water
Erlenmeyer flask, 250 ml
Graduated cylinder, 1 00 ml
Immersion oil
Inoculating loop and needle
Petri dishes, sterile (3)
Pipette, 10 ml, with bulb
Spatula
Stirring bar

Test tubes and caps (6)
Test tube rack
Wax pencil
Weigh paper

First Session

Media Preparation and Sterilization

1 . Following the directions on the bottle, prepare
1 ml of tryptic soy broth in a small flask or
beaker. Heat the mixture until the powder
dissolves, and then transfer 5 ml to each of two
tubes with a 10 ml pipette. Cap the tubes loosely,
and place them in a test tube rack.

2. Following the directions on the bottle, prepare
115 ml of tryptic soy agar in a 250 ml Erlenmeyer
flask. Heat the mixture until the powder dissolves
and the medium turns clear. Note: To keep the
powder from burning, swirl the flask contents
occasionally. Watch your flask closely, since the
medium will boil over if heated too long.

3. After the medium has become clear, use a 10 ml
pipette to transfer 5 ml to each of two tubes and
10 ml to each of two other tubes. Cap loosely,
and add these four tubes to the two already in
the test tube rack. Leave the remaining 85 ml
of tryptic soy agar in the flask, and cover with
foil. This volume will be used to pour plates
after sterilization.

4. Label your test tube rack and flask, and place
them in the autoclave for sterilization.

5. After sterilization, lean the two tubes with
5 ml of tryptic soy agar against a notebook
or similar object. When the medium cools,
these tubes will form agar slants. Let the
remaining tubes cool upright in the rack.
These tubes will form agar deeps.

6. Let the flask contents cool sufficiently to allow
handling without discomfort. When this has
occurred, pour three plates using the technique
depicted in figure 17.4. Do not pour hot agar
into a petri dish. This will cause excess
condensation on the lid of the petri dish and on
the agar surface. Excess moisture may allow the
culture to spread across the entire surface of the
agar, instead of forming discrete colonies.

7. When the agar has gelled in tubes and plates,
the media are ready to inoculate.

Alexander-Strete-Niles:
Lab Exercises in
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. Bacterial Cultivation

17. Preparation and
Inoculation of Growth
Media

122 Section III Bacterial Cultivation

(a) Remove foil top and flame opening of flask.

(b) Pour sterile medium into petri dish to fill bottom.
Allow the agar to cool and gel before moving dishes

Figure 17.4 Preparation of agar plates.

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

Bacterial Cultivation

17. Preparation and
Inoculation of Growth
Media

Preparation and Inoculation of Growth Media EXERCISE 17 123

Media Inoculation and Incubation

1 . After the different forms of media (broth, slants,
deeps, and plates) have been prepared, sterilized,
and cooled, they are ready for inoculation. Begin

by inoculating the two tubes of tryptic soy broth
with a culture of Escherichia coli. Use an
inoculating loop, and follow the procedure in
figure 17.5.

Culture

(a) Label the tube to be inoculated with the
microorganism used, the date, and your
name or initials.

(b) Take the broth culture in one hand

Culture

(d) Remove the plug or cap from the tube
by grasping it between the fingers of
the hand holding the inoculating loop.

(e) Flame the mouth (lip) of the broth
culture.

'■

(g) Introduce the loopful of culture by
immersing the loop into the sterile
broth. Stir lightly.

(h) Withdraw the loop, flame the lip, and
replace the plug or cap. Set the
inoculated tube in the test tube rack.

(f ) Insert the sterile loop into the broth
culture, and obtain a loopful of culture
Withdraw the loop, flame the mouth,
and replace the plug or cap. Set the
broth culture in a test tube rack. Pick
up the tube to be inoculated, remove
the plug or cap, and flame the mouth.

(i) Flame the inoculating loop again and
put it down. Incubate the inoculated
tube as directed.

Figure 17.5 Broth inoculation.

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

Bacterial Cultivation

17. Preparation and
Inoculation of Growth
Media

124 Section III Bacterial Cultivation

2. Inoculate the two tryptic soy agar slants with
Escherichia coli using an inoculating loop.
Follow the procedure outlined in figure 17.6.

3. Inoculate the two tryptic soy agar deeps with
Escherichia coli using an inoculating needle. The
inoculation of a deep is depicted in figure 17.7.

Culture

(a) Label the agar slant to be inoculated
with the microorganism to be used,
the date, and your name or initials.

(b) Take the broth culture in one hand

(

Culture

■

(d) Remove the plug or cap from the tube
by grasping it between the fingers of
the hand holding the inoculating loop.

(e) Flame the mouth of the broth culture

(f ) Obtain a loopful of the broth culture.
Withdraw the loop, flame the mouth,
and replace the plug or cap. Set the
tube down, and pick up the agar slant
to be inoculated. Remove the plug or
cap, and flame the mouth.

Agar slant

;

(g) Place the loop on the agar slant's
surface at its bottom. Move the loop
from side to side as you pull it upward
out of the tube.

(h) Withdraw the loop, flame the lip of the
tube, and replace the plug or cap.
Place the tube in a rack, and incubate
as directed.

(i) Flame the inoculating loop again, and
put it down.

Figure 17.6 Inoculation of an agar slant.

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

Bacterial Cultivation

17. Preparation and
Inoculation of Growth
Media

Inoculating
needle with
culture

Agar deep

Preparation and Inoculation of Growth Media EXERCISE 17 125

(a) Remove the plug or cap,
and flame the mouth of
the tube. Insert the
inoculating needle
without touching the
sides of the tube.

(b) Continue downward
until the needle nearly
reaches the bottom of
the tube.

Line of
inoculation

(c)

Slowly withdraw the
needle from the tube,
flame the opening, and
recap. Incubate the tube
as directed.

Figure 17.7 Stab technique for agar deep cultures. The inoculating needle is
sterilized and dipped into a broth culture before this step.

4. Inoculate the three plates of tryptic soy agar with
Escherichia coli using the method depicted in
figure 17.8. Spread the culture over several
quadrants of the plate using the streak-plate
method depicted in figure 17.9.

5. After inoculation, incubate plates and tubes for
48-72 hours in a laboratory incubator (figure
17.10) set at 35°C.

Second Session: Media Examination

1 . After incubation, examine cultures for growth.
Note: The assessment of growth in tubes and
plates can be aided by comparing inoculated
media with uninoculated media. Begin your
assessment with broth tubes. Broth tubes should
appear cloudy, or turbid, when compared to the
clear broth in uninoculated tubes.

2. Growth on slants should be evident extending
away from the line of inoculation.

3. In agar deeps, growth should occur along the
needle line of inoculation from top to bottom.

4. Growth on agar plates should appear as distinct
colonies by the second or third quadrant, each
colony having the same appearance. Good
separation of colonies is essential for two
reasons: (1) to confirm the presence of only one
species of bacteria (a pure culture); and (2) to
determine the specific characteristics of isolated
colonies. If colonies are not well separated, you
might consider inoculating another series of
plates in an attempt to improve your technique.

5. After examining all plates and tubes for
Escherichia coli growth, go back through and
inspect them for signs of contamination. Look for
any type of growth that appears different from
that of the culture you inoculated. The absence of
contamination indicates good aseptic technique.
Contamination in one or more tubes or plates
may indicate the need to review the basic aseptic
techniques in table 17.2.

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

. Bacterial Cultivation

17. Preparation and
Inoculation of Growth
Media

126 Section III Bacterial Cultivation

(a) Flame the opening of the broth tube

(b) Insert a sterile loop into the broth culture.

Figure 17.8 Inoculation of an agar plate

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

Bacterial Cultivation

17. Preparation and
Inoculation of Growth
Media

Preparation and Inoculation of Growth Media EXERCISE 17 127

Initial section inoculated

(a) Orient your plate as
depicted here.

(b) Lift the lid, and use a sterile
loop to make lines, or streaks,
across the agar as shown in
quadrant 1. Close the lid.

Quadrant 3

(d) Flame the loop, lift the lid,
and make streaks as shown
in quadrant 3. Close the lid.
Incubate the plate as directed

Figure 17.9 The streak-plate method.

Figure 17.10 A laboratory incubator. A temperature of
35 °C will encourage rapid bacterial growth.

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

Bacterial Cultivation

17. Preparation and
Inoculation of Growth
Media

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

Bacterial Cultivation

17. Preparation and
Inoculation of Growth
Media

Name

Lab Section

EXERCISE

Laboratory Report

Date

Preparation and Inoculation of Growth Media

1. Draw the growth results from your tubes and plates.

Broth tubes

Agar slants

Agar deeps

Growth (+ or -)

Contaminants?

Description
of growth

Growth (+ or -)

Contaminants?

Description

of growth
Good separation

of colonies?

Agar plates

129

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

Bacterial Cultivation

17. Preparation and
Inoculation of Growth
Media

130

Section III Bacterial Cultivation

2. Answer the following questions in the space provided,
a. Why is it essential that media be sterile prior to use?

b. Why must agar be cooled prior to pouring plates?

c. Why are the inoculating loop and needle flamed before and after use?

d. What is a contaminant? How does it gain entry into your culture? How do you keep a contaminant
from entering your lab culture?

e. How does one determine if growth has occurred in broth?

f . Why is streak-plating such an essential procedure in the isolation and characterization of bacteria?

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

Bacterial Cultivation

17. Preparation and
Inoculation of Growth
Media

Preparation and Inoculation of Growth Media EXERCISE 17 131

3. Answer the following questions based on these photographs:

a. Name of technique

Good separation of colonies?

(yes or no)

Pure culture? (yes or no)

How do you know?

b. Name of technique

Culture contaminant? (yes or no)

How can you tell?

c. Name of technique

Good separation of colonies?

(yes or no)

Pure culture? (yes or no)

d. Name of technique
Good separation of

colonies? (yes or no)

Pure culture? (yes or no)

How do you know?

Alexander-Strete-Niles:

III. Bacterial Cultivation

18. Culture

©The McGraw-Hill

Lab Exercises in
Organismal and Molecular
Microbiology

Characterization of
Bacteria

Companies, 2003

Culture Characterization of Bacteria

When a single bacterial culture is grown using different
forms of media (broth, slants, deeps, and plates), it dis-
plays a collective pattern of growth that is unique to
its species. This unique pattern of growth is referred
to as its culture characteristics. An organism's culture
characteristics can help distinguish it from other organ-
isms, since each bacterial species typically has a unique
pattern of growth. Although useful, culture character-
istics alone cannot be relied on to identify the many
species of bacteria. They must be combined with stain-
ing reactions and biochemical characteristics.

In this exercise, you will use different forms of
media to determine the culture characteristics of five
known bacteria. You will also be given one of these five
bacteria as an unknown to identify.

Materials

Cultures (24-48-hour broth)
Bacillus cereus
Micrococcus luteus
Proteus vulgaris
Pseudomonas aeruginosa
Staphylococcus epidermidis

Media

6 tubes tryptic soy broth
6 slants tryptic soy agar
6 deeps tryptic soy agar
6 plates tryptic soy agar
6 tubes motility test agar

Equipment

Incubator (set at 35 °C)

Miscellaneous supplies
Bunsen burner and striker
Inoculating loop and needle
Test tube rack
Wax pencil

First Session: Media Inoculation
and Incubation

1. Inoculate 6 tryptic soy broth tubes: 5 tubes with
the known cultures (Bacillus cereus, Micrococcus
luteus, Proteus vulgaris, Pseudomonas
aeruginosa, and Staphylococcus epidermidis)
and 1 tube with the unknown culture (one of

the previous five cultures, but designated by
number only).

2. Inoculate 6 tryptic soy agar slants: 5 slants
with the known cultures and 1 slant with the
unknown culture.

3. Inoculate 6 tryptic soy agar deeps: 5 deeps with
the known cultures and 1 deep with the unknown
culture. Use an inoculating needle and a straight
line of inoculation almost to the bottom.

4. Inoculate 6 motility test agar tubes: 5 tubes with
the known cultures and 1 tube with the unknown
culture. Use an inoculating needle and a straight
line of inoculation two-thirds of the way down.
Motility test agar is used to determine whether
or not a culture is motile. The composition of this
medium is listed in table 18.1.

Table 1 8.1 Components of Motility

Test Agar

Tryptose

10 g

Sodium chloride

Agar

Distilled water

1 ,000 ml

Final pH =

7.2

Source: The Difco Manual. Eleventh Edition. Difco Laboratories

133

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III. Bacterial Cultivation

18. Culture

©The McGraw-Hill

Lab Exercises in
Organismal and Molecular
Microbiology

Characterization of
Bacteria

Companies, 2003

134 Section III Bacterial Cultivation

5. Inoculate 6 tryptic soy agar plates: 5 plates with
the known cultures and 1 plate with the unknown
culture. Use an inoculating loop and the streak-
plate method.

6. Incubate all inoculated tubes and plates in a
35 °C incubator for 48-72 hours.

Second Session: Media Examination

1 . After incubation, examine all plates and tubes for
growth. Note: To aid in the interpretation of
growth, use an uninoculated plate or tube for
comparison. Begin your examination with the six
broth tubes. Of the growth patterns in broth

depicted in figure 18.1, determine which pattern
is displayed by each culture. Record your
determination in the table of the laboratory report.

2. Continue your examination of growth by
inspecting slants, deeps, motility test agar, and
plates. Again, consult the growth patterns in
figure 18.1 to determine which pattern is
displayed in the appropriate medium by each
culture. Record your results in the table of the
laboratory report.

3 . After inspecting your cultures and completing the
laboratory report table, determine which of the
five cultures you have as your unknown.

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III. Bacterial Cultivation

18. Culture

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Lab Exercises in
Organismal and Molecular
Microbiology

Characterization of
Bacteria

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Culture Characterization of Bacteria EXERCISE 18 135

C SRI ) Surface

growth

♦ . ♦

Solidified <
agar

Growth
throughout
) agar deep,
but greatest
at surface

Obligate aerobe Facultative anaerobe

Agar deep cultures

Sediment

*******

Pellicle Ring Turbid
Growth in broth media

♦♦ ♦ ♦

♦ ♦ • ♦ i

♦♦*•-♦

♦ •* ♦* * ♦

« ♦ ♦ ♦ ♦
♦ ♦ ♦ ♦ ,

♦ ♦ ♦ ♦ • *

* ♦ ♦ ♦ ♦ *

♦ . ♦ . •

Flocculent

Shape

Margin

Elevation

Size

Texture:

Appearance:
Pigmentation

Circular Rhizoid Irregular Filamentous Spindle

"■

Entire Undulate Lobate Curled Rhizoid Filamentous

Flat Raised Convex Pulvinate Umbonate

Pinpoint Small Moderate Large

Smooth or rough

Glistening (shiny) or dull

Nonpigmented (cream, tan, white)
Pigmented (purple, red, yellow)

Optical property: Opaque, translucent, transparent

Colonies on agar plates

Motile

Nonmotile

Motility test agar

•

.♦.-♦■

- ■.♦ *• .♦

Filiform Echinulate Beaded Effuse Arborescent Rhizoid

Growth on agar slants

Figure 18.1 Cultural characteristics of bacteria.

Alexander-Strete-Niles:

III. Bacterial Cultivation

18. Culture

©The McGraw-Hill

Lab Exercises in
Organismal and Molecular
Microbiology

Characterization of
Bacteria

Companies, 2003

Alexander-Strete-Niles:

III. Bacterial Cultivation

18. Culture

©The McGraw-Hill

Lab Exercises in
Organismal and Molecular
Microbiology

Characterization of
Bacteria

Companies, 2003

Name

Lab Section

EXERCISE

Laboratory Report

Date

Culture Characterization of Bacteria

1 . a. Fill in the following table from your observations of culture characteristics

Organism

Colony
morphology

Growth on
slants

Growth in
deeps

Growth in
broth

Motility
test agar

Bacillus
cere us

Micrococcus
luteus

Proteus
vulgaris

Pseudomonas
aeruginosa

Staphylococcus
epidermidis

Unknown
no.

b. Based on the results you recorded in the table, identify your unknown:

c. Which culture characteristic(s) were most useful to you in identifying your unknown?

d. Which organism has the following culture characteristics?

(1) water-soluble green pigment, forms pellicle in broth, and is motile:

(2) small-to-medium white colony, facultatively anaerobic, and nonmotile:

2. Does each culture appear to have its own unique culture characteristics? If so, explain how this could be
useful in identification.

137

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III. Bacterial Cultivation

18. Culture

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Lab Exercises in
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Microbiology

Characterization of
Bacteria

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138 Section III Bacterial Cultivation

3. Define these terms
a. colony

b. pigmentation

c. facultatively anaerobic

d. pellicle

4. Answer the following questions based on these photographs

a. Name this growth
pattern in broth.

b. Is there pigmentation?

In this exercise, displayed by

c. A streak-plate of which
organism in this exercise?

In this exercise,
displayed by

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III. Bacterial Cultivation

18. Culture

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Lab Exercises in
Organismal and Molecular
Microbiology

Characterization of
Bacteria

Companies, 2003

Culture Characterization of Bacteria EXERCISE 18

139

ml ^hjj

d. A culture characteristic of
which organism in this

exercise?

e. Name this growth pattern
in broth.

In this exercise, displayed
by

f. Is there pigmentation?

In this exercise, this colony
morphology displayed by

g. A streak-plate of which organism
in this exercise?

Alexander-Strete-Niles:

IV. Bacterial Identification

19. Biochemical Tests

©The McGraw-Hill

Lab Exercises in
Organismal and Molecular
Microbiology

Used to Identify Bacteria

Companies, 2003

Biochemical Tests Used

to Identify Bacteria

Although culture and staining characterization of bac-
teria provide a substantial amount of information, these
techniques are not sufficient by themselves for the iden-
tification of bacteria. The results of staining and cul-
turing must be combined with the results of
biochemical tests to definitively identify bacteria. Bio-
chemical tests evaluate the metabolic properties of a
bacterial isolate. After a number of biochemical tests
have been performed, the combination of test results
forms a biochemical pattern for an isolate, which is
unique for each species.

In this exercise, you will perform eight biochemi-
cal tests on known bacterial cultures. You will use two
cultures for each test, a culture known to have a positive
result and a culture known to have a negative result. This
will familiarize you with either test result, allowing you
to correctly interpret biochemical test results for the
nonclinical unknown you will identify in Exercise 20.

Cultures (24-48-hour agar or broth)
Alcaligenes faecalis
Enterobacter aerogenes
Enterococcus faecalis
Escherichia coli
Proteus vulgaris
Pseudomonas aeruginosa
Staphylococcus epidermidis

Media

1 plate tryptic soy agar

2 slants tryptic soy agar

6 tubes oxidation-fermentation (O-F) glucose

medium
2 tubes nitrate broth (with durham tube)
2 tubes methyl red-Voges Proskauer (MR-VP)

medium
4 tubes sulfide indole motility (SIM) medium
2 tubes lactose broth (with durham tube)

Equipment

Incubator (set at 35 °C)

Reagents

Hydrogen peroxide (3%)
Kovac's reagent
Methyl red (pH indicator)
Oxidase reagent

Miscellaneous supplies
Bunsen burner and striker
Inoculating loop and needle
Mineral oil (sterile)
Pasteur pipette with bulb
Test tube rack
Wax pencil

First Session: Inoculation
and Incubation

1 . Catalase test: Inoculate 2 tryptic soy agar slants,
one with Enterococcus faecalis, and the other
with Staphylococcus epidermidis. Use an
inoculating loop to make a back- and- forth streak
across the slant surface.

2. Denitrification test: Using an inoculating loop,
inoculate 2 nitrate broth tubes, one with
Alcaligenes faecalis, and the other with
Pseudomonas aeruginosa.

3. Hydrogen sulfide (H 2 S) production: Inoculate
2 SIM tubes, one with Escherichia coli, and
the other with Proteus vulgaris. Use an
inoculating needle, and stab the agar with a
single in-and-out motion.

4. Indole production: Inoculate 2 SIM tubes, one
with Enterobacter aerogenes, and the other with
Escherichia coli. Use an inoculating needle, and
stab the agar with a single in-and-out motion.

142

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IV. Bacterial Identification

19. Biochemical Tests

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Lab Exercises in
Organismal and Molecular
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Used to Identify Bacteria

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Biochemical Tests Used to Identify Bacteria EXERCISE 19 143

5. Lactose utilization: Using an inoculating loop,
inoculate 2 lactose broth tubes, one with
Escherichia coli, and the other with
Proteus vulgaris.

6. Methyl red test: Using an inoculating loop,
inoculate 2 MR-VP tubes, one with Enterobacter
aerogenes, and the other with Escherichia coli.

1 . Oxidase test: With a wax pencil, draw a line
down the center of a tryptic soy agar plate. Using
an inoculating loop, inoculate one half of the
plate with Escherichia coli and the other half
with Pseudomonas aeruginosa. Use a back-and-
forth streak across the surface of the agar.

8. Oxidation-fermentation (O-F) glucose test:

Using an inoculating needle, inoculate 2 tubes of
O-F glucose with Alcaligenes faecalis, 2 tubes
with Escherichia coli, and 2 tubes with
Pseudomonas aeruginosa. After inoculation,
cover one tube in each pair with a 2 cm layer
of sterile mineral oil. Note: Mineral oil can
be poured directly into the tube without using
a pipette.

9. Incubate all tubes and plates at 35°C for 24-48
hours, except MR-VP. MR-VP tubes require a
minimum of 72 hours of incubation.

Second Session: Reading Test Results

1 . Catalase test: Use a Pasteur pipette to place a few
drops of 3% hydrogen peroxide onto each slant
culture. Watch for immediate signs of bubbling,
which represent a positive test; the absence of
bubbles is a negative test (figure 19.1). A slide
test can be done by mixing a small amount of
culture into a drop of water on a glass slide. The
hydrogen peroxide is then added to the drop.

Expected results: Staphylococcus epidermidis is
catalase-positive, while Enterococcus faecalis is
catalase-negative.

2. Denitrification test: Note the small inverted tube
in the bottom of the medium. This tube, called a
durham tube, is designed to collect gas. Read this
test by looking for gas bubbles in the durham

Positive test:

added H 2 2 ► H 2 + 2

(bubbles)

Negative test:

no catalase

added H 2 2 ► H 2 2

(no bubbles)

Example: Example:

Staphylococcus epidermidis Enterococcus faecalis

Test
results
in tubes

Positive
test result
on a slide

Figure 19.1 Catalase test: reactions and results of
positive and negative tests.

tube (nothing needs to be added). Nitrate
broth contains potassium nitrate (table 19.1).
Denitrification by bacteria converts the nitrate to
nitrogen gas. Gas bubbles in the durham tube,
therefore, represent a positive test (figure 19.2).
The absence of bubbles represents a negative test

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IV. Bacterial Identification

19. Biochemical Tests

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Used to Identify Bacteria

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144

Section IV Bacterial Identification

Table 19.1 Composition

of Biochemical

Test Medio

Nitrate broth

Peptone

Beef extract

Potassium nitrate

Distilled water

1,000 ml

Final pH = 7.0

SIM medium

Peptone

30 g

Beef extract

Peptonized iron

0.2 g

Sodium thio sulfate

0.02 g

Agar

Distilled water

1,000 ml

Final pH = 7.3

Lactose broth

Beef extract

Proteose peptone

10 g

Sodium chloride

Lactose

Phenol red

0.018 g

Distilled water

1,000 ml

Final pH = 7.4

MR-VP medium

Peptone

Dextrose

Dipotassium phosphate

Distilled water

1,000 ml

Final pH = 6.9

Oxidation-fermentation

(O-F) glucose medium

Glucose

10 g

Tryptone

Sodium chloride

Dipotassium phosphate

0.3 g

Bromthymol blue

0.08 g

Agar

Distilled water

1,000 ml

Final pH = 6.8

Positive test:

Negative test:

nitrate reductase

3 iN 2

(bubbles in durham tube)

Example:

Pseudomonas aeruginosa

N 2 (gas) N0 3

no nitrate
reductase

* NO3 (no gas)

(no bubbles in durham tube)

Example:

Alcaligenes faecalis

Figure 19.2 Denitrification: positive and negative
test results.

Source: The Difco Manual. Eleventh Edition. Difco Laboratories

Expected results: Pseudomonas aeruginosa ^
positive, while Alcaligenes faecalis is negative.

3. Hydrogen sulfide (H 2 S) production: Examine
each SIM tube for the presence of a black color
(nothing needs to be added) . A black color
indicates the production of H 2 S, which combines
with the peptonized iron in the SIM medium
(table 19.1). The result is FeS, which causes a
blackening of the medium and represents a
positive test (figure 19.3). The absence of a black
color is a negative test.

Expected results: Proteus vulgaris is positive,
while Escherichia coli is negative.

4. Indole production: Use a dropper to place 5
drops of Ko vac's reagent onto the top of the SIM
agar in each tube. If the amino acid tryptophan
has been broken down by the enzyme
tryptophanase to form indole, the Kovac's reagent
will combine with the indole to form a red color.
A red color in the Kovac's reagent at the top of
the agar represents a positive test (figure 19.4).

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IV. Bacterial Identification

19. Biochemical Tests

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Lab Exercises in
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Used to Identify Bacteria

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Biochemical Tests Used to Identify Bacteria EXERCISE 19 145

Positive test:

cysteine desulfurase

cysteine ► NH 3 + pyruvic acid + H 2 S

H 2 S + FeSQ 4

* FeS + H 2 S0 4

(blackening of medium)

Example: Proteus vulgaris

no cysteine desulfurase

Negative test: cysteine ► cysteine

(no blackening of medium)

Example: Escherichia coli

Figure 19.3 Hydrogen sulfide (H 2 S) production: positive and negative test results.

tryptophanase . .

Positive test: tryptophan ► NH 3 + pyruvic acid + indole

indole + added Kovac's reagent = red color

Example: Escherichia coli

no tryptophanase

Negative test: tryptophan ► tryptophan

tryptophan + added Kovac's reagent = no red color

Example: Enterobacter aerogenes

Figure 19.4 Indole production: reactions and results for positive and negative tests.

No color change in the Kovac's reagent is a
negative test.

Expected results: Escherichia coli is
indole-positive, while Enterobacter aerogenes
is indole-negative.

5. Lactose utilization: When examining these tubes,
look for a color change in the broth and gas in the
durham tube (nothing needs to be added). Lactose
broth contains the sugar lactose and the pH
indicator phenol red (table 19.1). When lactose is

Alexander-Strete-Niles:

IV. Bacterial Identification

19. Biochemical Tests

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Used to Identify Bacteria

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146

SECTION IV Bacterial Identification

Positive test: lactose

(red)

acids (pH decreases) and gas

(yellow and bubbles in durham tube)

Example: Escherichia coli (acid and gas)

Negative test: lactose

(red)

lactose (pH unchanged and no gas)
(red and no bubbles in durham tube)

Example: Proteus vulgaris (no acid or gas)

Figure 19.5 Lactose utilization: possible reactions and results.

utilized, acids or acids and gas are produced. The
acid causes the pH to decrease, turning the
phenol red from red to yellow. The gas collects in
the durham tube. Therefore, a yellow color or a
yellow color and bubbles in the durham tube
represent a positive test (figure 19.5). No color
change and no bubbles in the durham tube
represent a negative test.

Expected results: Escherichia coli is positive,
while Proteus vulgaris is negative.

6 . Methyl red test: Using a Pasteur pipette, add 1
drops of methyl red pH indicator to each tube.
Swirl the tube gently to mix the drops into the
broth. Examine each tube for color change.
Bacteria that produce many acids from the
breakdown of dextrose (glucose) in the MR-VP
medium (table 19.1) cause the pH to drop to 4.2.
At this pH, methyl red is red. A red color
represents a positive test (figure 19.6). Bacteria
that produce fewer acids from the breakdown of
glucose drop the pH to only 6.0. At 6.0, methyl
red is yellow. A yellow color represents a
negative test.

Expected results: Escherichia coli is methyl-
red-positive, while Enterobacter aerogenes is
methyl-red- negative.

7. Oxidase test: Drop 1-2 drops of oxidase reagent
onto colonies of both cultures. Watch for a
gradual color change from pink, to light purple,
and then to dark purple within 10-30 seconds.
Such a color change indicates the presence of the
respiratory enzyme cytochrome c oxidase and
represents a positive test (figure 19.7). No color
change in this period is a negative test.

Expected results: Pseudomonas aeruginosa is
oxidase-positive, while Escherichia coli is
oxidase-negative.

8 . Oxidation-fermentation (O-F) glucose test: In

these tubes, you will look for color changes in
the medium (nothing needs to be added). O-F
glucose medium contains the sugar glucose and
the pH indicator bromthymol blue (table 19.1).
This pH indicator is green at the initial pH of 6.8,
but turns to yellow at a pH of 6.0. If glucose is
utilized, acids are produced and the pH drops,

Alexander-Strete-Niles:

IV. Bacterial Identification

19. Biochemical Tests

©The McGraw-Hill

Lab Exercises in
Organismal and Molecular
Microbiology

Used to Identify Bacteria

Companies, 2003

Biochemical Tests Used to Identify Bacteria EXERCISE 19 147

Positive test: glucose

> pyruvic acid ( 1 day)

> lactic, acetic, and formic acids (2-5 days)

pyruvic acid

many acids (pH 4.2) + added methyl red = red color

Example: Escherichia coli

Negative test: glucose

- pyruvic acid ( 1 day)

- neutral end products (2-5 days)

pyruvic acid

neutral end products (pH 6.0) + added methyl red = yellow color

Example: Enterobacter aerogenes

Figure 19.6 Methyl red test: reactions and results for a positive and negative test.

Positive test: cytochrome c (reduced) + ^2

cytochrome oxidase

* cytochrome c (oxidized) + H 20

cytochrome c (oxidized) + oxidase reagent (re duced) > cytochrome c (re duced) + oxidase reagent (ox idized)

(colorless) (dark purple)

Example: Pseudomonas aeruginosa

Negative test:

no cytochrome oxidase

cytochrome c (re duced) + °2 > cytochrome c (re duced) + °2

cytochrome c (re duced) + oxidase reagent (re d U ced) * cytochrome c (re duced) + oxidase reagent (re duced)

(colorless) (colorless)

Example: Escherichia coli

Oxidase-positive test result

Figure 19.7 Oxidase test: reactions and results of positive and negative tests

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IV. Bacterial Identification

19. Biochemical Tests

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Lab Exercises in
Organismal and Molecular
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Used to Identify Bacteria

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148

Section IV Bacterial Identification

glucose (with oil)
(green)

glucose (open tube without oil)
(green)

ino reaction! glucose >
(inert) (green)

glucose *
(green)

Example: Alcaligenes faecalis

Oxidation-fermentation:

glucose

— ► acids, pH decreases (with oil)

(facultative anaerobe)

(green)

(yellow)

glucose

— ► acids, pH decreases (open tube without oil)

(green)

(yellow)

Example: Escherichia coli

Oxidation:

(aerobe)

glucose
(green)

- glucose (with oil)

(green)

- acids, pH decreases (open tube without oil)

(yellow)

Example: Pseudomonas aeruginosa

Figure 19.8 Oxidation-fermentation (O-F) glucose test: possible reactions and results

causing the bromthymol blue to turn from green
to yellow. If both tubes (with and without oil)
turn yellow, the test organism is considered a
facultative anaerobe, able to use glucose in the
presence or absence of oxygen (figure 19.8). If
only the tube without oil turns yellow, the test
organism is considered an aerobe, able to use

glucose only when oxygen is present. No change
in either tube indicates that the test organism is
unable to utilize glucose.

Expected results: Escherichia coli is a facultative
anaerobe, Pseudomonas aeruginosa is an
aerobe, mid. Alcaligenes faecalis is nonreactive
(inert) on glucose.

Alexander-Strete-Niles:

IV. Bacterial Identification

19. Biochemical Tests

©The McGraw-Hill

Lab Exercises in
Organismal and Molecular
Microbiology

Used to Identify Bacteria

Companies, 2003

Name

Lab Section

EXERCISE

Laboratory Report

Date

Biochemical Tests Used to Identify Bacteria

Record your results for the biochemical tests.

Biochemical test

Reagent added

Observations

Interpretation

1. Catalase test:

Enterococcus faecalis

Staphylococcus epidermidis

2. Denitrification test:
Alcaligenes faecalis

Pseudomonas aeruginosa

3. H 2 S production:
Escherichia coli

Proteus vulgaris

4. Indole production:
Enterobacter aerogenes

Escherichia coli

5. Lactose utilization:
Escherichia coli

Proteus vulgaris

6. Methyl red test:

Enterobacter aerogenes

Escherichia coli

7. Oxidase test:
Escherichia coli

Pseudomonas aeruginosa

8. O-F glucose test:
Alcaligenes faecalis

Pseudomonas aeruginosa

Escherichia coli

149

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

IV. Bacterial Identification

20. Application:
Identification of a
Nonclinical Bacterial
Unknown

Application: Identification of a
Nonclinical Bacterial Unknown

Background

Previous exercises have covered basic aspects of micro-
biology, including microscopic observation, staining, cul-
tivation, and biochemical testing. In this exercise, you will
apply what you have learned about these techniques to the
identification of a nonclinical bacterial unknown.

Materials

Cultures (24-48-hour broth)
Alcali genes faecalis
Bacillus cere us
Enterobacter aerogenes
Enterococcus faecalis
Escherichia coli
Micrococcus lute us
Mycobacterium phlei
Neisseria sicca
Proteus vulgaris
Pseudomonas aeruginosa
Serratia marcescens
Staphylococcus epidermidis

All agents in red are BSL2 bacteria.

Stains

Gram stain
Crystal violet
Gram's iodine
Ethanol (95%)
Safranin

Acid-fast stain
Carbolfuchsin
Acid-alcohol
Methylene blue

Spore stain

Malachite green
Safranin

Tryptic soy agar slants

O-F glucose tubes

Nitrate broth tubes (with durham tube)

MR-VP tubes

SIM tubes

Lactose broth tubes (with durham tube)

Equipment

Hot plate (optional)
Incubator (set at 35 °C)
Light microscope

Reagents

Hydrogen peroxide (3%)
Kovac's reagent
Methyl red (pH indicator)
Oxidase reagent

Miscellaneous supplies
Bibulous paper
Bunsen burner and striker
Clothespin

Disposable gloves (optional)
Egg albumin solution
Glass slides
Immersion oil
Inoculating loop and needle
Lens paper
Mineral oil (sterile)
Pasteur pipette with bulb
Staining tray
Test tube rack
Wash bottle with tap water
Wax pencil

Media

Tryptic soy agar plates

1 . You will select an unknown bacterial culture, or
one will be assigned to you. In either case, be
sure to record in the laboratory report the number
assigned to your unknown.

2. Do a streak-plate of your unknown (see Exercise
17). After incubation, examine your streak-plate
to make sure that you have a pure culture free of
contamination. ^

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

IV. Bacterial Identification

20. Application:
Identification of a
Nonclinical Bacterial
Unknown

152 SECTION IV Bacterial Identification

3. Examine the streak-plate to determine the colony
characteristics of your unknown culture (see
Exercise 18). Also examine the broth tube your
unknown was cultured in to determine its
characteristics (see Exercise 18).

4. Do a Gram stain of an 18-24-hour culture
to determine cell morphology and Gram
reaction (see Exercise 11). Cell shape and
arrangement can be verified with either a
negative stain (see Exercise 9) or a simple
stain (see Exercise 10).

5. Examine the identification scheme in figure 20.1
to determine the test to be done next. If a spore

stain is required, consult Exercise 13; if an acid-
fast stain is necessary, see Exercise 12; if
biochemical tests are needed, you can find them
in Exercise 19. Note: Inoculate a new plate each
week to keep your culture viable.

6. Continue with your tests until your unknown has
been identified. Be sure to record the results of
all tests and the identity of your unknown in the
laboratory report.

7. Your laboratory instructor may wish to see all
results when you are finished. Therefore, be sure
to keep all slides, plates, and tubes until
examined by your laboratory instructor.

Gram stain

Coccus shape

Rod shape

Gram-negative

Gram-positive

Gram-negative

Gram-positive

Diplococci,

oxidase (+)

{Neisseria

sicca)

Catalase

O-F glucose and oxidase

Spore stain

O-F glucose

Aerobic

Cells in

short chains

(Enterococcus

faecalis)

Facultatively
anaerobic

Yellow colonies,

cells in tetrads

(Micrococcus

lute us)

Aerobic / inert and
oxidase (+)

Denitrification (+) Denitrification (-)

(Pseudomonas
aeruginosa)

(Alcaligenes
faecalis)

Acid-fast,

cells in cords

(Mycobacterium

phlei)

Non-acid-fast,

cells in chains

(Bacillus cereus)

White colonies,

cells in clusters

(Staphylococcus

epidermidis)

Facultatively anaerobic
and oxidase (-)

Methyl red and indole

Lactose

Figure 20.1 Identification
scheme for 12 bacterial
unknowns. You can use this
scheme when identifying your
nonclinical bacterial unknown.

Red colonies
at 22° C

(Serratia

marcescens)

Nonpigmented colonies

(Enterobacter

aero genes)

Lactose (+)

(Escherichia

coli)

Lactose (-)
(Proteus
vulgaris)

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

IV. Bacterial Identification

20. Application:
Identification of a
Nonclinical Bacterial
Unknown

Name

Lab Section

EXERCISE

Laboratory Report

Date

Application: Identification of a Nonclinical Bacterial Unknown

Unknown no.

1 . Follow the identification scheme in figure 20. 1 to identify your nonclinical bacterial unknown
Be sure to perform only the tests required to identify your unknown.

2. Record your results for the required tests.

Procedure

Observations

Results

Culture characteristics
Broth

Agar

Staining characteristics
Cell shape

Cell arrangement

Gram stain

Acid-fast stain

Spore stain

Biochemical characteristics
Catalase test

Denitrification test

H 2 S production

Indole production

Lactose utilization

Methyl red test

Oxidase test

O-F glucose test

3. After following the scheme in figure 20.1 and recording the results for the required tests in the
preceding table, I conclude that my unknown is

153

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V. Medical Microbiology

21. Isolation and

©The McGraw-Hill

Lab Exercises in
Organismal and Molecular
Microbiology

Identification of
Staphylococci from the
Skin

Companies, 2003

solation and Identification of

Staphylococci from the Skin

Normal Flora of Human Skin

Many regions of the body, including the skin, have a
usual population of bacteria referred to as residents or
normal flora. Particularly common on the skin are the
Gram-positive cocci, including nonpathogenic Staphy-
lococcus epidermidis and species of Micrococcus. In
addition to these, Gram-positive pleomorphic rods,
called diphtheroids, are also found.

Pathogens of Human Skin

Staphylococcus aureus is a normal resident of the nasal
membranes, but can be transferred to the skin, where
it is considered a transient. S. aureus is considered a
pathogen because it causes skin infections such as
boils, abscesses, carbuncles, impetigo, and scalded skin
syndrome. In addition, exotoxin-producing strains of
S. aureus cause food poisoning and toxic shock.

Identification of Skin Isolates

The Gram stain is used to determine the morphology
and Gram reaction of skin isolates. The catalase test
is used to differentiate the Gram-positive staphylococci
and micrococci, which are catalase-positive (table 21.1),

from the Gram-positive streptococci, which are
catalase-negative. Catalase-positive, Gram-positive cocci
are differentiated using the oxidation-fermentation
(O-F) glucose test (see Exercise 19), where the staphy-
lococci are facultatively anaerobic (table 21.1). The pres-
ence of staphylococci is verified by growth on mannitol
salt agar (MSA), since only these bacteria can tolerate
the 7.5% salt content of the medium (table 21.2).

The identification of Staphylococcus aureus can be
completed with the tests just described plus three addi-
tional tests. S. aureus ferments mannitol in MSA to pro-
duce acids that turn the medium from red to yellow. In
addition, S. aureus produces coagulase, an enzyme that
clots blood plasma, and hemolysins, enzymes that lyse
red blood cells. The presence of the former enzyme is
detected by a coagulase test, while the latter enzymes
are detected with blood agar.

Detecting Penicillinase-Producing
Staphylococci

The enzyme penicillinase opens the beta-lactam ring of
the penicillin molecule, resulting in harmless penicilloic
acid (figure 21.1). Therefore, bacteria able to produce
penicillinase can break down penicillin and as a result
are penicillin-resistant. With the extensive use of
penicillin over 50 years, many bacteria are penicillin-

Table 21.1 Characteristics of Common Gram-positive Cocci from the Skin

Characteristic

Micrococcus

Staphylococcus epidermidis

Staphylococcus aureus

Pigment on agar

Bright yellow

White

Light to golden yellow

Catalase

(+)

O-F glucose

Aerobic

Facultatively anaerobic

Mannitol fermentation

(-)

(+>

Coagulase

(-)

(+>

Hemolysis on blood agar

None

Beta-hemolysis

156

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V. Medical Microbiology

21. Isolation and

©The McGraw-Hill

Lab Exercises in
Organismal and Molecular
Microbiology

Identification of
Staphylococci from the
Skin

Companies, 2003

Isolation and Identification of Staphylococci from the Skin EXERCISE 21

157

Table 21.2 The

Composition

of Mannitol

Salt

Agar (MSA)

Proteose peptone

10 g

Beef extract

D-mannitol

10 g

Sodium chloride

75 g

Phenol red

0.025 g

Agar

15 g

Distilled water

1 ,000 ml

Final pH

7.4

Source: The Difco Manual. Eleventh Edition. Difco Laboratories

resistant, including the majority of staphylococci. Iso-
lating staphylococci from the skin offers an excellent
opportunity to demonstrate penicillin resistance. This
can be easily done by using nitrocefin-impregnated
disks. Nitrocefin has a beta-lactam ring similar to that
of penicillin. When the ring of nitrocefin is opened by
penicillinase, the molecule turns red. Therefore, the
appearance of a red color on a nitrocefin dry slide after
the addition of a staphylococcal skin isolate is indica-
tive of the enzyme penicillinase.

Cultures (24-48-hour agar)
Staphylococcus aureus

Staphylococcus epidermidis

All agents in red are BSL2 bacteria

Media

Blood agar plates (tryptic soy agar with 5%

sheep blood)
O-F glucose tubes
Mannitol salt agar (MSA) plates
Tryptic soy agar plates
Tryptic soy broth tubes

Chemicals and reagents

Blood plasma (rabbit; for coagulase test)
Gram-stain reagents
Hydrogen peroxide (for catalase test)
Nitrocefin dry slides
Rapid latex agglutination test kit (for
coagulase test)

Equipment

Incubator (35 °C)
Light microscope

Miscellaneous supplies
Bibulous paper

Biohazard bag (or similar container)
Bottle with tap water
Bunsen burner and striker
Cotton-tipped swabs, sterile
Disposable gloves
Glass microscope slides
Immersion oil
Inoculating loop and needle
Lens paper
Mineral oil, sterile
Pasteur pipette with bulb
Pipette, 1 ml
Test tube
Test tube rack
Wax pencil

Beta-lactam ring

R-C-NH-CH-CH

Penicillinase

0=C

■N-

. CH 3
CH-COOH

Penicillin (effective)

R-C-NH-CH-CH

o=c

N-

. CH 3
CH-COOH

OH H

Penicilloic acid (ineffective)

Figure 21.1 Penicillinase produces resistance to penicillin by breaking a bond (arrow) in
the beta-lactam ring of penicillin, resulting in penicilloic acid, a molecule with no effect on
bacterial growth.

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V. Medical Microbiology

21. Isolation and

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Lab Exercises in
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Microbiology

Identification of
Staphylococci from the
Skin

Companies, 2003

158 Section V Medical Microbiology

Procedure

First Session: Isolation of Bacteria
from the Skin

1. Following the procedure shown in figure 21.2,
dip a sterile cotton-tipped swab into a tube of
tryptic soy broth. Blot the excess fluid against the
side of the tube.

2. Select a portion of your arm, face, or leg. Rub
the swab back and forth across a 5-square-
centimeter area.

3 . Use the swab to inoculate a tryptic soy agar
(TSA) plate. Rub the swab back and forth over
one area of the plate near the edge (figure 21.2c).
Note: Dispose of the swab in a biohazard bag.
With a sterile loop, cross over the swabbed area
to spread the bacteria across the plate surface.
Repeat this spreading process with the loop in
two more quadrants as depicted in figure 21.2c, /

4. Place the TSA plate into a 35°C incubator.

Second Session

Selection of Skin Isolates

1 . After 24-48 hours, examine the inoculated plate.
Based on colony morphology, determine the total

number of different bacterial types on TSA.
Record this number in the laboratory report.

2. Select two different bacteria from the plate that
are most common — that is, the bacterial types
with the greatest number of colonies. Number
these #1 and #2, and record their colony
morphology along with the colony morphology
of the common skin isolates, Staphylococcus
epidermidis and S. aureus.

Identification of Skin Isolates

The two common skin isolates will be identified using
the tests specified in figure 21.3. You will also test
Staphylococcus epidermidis and Staphylococcus aureus
in conjunction with your unknowns.

1 . Gram-stain your unknown skin isolates and the
two known cultures. Record your results in the
laboratory report.

2. If one or both of your unknown isolates are
Gram-positive cocci, continue your identification
by doing a catalase test as follows: Use a sterile
loop to deposit some cells from the unknown
colony into a drop of water on a glass slide. Add
a drop of hydrogen peroxide. Watch for bubbles,
indicative of a positive test. Do the same for your
two known cultures.

(a) Moisten a sterile cotton-tipped swab in
tryptic soy broth.

(b) Swab an area of your skin

(d) Use a sterile loop to spread the
inoculum into quadrant 1 .

(e) Use a loop to spread into quadrant 2

(f ) Use a loop to spread into quadrant 3

Figure 21.2 Isolation of bacteria from the skin.

Alexander-Strete-Niles:

V. Medical Microbiology

21. Isolation and

©The McGraw-Hill

Lab Exercises in
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Microbiology

Identification of
Staphylococci from the
Skin

Companies, 2003

Isolation and Identification of Staphylococci from the Skin EXERCISE 21

159

Common isolates from the skin

Gram stain

Gram-positive rods

Gram-positive cocci

Catalase

Regular rods

Irregular rods

Presumptive

Bacillus (large,

white to tan

colonies with dull

surface)

Presumptive
Coryne bacterium

(large, dry,
wrinkled colonies)

O-F glucose

Presumptive

Streptococcus
or Enterococcus

Facultatively anaerobic

Aerobic

Presumptive

Staphylococcus

Presumptive

Micrococcus

Mannitol salt agar

Antibiotic
resistant?

Presumptive

Staphylococcus
aureus

Presumptive

Staphylococcus
epidermidis

Antibiotic
resistant?

Coagulase

Coagulase-positive staphylococci
(confirmed Staphylococcus aureus)

Coagulase-negative staphylococci

Figure 21.3 Identification scheme for common isolates from the skin.

3. If one or both of your unknown isolates is a
Gram-positive coccus and catalase-positive,
inoculate a mannitol salt agar (MSA) plate,

a blood agar plate, and a pair of O-F glucose
tubes (cover the medium in one tube with
sterile mineral oil). Do the same for your
two known cultures.

4. Incubate plates and tubes at 35 °C.

Third Session

Identification of Staphylococci

1 . After 24-48 hours, inspect each O-F tube for
color change and each MSA plate for growth.
The presence of growth on MSA and a color
change from red to yellow in both O-F glucose
tubes is indicative of staphylococci. If one or

Alexander-Strete-Niles:

V. Medical Microbiology

21. Isolation and

©The McGraw-Hill

Lab Exercises in
Organismal and Molecular
Microbiology

Identification of
Staphylococci from the
Skin

Companies, 2003

160 Section V Medical Microbiology

more of your unknown isolates has these test
results, you have confirmed the isolation of
staphylococci from your skin. Both known
cultures should yield these tests results.

2. To determine whether your isolate is the
nonpathogenic Staphylococcus epidermidis or the
pathogenic Staphylococcus aureus, examine the
MSA plate for color change and the blood agar
plate for hemolysis. S. aureus ferments mannitol
to acids, yielding a color change from red to
yellow. S. aureus also produces hemolysins,
which lyse red blood cells, causing a clear zone
of beta-hemolysis around the colonies. The
nonpathogenic S. epidermidis produces no color
change on MSA and no clear zone around
colonies on blood agar.

3. To confirm the presence of S. aureus for those
organisms that ferment mannitol and are beta-
hemolytic, perform a coagulase test. This can be
done using one of the following two methods. A
positive result using either test provides
confirmation of S. aureus.

Rabbit Plasma: Mix a loopful of the organism
into 0.5 ml of rehydrated rabbit plasma in a test
tube. Incubate the tube at 35 °C for 4 hours. After
incubation, examine the plasma for clotting by
tilting the tube to the side. Plasma that has clotted
will not run, indicating a positive test.

Latex agglutination: A rapid latex agglutination
test kit can detect coagulase. Its use is outlined in
figure 21.4.

Determination of Penicillin Resistance

1 . To ascertain if your staphylococci are penicillin-
resistant, take a loopful of culture from the MSA
or blood agar plate, and rub it onto a moistened
nitrocefin disk.

2. Examine the disk for color change. Penicillinase-
producing staphylococci will yield a red color as
the beta-lactam ring of nitrocefin is opened.
Staphylococci that are penicillinase-negative
produce no color change. Record your results in
the laboratory report.

(a) Add one drop of latex reagent.

(b) Select an isolated colony, and pick it up (c) Mix the organism into the latex reagent,
with a stick.

Positive test

Negative test

Clumps

No clumps

(d) Rotate the card gently after inoculation. (e) Examine the card for black clumps (agglutination)

Figure 21.4 Use of the rapid latex agglutination test to detect coagulase.

Alexander-Strete-Niles:

V. Medical Microbiology

21. Isolation and

©The McGraw-Hill

Lab Exercises in
Organismal and Molecular
Microbiology

Identification of
Staphylococci from the
Skin

Companies, 2003

Name

Lab Section

EXERCISE

Laboratory Report

Date

solation and Identification of Staphylococci from the Skin

1 . Total number of bacterial types from the skin on TS A =

2. Identification of skin isolates

a. Identification of staphylococci

Common skin
isolates

Colony
morphology

Cell morphology
and Gram reaction

Catalase
test

* Results

indicative of

staphylococci?

Known cultures:
S. epidermidis

S. aureus

:[-

If yes, continue with (b); if no, stop here

b. Confirmation of staphylococci

Common skin isolates

O-F glucose

(facultatively

anaerobic?)

Growth on MSA?

* Confirmed
staphylococci?

Known cultures:
S. epidermidis

S. aureus

If yes, continue to (c); if no, stop here.

c. Differentiation of staphylococci

Common skin
isolates

Yellow on
MSA?

Beta-hemolysis
on blood agar?

Coagulase?

Indicative of
S. epidermidis
or S. aureus?

Known cultures:
S. epidermidis

S. aureus

161

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21. Isolation and

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Lab Exercises in
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Microbiology

Identification of
Staphylococci from the
Skin

Companies, 2003

162 Section V Medical Microbiology

d. Did you isolate nonpathogenic Staphylococcus epidermidis from your skin? Is this a common result?
Explain.

e. Did you isolate pathogenic S. aureus from your skin? Is this a common result? Explain.

3. Penicillin resistance

a. Was your staphylococcal isolate penicillin-resistant (i.e., produced red color change on
nitrocefin)?

b. For your lab section, determine how many staphylococcal isolates were tested for penicillin resistance
and how many of these were resistant. Calculate the percentage of penicillin-resistant isolates.

Total tested =

Total penicillin-resistant =

Percent penicillin-resistant =

Was this percentage expected? Explain.
4. Answer the following questions based on these photographs

a. How many different bacterial b. Are these results on MSA

types are on this skin plate?

indicative of staphylococci?
(yes or no)

c. Are these results on blood agar
indicative of S. epidermidis or
S. aureus?

How do you know?

Which isolate (on left or right)
is S. aureus?

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V. Medical Microbiology

22. Isolation and

©The McGraw-Hill

Lab Exercises in
Organismal and Molecular
Microbiology

Identification of
Streptococci from the
Throat

Companies, 2003

solation and Identification
of Streptococci from the Throat

Normal Flora of the Throat

The human throat is a region of the body that has a
resident, or normal, bacterial flora. The predominant
throat residents are species of streptococci. The most
common streptococci are the nonpathogenic viridans
streptococci. An opportunistically pathogenic species
of streptococci, Streptococcus pneumoniae, the
causative agent of pneumococcal pneumonia, is found
in the throat of 30-70% of normal individuals. It can
enter the lungs to cause pneumonia when an individ-
ual's resistance is weakened by a primary infection,
such as influenza. Other resident members of the throat
flora include species of staphylococci, including in
some cases Staphylococcus aureus, species of Neisse-
ria, and diphtheroids.

Pathogens of the Throat

The pathogenic species Streptococcus pyogenes is not
considered a normal member of the resident throat flora.

However, this organism can enter the throat through the
air via aerosol droplets from an infected individual.
Once in the throat, it can cause strep throat, a condi-
tion characterized by a sore throat, high fever, and a red,
inflamed appearance at the back of the throat.

Identification of Throat Isolates

The Gram stain is used to determine the morphology
and Gram reaction of throat isolates. The catalase test
is used to differentiate the streptococci, which are
catalase-negative, from the staphylococci/micrococci,
which are catalase-positive. The Gram-positive cocci
that are catalase-negative are considered presumptive
species of streptococci.

Streptococci common in the throat are differenti-
ated based on the tests listed in table 22.1. For example,
certain streptococci produce hemolysins that com-
pletely lyse red blood cells, resulting in a clear zone
around colonies on blood agar. This reaction, called
beta-hemolysis, differentiates the beta-hemolytic strep-
tococci from the alpha-hemolytic streptococci. These
latter streptococci produce hemolysins that only partially

Table 22.1 Differentiation of

Species

Streptococci Commonly Found

in the Throat

Strep name

Hemolyj

sis

Bacitracin-susceptible

Optochin-susceptible

Group A

{Streptococcus
pyogenes)

beta

(+)

N/A

Non-group A,
beta-hemolytic

beta

(-)

N/A

Streptococcus
pneumoniae

alpha

N/A

(+)

Viridans
streptococci

alpha

N/A

(-)

163

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Lab Exercises in
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Identification of
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Throat

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164 Section V Medical Microbiology

lyse red blood cells, producing a green color around
colonies on blood agar. This reaction is called alpha-
hemolysis. The beta-hemolytic streptococci are further
differentiated based on their susceptibility to the antibi-
otic bacitracin. Bacitracin- susceptible, beta-hemolytic
streptococci are pathogenic and referred to as group A
streptococci, or Streptococcus pyogenes. The alpha-
hemolytic streptococci are further differentiated based
on their susceptibility to ethy lhydrocupreine (optochin) .
Optochin-susceptible, alpha-hemolytic streptococci are
considered presumptive Streptococcus pneumoniae,
while optochin-resistant, alpha-hemolytic streptococci
are considered nonpathogenic viridans streptococci.

Cultures (24-48-hour on blood agar)
Streptococcus pneumoniae
Streptococcus pyogenes

AH agents in red are BSL2 bacteria.

Media

Blood agar plates (tryptic soy agar with
5% sheep blood)

Chemicals and reagents
Bacitracin (A) disks
Gram- stain reagents
Hydrogen peroxide (for catalase test)
Optochin disks

Equipment

Incubator (35 °C)
Light microscope

Miscellaneous supplies
Bibulous paper

Biohazard bag (for waste disposal)
Bottle with tap water
Bunsen burner and striker
Cotton- tipped swab, sterile
Disposable gloves
Forceps

Glass microscope slides
Immersion oil
Inoculating loop
Lens paper
Ruler (mm)
Tongue depressor
Wax pencil

First Session: Isolation of Bacteria
from the Throat

1 . After putting on disposable gloves, take a sterile
cotton-tipped swab in your right hand and a
tongue depressor in your left hand, or vice versa
if left-handed.

2. Hold down the tongue of your lab partner with
the tongue depressor while moving the cotton-
tipped end of the swab toward the back of the
throat. Do not touch any other part of the
mouth. Touch the swab to the back of the throat.
Rub the cotton-tipped end over the back of the
throat as shown in figure 22. 1 . Withdraw the
swab from the mouth without touching any other
surface. Give the swab to your lab partner so that

Sterile swab

Back of the throat

Tongue depressor

(a)

(b)

Figure 22.1 Procedure for obtaining a throat swab,
(a) Side view, (b) Front view.

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22. Isolation and

©The McGraw-Hill

Lab Exercises in
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Identification of
Streptococci from the
Throat

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Isolation and Identification of Streptococci from the Throat EXERCISE 22 165

he or she can use it to inoculate a blood agar
plate. Discard the tongue depressor in a biohazard
bag or similar container.

3 . To inoculate a blood agar plate, rub the swab
back and forth over one area of the plate near the
edge (figure 22.2a). Dispose of the swab in a
biohazard bag or similar container. With a sterile
loop, cross the swabbed area to spread the
bacteria. Repeat this spreading process a second
and third time as depicted (figure 22.2c, d). Label
your plate. Dispose of the gloves in a biohazard
bag or similar container.

4. Place the inoculated plate into a 35°C incubator.

Second Session

Selection of Throat Isolates

1. After 24-48 hours, examine your throat culture
plate. Determine the total number of different
bacterial types. Record this number in the
laboratory report.

2. Examine your plates for signs of hemolysis.
Look for a green discoloration, indicative of
alpha-hemolysis, and clearing around colonies,

(a) Area of the plate
initially swabbed

(b)

Initial inoculum
spread with ^
loop into
quadrant 1

(d) Inoculum

spread with

loop into
quadrant 3

Figure 22.2 Steps in the inoculation of a blood agar
plate with a throat culture.

indicative of beta-hemolysis. Record the presence
of these reactions.

3. Select from the plate one common bacterial type
that is alpha-hemolytic, and one common bacterial
type that is beta-hemolytic. If there are no
beta-hemolytic colonies, then select two alpha-
hemolytic types. Number these #1 and #2, and
record their colony morphology and hemolytic
reaction. Do the same for two known cultures,
Streptococcus pneumoniae and S. pyogenes.

Identification of Throat Isolates

The two common throat isolates will be identified using
the tests specified in figure 22.3. You will also test
Streptococcus pneumoniae and Streptococcus pyogenes
in conjunction with your unknowns.

1 . Gram-stain your unknown throat isolates and the
two known cultures. Record your results in the
laboratory report.

2. If one or both of your unknowns are Gram-
positive cocci, continue your identification by
doing a catalase test as follows: Use a sterile loop
to place some cells from the unknown culture
into a drop of water on a glass slide. Add a drop
of hydrogen peroxide. Watch for bubbles,
indicative of a positive test. The absence of
bubbles indicates a negative test. Do a catalase
test for your two known cultures as well. Record
your results.

3. If one or both unknowns is a catalase-negative,
Gram-positive coccus, then you have isolated
presumptive streptococci. If so, continue on to
step 4 for your unknown and known cultures.

4. Do a separate streak-plate on blood agar for each
culture. Using sterile forceps, place a 0.04 U
bacitracin (A) disk in streak quadrants 1 and 2
for beta-hemolytic streptococci. Using sterile
forceps, place an optochin disk in streak
quadrants 1 and 2 for alpha-hemolytic
streptococci. Incubate the plates at 35 °C.

Alexander-Strete-Niles:

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22. Isolation and

©The McGraw-Hill

Lab Exercises in
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Microbiology

Identification of
Streptococci from the
Throat

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166 Section V Medical Microbiology

alpha- and beta-hemolytic colonies on blood agar

Gram stain

Gram-negative rods or cocci,
or Gram-positive rods

Gram-positive cocci

Catalase

Non-streptococci/
staphylococci

Presumptive streptococci

Presumptive staphylococci

or micrococci (possibly

Staphylococcus aureus if

beta-hemolytic)

alpha-hemolysis

beta-hemolysis

Optochin susceptibility

Bacitracin susceptibility

Presumptive

Streptococcus
pneumoniae

Presumptive

viridans
streptococci

Group A

beta-hemolytic

streptococci

(presumptive

Streptococcus

pyogenes)

Non-group A

beta-hemolytic

streptococci

Figure 22.3 Identification scheme for common streptococci from the throat.

Third Session

Identification of Streptococci

1. After 24-48 hours, examine the blood agar plates
streaked with beta-hemolytic streptococci. If the
growth of beta-hemolytic streptococci is absent
around the bacitracin disk, indicating
susceptibility, your isolate is Streptococcus
pyogenes, the causative agent of strep throat. The
presence of this organism does not necessarily
indicate an active case of strep throat, since it
may occur normally in low numbers in some
individuals. However, a large number of colonies
of this isolate on your original plate (hundreds)
indicates an active case of strep throat.

2. After 24-48 hours, examine the blood agar plates
streaked with alpha-hemolytic streptococci. If a
zone of inhibition greater than or equal to 14 mm
occurs around the optochin disk, this indicates
susceptibility and the occurrence of Streptococcus
pneumoniae, the causative agent of pneumococcal
pneumonia. The presence of this organism in the
throat is considered normal in many individuals,
and does not indicate an active case of
pneumonia. If there is no zone of inhibition or if
the zone around the optochin disk measures less
than 14 mm, your isolate is resistant to optochin
and a member of the viridans streptococci, a
nonpathogenic group that represents the most
common form of streptococci in the throat.

Alexander-Strete-Niles:

V. Medical Microbiology

22. Isolation and

©The McGraw-Hill

Lab Exercises in
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Microbiology

Identification of
Streptococci from the
Throat

Companies, 2003

Name

Lab Section

EXERCISE

Laboratory Report

Date

solation and Identification of Streptococci from the Throat

1 . Total number of bacterial types from your throat =

2. Hemolysis on blood agar plates:

alpha-hemolysis present? (yes or no)

beta-hemolysis present? (yes or no)

3. Throat isolates

a. Identification of streptococci

Hemolytic
isolates

Colony
morphology

Type of

hemolysis

(alpha or beta)

Cell morphology

and Gram

reaction

Catalase

* Results
indicative of
streptococci?

Known
cultures:
Streptococcus
pneumoniae

Streptococcus
pyogenes

*If yes, continue with (b) and (c); if no, stop here.

b. Differentiation of beta-hemolytic streptococci

Beta-hemolytic throat isolate

Bacitracin susceptibility

Identification

Which # or #'s?

Known culture:
Streptococcus pyogenes

c. Differentiation of alpha-hemolytic streptococci

Alpha-hemolytic throat isolate

Optochin susceptibility

Identification

Which # or #'s?

Known culture:
Streptococcus pneumoniae

167

Alexander-Strete-Niles:

V. Medical Microbiology

22. Isolation and

©The McGraw-Hill

Lab Exercises in
Organismal and Molecular
Microbiology

Identification of
Streptococci from the
Throat

Companies, 2003

168 Section V Medical Microbiology

4. Did you isolate nonpathogenic viridans streptococci from your throat? Is this a common result? Explain

5. Did you isolate S. pneumoniae from your throat? Is this a common result? Explain.

6. Did you isolate S. pyogenes from your throat? Is this a common result? Explain

7. Answer the following questions based on these photographs:

a. Does this individual have strep throat?

How do you know?

c. This Gram-positive, catalase-positive
coccus is beta-hemolytic on blood agar.
Does the presence of this isolate in the

throat indicate strep throat?

Explain.

b. Is the colony morphology and hemolysis of
this throat isolate consistent with Streptococcus

pyogenes!.

Explain.

d. This isolate is a Gram-positive coccus,
catalase-negative, and optochin-resistant
Is this isolate part of the normal throat

flora?

Explain

Alexander-Strete-Niles:

V. Medical Microbiology

23. Iden. of Ent. Bacteria,

©The McGraw-Hill

Lab Exercises in
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Microbiology

Enterotubes, a Rapid
System to Iden. Ent.
Bacteria

Companies, 2003

A. Identification of Enteric Bacteria,
ncluding the Intestinal Pathogens
Salmonella and Shigella

B. Enterotubes, a Rapid Test System
to Identify Enteric Bacteria

Normal Flora of the Intestinal Tract

The large intestine offers an ideal environment for the
survival of a large number of resident bacteria. The
most common intestinal bacteria are members of the
family Enterobacteriaceae . These bacteria are often
referred to as enterics and include normally nonpath-
ogenic species, such as Escherichia, Enterobacter,
Klebsiella, Proteus, and Citrobacter. Non-enteric bac-
teria are also common in the intestine, including En-
terococcus (Streptococcus) faecalis, Pseudomonas
aeruginosa, and Staphylococcus aureus.

Pathogens of the Intestinal Tract

While the nonpathogenic enterics and non-enterics are
always present in large numbers, there are several bac-
teria that invade the intestinal tract after being ingested
in contaminated food or water. Among these intestinal
invaders are two genera of enteric bacteria, Salmonella
and Shigella. Although they share characteristics with
nonpathogenic enterics, these enterics are considered
pathogens and not normal residents. Salmonella, when
ingested, causes the intestinal disease salmonellosis,
while Shigella, when ingested, causes the intestinal dis-
ease called shigellosis, or bacterial dysentery.

Identification of Intestinal Bacteria

Enterics are Gram-negative rods, facultatively anaer-
obic, and oxidase-negative. A key trait used to differ-
entiate enterics is lactose utilization, which is easily
determined on MacConkey agar (table 23.1). Enter-
ics that utilize lactose are called lactose fermenters;
they turn the medium red and include nonpathogenic
enterics such as Escherichia and Enterobacter. Enter-
ics that do not utilize lactose are called lactose non-
fermenters; they produce no color change on MacConkey

Table 23.1 Composition of MacConkey

Agar (MAC)

Peptone

17 g

Proteose peptone

Lactose

10 g

Bile salts

1.5 g

Sodium chloride

Neutral red

0.03 g

Crystal violet

0.001 g

Agar

13.5 g

Distilled water

1 ,000 ml

Final pH

7.1

Source: The Difco Manual. Eleventh Edition. Difco Laboratories

agar and include the nonpathogenic enteric Proteus and
the pathogenic enterics Salmonella and Shigella.
Therefore, lactose fermentation is a critical test for dis-
tinguishing nonpathogenic enterics from pathogenic
enterics. The medium triple sugar iron (TSI) agar
(table 23.2) is useful in distinguishing lactose non-
fermenters (table 23.3), while MR-VP medium and
SIM medium (see Exercise 19) are useful for distin-
guishing lactose fermenters.

Enterotube, a Rapid Test System
to Identify Enteric Bacteria

Recently, rapid test systems have been commercially
developed for the identification of enteric bacteria.
These systems incorporate a large number of tests into
a single unit. All tests are inoculated at once, and the
results, which are obtained in 24-48 hours, provide suf-
ficient information for the identification of an isolate.

169

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Lab Exercises in
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Microbiology

Enterotubes, a Rapid
System to Iden. Ent.
Bacteria

Companies, 2003

170 Section V Medical Microbiology

Table 23.2 Composition

Triple Sugar

Iron

(TSI) Agar

Beef extract

Ferrous sulfate

0.2 g

Yeast extract

Sodium chloride

Peptone

15 g

Sodium thio sulfate

0.3 g

Proteose peptone

Phenol red

0.024 g

Dextrose

Agar

12 g

Lactose

10 g

Distilled water

1 ,000 ml

Sucrose

10 g

Final pH

7.4

Source: The Difco Manual. Eleventh Edition. Difco Laboratories

Table 23.3 Differentiation of Three Lactose Non-fermenting Enterics Using Triple Sugar Iron Agar

Enteric

Slant color

Butt color

H 2 S

Designation

(lactose and/or

(glucose fermentation)

production

sucrose fermentation)

Proteus vulgaris

Yellow

Black

A/A, H 2 S

(+)

Salmonella

Red

Yellow

Black

Alk/A, H 2 S

typhimurium

(-)

(+>

(+)

Shigella

Red

Yellow

No black

Alk/A

flexneri

(-)

(+)

(-)

One such test system, depicted in figure 23.1a, is
called the Enterotube® II. It contains 12 compart-
ments in a single unit that accommodates 15 bio-
chemical tests. The compartments are all inoculated
at once by pulling an inoculating wire through the
unit. In 18-24 hours, the color changes in compart-
ments are noted, and a test is scored as either posi-
tive or negative (figure 23.1b). Positive tests are used
to determine a 5 -digit identification number, which
identifies the unknown (figure 23.1c).

Materials

Cultures (24-48 hour on agar)

Enterobacter aerogenes, a nonpathogenic

enteric
Enterococcus {Streptococcus) faecalis,

a non-enteric
Escherichia coli, a nonpathogenic enteric
Proteus vulgaris, a nonpathogenic enteric
P seudomonas aeruginosa, a non-enteric
Salmonella typhimurium, a pathogenic enteric

SAFETY

Shigella flexneri, a pathogenic enteric
Staphylococcus aureus, a non-enteric

All agents in red are BSL2 bacteria

Media

Enterotube® II

MacConkey (MAC) agar plates

MR-VP medium tubes

O-F glucose tubes

SIM medium tubes

Tryptic soy agar (TSA) plates

Triple sugar iron (TSI) agar tubes

Chemicals and reagents
Gram- stain reagents
Hydrogen peroxide (for catalase test)
Kovac's reagent (for indole test)
Methyl red pH indicator (for methyl red test)
Mineral oil
Oxidase reagent

Alexander-Strete-Niles:

V. Medical Microbiology

23. Iden. of Ent. Bacteria,

©The McGraw-Hill

Lab Exercises in
Organismal and Molecular
Microbiology

Enterotubes, a Rapid
System to Iden. Ent.
Bacteria

Companies, 2003

A. Identification of Enteric Bacteria; B. Enterotubes EXERCISE 23 171

(a) The Enterotube II unit. The 12 compartments are inoculated with the enclosed
inoculating wire.

.Nega finer

L'Axzr.am L.v»nii Qn illiiii: InddC Ad&"lfc4 Laptop V:il:innw Sr?WljpJ P'ni?»=S".*T PA ' -hm £h-HlH

.HDBliVL'

i=ii:cn5c Lysine Ornllhlne 1-^5 ■"rUunlfcii l.y^lu-w AnU.iiii.^y .-.uiifoj Pjoa

DiUAtu

Gai PnlJutTw

Inrinfc

Note: VF Jlttz&d a& oonflrroalDiy fcesl o*ilj«

(b) After incubation, each compartment is examined for color change to indicate a
positive or negative test result.

ENTEROTUBE* II

°l

■ ■

*t\M

■a

"jTj

iff

M i

sis

21
I

ID Value

ia±UL£±JL±.

1 J [4 + 2 + 1

r y

4 +

2 + 1 j [ 4 + 2 + 1 j

Culture Number cv-P&torf Nome

&ate

Organism Hendned

Figure 23.1 An outline of the Enterotube II procedure.

Equipment

Incubator (35 °C)
Light microscope

Miscellaneous supplies
Bibulous paper
Bottle with tap water
Bunsen burner and striker
Disposable gloves

Glass microscope slides
Immersion oil
Inoculating loop
Inoculating needle
Lens paper

Pasteur pipette with bulb
Test tube rack
Wax pencil

Alexander-Strete-Niles:

V. Medical Microbiology

23. Iden. of Ent. Bacteria,

©The McGraw-Hill

Lab Exercises in
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Enterotubes, a Rapid
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Bacteria

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172 Section V Medical Microbiology

Procedure

First Session: Identification
of Intestinal Bacteria

You will be assigned three unknown cultures to iden-
tify: a non-enteric, a nonpathogenic enteric, and a path-
ogenic enteric. You will first need to determine which
of the three are enterics by following the identification
scheme in figure 23.2.

1 . Do a Gram stain on each of your three unknown
cultures. Record your results in the laboratory
report. If one of your cultures is a Gram-positive
coccus, do a catalase test to complete your
identification of this non-enteric (figure 23.2).

2. For Gram-negative rods, inoculate two O-F
glucose tubes (cover the medium in one tube
with sterile mineral oil) and a tryptic soy agar
(TSA) plate. Place the tubes and plate in a

35 °C incubator.

Intestinal bacteria

Gram stain

Gram-positive

Gram-negative rods

O-F test and
oxidase

Non-enteric

Catalase

Facultatively

anaerobic and

oxidase (-)

Aerobic and
oxidase (+)

Enterics

Non-enteric

{Enterococcus
faecalis)

Non-enteric

{Staphylococcus

aureus)

Non-enteric

(Pseudomonas

aeruginosa)

Lactose fermentation on
MacConkey agar

Methyl red and/or indole

A/A, H 2 S

Alk/A, H 2 S

Escherichia coli

Enterobacter
aero genes

Proteus
vulgaris

Salmonella
typhimurium

Figure 23.2 Identification scheme for non-enterics, nonpathogenic enterics, and pathogenic enterics.

Alexander-Strete-Niles:

V. Medical Microbiology

23. Iden. of Ent. Bacteria,

©The McGraw-Hill

Lab Exercises in
Organismal and Molecular
Microbiology

Enterotubes, a Rapid
System to Iden. Ent.
Bacteria

Companies, 2003

A. Identification of Enteric Bacteria; B. Enterotubes EXERCISE 23 173

Second Session: Identification
of Enterics

1. After 24-48 hours, examine the O-F glucose
tubes for color changes. Enterics are facultatively
anaerobic and will turn both tubes yellow. Now
place a drop of oxidase reagent on colonies of the
TSA plate. Enterics are oxidase-negative, so
there should be no color change. Oxidase-positive
colonies turn purple. Record your results for
both tests.

2. You have now identified your non-enteric,
whether Gram-positive or Gram-negative.

3. Use figure 23.2 to identify one of your enterics.
For this, inoculate a MacConkey agar plate, and
incubate at 35°C.

4. Use the Enterotube® II stepwise procedure
outlined in figure 23.3 to identify the
other enteric.

Third Session: Differentiation
of Enterics

1 . After 24-48 hours, examine your MacConkey
agar plate for color changes. Lactose fermenters
will appear red, while non-fermenters will appear
colorless. If positive for lactose fermentation,
inoculate an MR-VP medium tube and/or a SIM
medium tube. If negative for lactose fermentation,
inoculate a TSI agar tube with an inoculating

needle. Stab the butt first, and then streak the
slant. Incubate inoculated tubes at 35 °C.

2. After 18-24 hours, examine the compartments of
the Enterotube® II, and record positive and
negative tests using the information provided in
table 23.4. Use positive test results to calculate
your identification number (see example in
figure 23.3). Consult the information booklet
provided to find the bacterium that matches
this code number.

Fourth Session: Differentiation
of Enterics (continued)

1. Lactose fermenters : After 72 hours, add 10 drops
of methyl red pH indicator to your MR-VP
medium tube. After mixing, examine for color
change (a red color is positive, while a yellow
color is negative). Add 5 drops of Kovac's
reagent to your SIM medium tube. If the reagent
turns red, the culture is indole-positive; if it
remains yellow, the culture is indole- negative.
Record these results and your identification.

2. Lactose non-fermenters: After 24 hours,
examine the TSI agar tube. Record the reactions
for the slant and butt and whether or not
hydrogen sulfide was produced. Refer to table
23.3 for help in interpreting your results.
Record your identification.

Alexander-Strete-Niles:

V. Medical Microbiology

23. Iden. of Ent. Bacteria,

©The McGraw-Hill

Lab Exercises in
Organismal and Molecular
Microbiology

Enterotubes, a Rapid
System to Iden. Ent.
Bacteria

Companies, 2003

174 Section V Medical Microbiology

■

(a) Remove organisms from a well-islolated colony. Avoid
touching the agar with the wire. To prevent damaging
Enterotube II media, do not heat- sterilize the inoculating wire

(b) Inoculate each compartment by first twisting the wire and then
withdrawing it all the way out through the 12 compartments,
using a turning movement.

♦

: _=

♦

(c) Reinsert the wire (without sterilizing), using a turning motion
through all 1 2 compartments until the notch on the wire is
aligned with the opening of the tube.

•-'

tiiK.

■

(d) Break the wire at the notch by bending. The portion of the wire
remaining in the tube maintains anaerobic conditions essential
for true fermentation.

.-■■

■

(e) Punch holes with broken-off part of wire through the thin
plastic covering over depressions on sides of the last eight
compartments (adonitol through citrate). Replace caps, and
incubate at 35°C for 18-24 hours.

(f ) After interpreting and recording positive results on the sides of
the tube, perform the indole test by injecting 1 or 2 drops of
Kovac's reagent into the H 2 S/indole compartment.

O
R

H 2 S N
D

+ 1

A
D
O

L
A
C

A
R
A
B

R
B

U
L

©+ 2 +QA4 +© + (D/\@+© +

U
R

(g) Perform the Voges-Proskauer test, if needed for confirmation,
by injecting the reagents into the H 2 S/indole compartment.
After encircling the numbers of the positive tests on the
laboratory report, total up the numbers of each bracketed series
to determine the 5-digit code number. Refer to the Enterotube II
Interpretation Guide for identification of the unknown by using
the code number.

Figure 23.3 Steps in the Enterotube II procedure.

Alexander-Strete-Niles:

V. Medical Microbiology

23. Iden. of Ent. Bacteria,

©The McGraw-Hill

Lab Exercises in
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Enterotubes, a Rapid
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A. Identification of Enteric Bacteria; B. Enterotubes EXERCISE 23 175

Table 23.4 Enrerotube II Reactions

Results I

Summary

Compartment

Medium/Test

Description and Interpretation of Tests

Positive

Negative

Glucose (GLU)

Tests for glucose fermentation. A shift in pH is
indicated by a change in color of the medium
from red to yellow, reflecting the production of
acidic fermentation by-products. A change in the
color of the medium from red to yellow should be
interpreted as a positive reaction. Orange should be
interpreted as negative.

Yellow

Red/orange

Gas production

Gas from fermentation is indicated as a definite

Separation

(GAS)

separation of the wax overlay from the surface
of the culture medium. Bubbles in the culture
medium should not be interpreted as evidence of
gas production.

of wax

separation
of wax

Lysine

decarboxylation

(LYS)

Measures the ability of bacteria to decarboxylate
lysine to produce the alkaline by-product cadaverine.
Any shift in the color of the culture medium
from yellow to purple should be interpreted as a
positive reaction. The medium should remain
yellow if decarboxylation does not take place.

Purple

Yellow

Ornithine

decarboxylation

(ORN)

Measures the ability of bacteria to decarboxylate
ornithine to produce the alkaline by-product
putresine. Any shift in the color of the culture
medium from yellow to purple should be interpreted
as a positive reaction. The medium should remain
yellow if decarboxylation does not take place.

Purple

Yellow

H 2 S production

H 2 S is produced from the metabolism of sulfur

Black

(H 2 S)

containing compounds (e.g., thiosulfate and amino
acids) in the culture medium. Ferrous (Fe 2+ ) ions
in the medium react with the H 2 S to produce the
black precipitate (FeS). Any blackening of the
medium indicates that H 2 S has been produced.

change

Indole

Indole is produced when tryptophan is degraded

Red

formation

by the enzyme tryptophanase. After injection

change

(IND)

of Kovac's reagent into the medium (after 18-24
hours of incubation), any indole present will react
with the reagent to produce a pink-red color.

Adonitol

Tests for adonitol fermentation. A shift in pH is

Yellow

Red/

(ADON)

indicated by a change in the color of the medium from
red to yellow, reflecting the production of acidic
fermentation by-products. A change in the color of
the medium from red to yellow should be interpreted
as a positive reaction. Orange should be interpreted
as negative.

orange

Lactose

Tests for lactose fermentation. A shift in pH is

Yellow

Red/

(LAC)

orange

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176 Section V Medical Microbiology

Table 23.4 Enterotube II Reactions (continued)

Compartment Medium/Test Description and Interpretation of Tests

Results Summary
Positive Negative

Arabinose
(ARAB)

Sorbitol
(SORB)

Voges-

Proskauer

(VP)

Dulcitol
(DUL)

Phenylalanine
deaminase (PA)

Urea (UREA)

Citrate
(CIT)

Tests for arabinose fermentation. A shift in pH is
indicated by a change in the color of the medium from
red to yellow, reflecting the production of acidic
fermentation by-products. A change in the color of
the medium from red to yellow should be interpreted
as a positive reaction. Orange should be interpreted
as negative.

Tests for sorbitol fermentation. A shift in pH is
indicated by a change in the color of the medium from
red to yellow, reflecting the production of acidic
fermentation by-products. A change in the color of
the medium from red to yellow should be interpreted
as a positive reaction. Orange should be interpreted
as negative.

Tests for the production of acetoin, an intermediate
in the 2,3-butanediol fermentation pathway. Acetoin
is detected by the injection of 2 drops of solution
containing 20% KOH and 0.3% creatine and 3 drops
of alpha-napthol solution (5% wt/vol alpha-napthol in
absolute ethanol). The development of a pink-red color
1 to 20 minutes after the addition of the alpha-
napthol solution indicates that acetoin was produced.

Tests for dulcitol fermentation. A shift in pH is indicated
by a change in the color of the medium from green to
yellow, reflecting the production of acidic fermentation
by-products. A change in the color of the medium to
yellow or pale yellow should be interpreted as a
positive reaction.

Test for the formation of pyruvic acid from the
deamination of phenylalanine. Pyruvic acid reacts with
Fe 3+ in the medium to cause a gray to black discoloration.

Test for urease production. Hydrolysis of urea results in
the production of ammonium, which makes the medium
alkaline and causes a color change from yellow to red-
purple. Light pink and other shades of red should be
interpreted as positive.

Test for the ability of certain bacteria to use citrate as
the sole source of carbon. Utilization of citrate results in
the production of alkaline metabolites, which turn the
pH indicator in the culture medium from green to
royal blue. Any intensity of blue should be interpreted
as positive.

Yellow

Red/
orange

Yellow

Red/
orange

Pink

Colorless

Yellow

Green

Black

Yellow

Red-
violet

Yellow

Blue

Green

Source: Becton Dickinson Microbiology Systems, Cockeysville, MD 21030.

Alexander-Strete-Niles:

V. Medical Microbiology

23. Iden. of Ent. Bacteria,

©The McGraw-Hill

Lab Exercises in
Organismal and Molecular
Microbiology

Enterotubes, a Rapid
System to Iden. Ent.
Bacteria

Companies, 2003

EXERCISE

Laboratory Report

Name

Date

Lab Section

A. Identification of Enteric Bacteria, Including the Intestina
Pathogens Salmonella and Shigella

B. Enterotubes, A Rapid Test System to Identify Enteric Bacteria

1. Unknown nos.

2. Record the results of your tests for the three unknown cultures

Unknown no.

Cell
morphology

Gram
reaction

Catalase
(if Gram-
positive coccus)

O-F test

Oxidase

Enteric?

3. Identify the non-enteric based on your data in the preceding table

4. Identify an enteric using figure 23.2.

Unknown no.

Lactose
fermentation
on MAC agar

Methyl red

Indole

TSI

Identification

5. Identify an enteric using Enterotube® II.

Unknown no.

Code no. obtained

Identification

177

Alexander-Strete-Niles:

V. Medical Microbiology

23. Iden. of Ent. Bacteria,

©The McGraw-Hill

Lab Exercises in
Organismal and Molecular
Microbiology

Enterotubes, a Rapid
System to Iden. Ent.
Bacteria

Companies, 2003

178 Section V Medical Microbiology

6. Answer the following questions based on these photographs

a. The culture on the left is growing on MAC
agar. Is the culture a lactose fermenter?

b. Which TSI agar tube is indicative of the
results of Shigella flexneril

How do you know?

Alexander-Strete-Niles:

V. Medical Microbiology

24. Isolation and

©The McGraw-Hill

Lab Exercises in
Organismal and Molecular
Microbiology

Identification of Bacteria
from the Urinary Tract

Companies, 2003

solation and Identification of Bacteria
from the Urinary Tract

Normal Flora of the Urinary Tract

The urinary tract is normally sterile, but the urine
released from it can become contaminated by bacteria
that inhabit the distal end of the urethra and the exter-
nal genitalia. Even so, the number of bacteria in urine
is typically low (i.e., ranging from to 10,000 bacte-
ria/ml). This range is considered normal.

Pathogens of the Urinary Tract

When bacteria invade the urinary tract and cause a uri-
nary tract infection (UTI), their presence is reflected in
extremely high numbers in urine (i.e., in excess of
100,000 bacteria/ml of urine). Bacteria capable of invad-
ing the urinary tract and causing UTIs include Escherichia
coli, Enterobacter aerogenes, Pseudomonas aeruginosa,
Proteus vulgaris, Enterococcus faecalis, and Staphylo-
coccus saprophyticus.

Identification of Urinary Tract Isolates

A Gram stain is done on an isolate from urine that num-
bers in excess of 100,000 bacteria/ml. If a Gram-
positive coccus is found, a catalase test will determine
whether it is a staphylococcus (positive) or streptococ-
cus/enterococcus (negative). If the culture is catalase-
negative, the use of BEA, or bile esculin agar (table
24.1), will confirm the group D streptococci, since only
these bacteria can tolerate the high bile content of this
agar while hydrolyzing esculin, a reaction that yields a
dark brown color.

A Gram-negative rod can be tested for oxidase. A
positive reaction may indicate Pseudomonas aerugi-
nosa. A negative oxidase test is indicative of the enteric
bacteria, such as Proteus vulgaris, Escherichia coli, and
Enterobacter aerogenes. These bacteria can be differ-
entiated by triple sugar iron (TSI) agar (see Exercise
23), the methyl red test (see Exercise 19), and the indole
test (see Exercise 19).

Table 24.1 The composition of Bile

Esculin Agar (BEA)

Beef extract

Peptone

Esculin

Oxgall

Ferric citrate

Agar

Distilled water

Final pH

3g
5g

lg
40 g

0.5 g

15 g
1 ,000 ml

6.6

Source: The Difco Manual. Eleventh Edition. Difoo Laboratories

Materials

Cultures (on agar plates)
Enterobacter aerogenes
Enterococcus faecalis
Escherichia coli
Proteus vulgaris
Pseudomonas aeruginosa
Staphylococcus saprophyticus

Media

Bile esculin agar (BEA) tubes

MR-VP medium tubes

SIM medium tubes

Triple sugar iron (TSI) agar tubes

Tryptic soy agar (TSA) plates

Chemicals and reagents
Gram-stain reagents
Hydrogen peroxide (for catalase test)
Kovac's reagent
Methyl red pH indicator
Oxidase reagent

Equipment

Incubator (35 °C)
Light microscope

179

Alexander-Strete-Niles:

V. Medical Microbiology

24. Isolation and

©The McGraw-Hill

Lab Exercises in
Organismal and Molecular
Microbiology

Identification of Bacteria
from the Urinary Tract

Companies, 2003

180 Section V Medical Microbiology

Miscellaneous supplies
Bibulous paper
Biohazard bag
Bottle of tap water
Bunsen burner and striker
Container and bag (to collect urine)
Disposable gloves
Glass microscope slides
Immersion oil
Inoculating loop (standard)
Inoculating loop (5 jil)
Inoculating needle
Lens paper

Pasteur pipette with bulb
Test tube rack

Towelette (to clean urethral opening)
Wax pencil

First Session: Collection
and Inoculation of Urine

1 . Wash your hands. Use an antiseptic towelette to
clean around the opening of the urethra.

2. Collect a midstream sample of urine in a clean,
plastic container. Put the lid on, and close tightly.
Place the container in a plastic bag. Store in the
refrigerator if the urine will not be cultured
within 1-2 hours.

.CAUTION

This step should be done
wearing gloves under a safety
hood or behind a plastic shield
placed on the countertop.

3 . When ready to culture, mix the urine, and then
dip a 5 jil inoculating loop into the fluid. Streak
the 5 jil of urine obtained onto a tryptic soy agar
(TSA) plate using the method depicted in figure
24. 1 . Repeat this process for a second plate.
Number the plates #1 and #2. Discard the
remaining urine in the restroom, and then deposit
the gloves, urine container, bag, and loop in a
biohazard bag or similar waste container.

4. Place both plates in a 35°C incubator.

(b)

(a)

Figure 24.1 Inoculation of a TSA plate with urine,
(a) Initial line streak, (b) Back-and-forth streak across
initial line streak.

Second Session: Isolation and
Identification of Urinary Tract Bacteria

1 . After 48-72 hours, examine your culture plates.
Count the number of bacterial colonies on each
plate, average this number, and then use this
average to calculate the number of bacteria per
milliliter of urine. (To do this, multiply the
average number of bacterial colonies by a
factor of 200.) Record your results in the
laboratory report.

2. If any of the bacteria on your plate exceeded
100,000 per milliliter of urine, you may have a
UTI caused by this organism. Identify this
organism and one assigned unknown culture
using the scheme depicted in figure 24.2. If you
do not have a UTI, then you will identify two
assigned unknown cultures using this scheme.

3. Do a Gram stain of your two cultures. Record
their morphology and Gram reaction. For Gram-
positive cocci, do a catalase test. If catalase-
positive, the culture is Staphylococcus. If
catalase-negative, inoculate a BE A tube and
place it in a 35 °C incubator. For Gram- negative
rods, do an oxidase test. If oxidase-positive, the
culture is Pseudomonas. If oxidase-negative,
inoculate a TSI agar tube, a MR-VP medium
tube, and/or a SIM medium tube. Place these
tubes in a 35 °C incubator.

Alexander-Strete-Niles:

V. Medical Microbiology

24. Isolation and

©The McGraw-Hill

Lab Exercises in
Organismal and Molecular
Microbiology

Identification of Bacteria
from the Urinary Tract

Companies, 2003

Isolation and Identification of Bacteria from the Urinary Tract EXERCISE 24 181

Common isolate from urine plate/unknown culture

Gram stain

Gram-positive cocci

Gram-negative rod

Catalase

Oxidase

Bile esculin

Presumptive

Staphylococcus
saprophyticus

Presumptive

Pseudomonas
aeruginosa

Non-group D
streptococci

Group D

streptococci

(presumptive

Enterococcus

faecalis)

A/A, H 2 S

Proteus
vulgaris

Methyl red and/or indole

Escherichia
coli

Enterobacter
aero genes

Figure 24.2 Identification scheme for bacteria that commonly cause urinary tract infections

Third Session: Identification of Urinary
Tract Bacteria (continued)

1 . After 24-48 hours, examine your BEA slant
for the presence of a dark brown color,
indicative of group D streptococci. The absence
of a dark brown color indicates a non-group

D streptococcus.

2. After 24-48 hours, examine the TSI agar tube. A
yellow butt and slant with a black discoloration is
indicative of Proteus vulgaris. If there is no black
discoloration, examine your MR-VP medium
tube using step 3 and/or your SIM medium tube
using step 4.

3. After 72 hours, add 10 drops of methyl red pH
indicator to the MR-VP medium tube, and mix.
A red color indicates a positive methyl red test,
while a yellow color indicates a negative methyl
red test.

4. After 24-48 hours, add 5 drops of Kovac's
reagent to your SIM medium tube, and watch for
the development of a red color in the reagent, a
positive indole test. No color change is a negative
indole test.

Alexander-Strete-Niles:

V. Medical Microbiology

24. Isolation and

©The McGraw-Hill

Lab Exercises in
Organismal and Molecular
Microbiology

Identification of Bacteria
from the Urinary Tract

Companies, 2003

Alexander-Strete-Niles:

V. Medical Microbiology

24. Isolation and

©The McGraw-Hill

Lab Exercises in
Organismal and Molecular
Microbiology

Identification of Bacteria
from the Urinary Tract

Companies, 2003

Name

Lab Section

EXERCISE

Laboratory Report

Date

solation and Identification of Bacteria from the Urinary Tract

1 . Record the number of bacteria in the urine sample.

Plate

Number of bacteria/5 ul of urine

.bacteria/5 jul x 200 =

.bacteria/ml

Average =

2. Did the average number of bacteria/ml urine fall within the normal range of 0-10,000 bacteria/ml?
If yes, how do you account for these bacteria in urine?

3. Did the average number of bacteria/ml urine exceed 100,000 bacteria/ml? If yes, what does this
number indicate?

4. Identify the urinary tract bacteria in your sample.

Test

UTI isolate or unknown #1

Unknown #2

Cell morphology

Gram reaction

Catalase

BEA

Oxidase

TSI

Methyl red

Indole

UTI isolate or unknown #1

Unknown #2

Identification:

183

Alexander-Strete-Niles:

V. Medical Microbiology

24. Isolation and

©The McGraw-Hill

Lab Exercises in
Organismal and Molecular
Microbiology

Identification of Bacteria
from the Urinary Tract

Companies, 2003

184 Section V Medical Microbiology

5. Answer the following questions based on these photographs

a. This TSA plate was inoculated with

5 jil of urine. Is a UTI indicated?

Explain.

b. This organism, responsible for a UTI,
gave the above reaction on BEA. What
organism is indicated?

Alexander-Strete-Niles:

V. Medical Microbiology

25. Assessing Antibiotic

©The McGraw-Hill

Lab Exercises in
Organismal and Molecular
Microbiology

Effectiveness: The
Kirby-Bauer Method

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Assessing Antibiotic Effectiveness:
The Kirby-Bauer Method

Among the various chemical agents used to control micro-
bial growth, antibiotics are unique because they are selec-
tive in their action — that is, they specifically target
bacterial cells. For this reason, they can be introduced into
the human body to treat disease with minimal effects on
human cells. Since Alexander Fleming discovered peni-
cillin produced by a mold in his laboratory over 60 years
ago, antibiotics have become a standard method used by
physicians to treat bacterial diseases.

Types of Antibiotics

Since the discovery of penicillin, many other useful
antibiotics have been developed. Each antibiotic has a
specific mechanism of action against bacteria. In some
cases, the action is broad-spectrum, or effective
against a wide variety of bacteria. In others, the action
is narrow-spectrum, or effective against only certain
bacteria. Table 25.1 lists 10 selected antibiotics, their
effect on cells, and their spectrum of activity.

Table 25.1 Antibiotics Used to Treat Bacterial Infections

Antibiotic

Effect on cells

Spectrum of activity

Ampicillin

Inhibits cell wall synthesis

Broad-spectrum antibiotic [effective against
Gram (+) cocci and some Gram (-) bacteria]

Bacitracin

Inhibits cell wall synthesis

Narrow- spectrum antibiotic [effective against
Gram (+) bacteria]

Chloramphenicol

Inhibits protein synthesis

Broad-spectrum antibiotic [effective against Gram
(+) and Gram (-) bacteria]

Erythromycin

Inhibits protein synthesis

Narrow- spectrum antibiotic [effective against
Gram (+) bacteria]

Gentamicin

Inhibits protein synthesis

Broad- spectrum antibiotic [effective against Gram
(+) and Gram (-) bacteria]

Penicillin G

Inhibits cell wall synthesis

Narrow- spectrum antibiotic [effective against
Gram (+) cocci]

Polymyxin B

Disrupts cell membrane

Narrow- spectrum antibiotic [effective against
Gram (-) rods]

Streptomycin

Inhibits protein synthesis

Broad- spectrum antibiotic [effective against Gram
(+) and Gram (-) bacteria]

Tetracycline

Inhibits protein synthesis

Broad-spectrum antibiotic [effective against Gram
(+) and Gram (-) bacteria]

Vancomycin

Inhibits cell wall synthesis

Narrow- spectrum antibiotic [effective against
Gram (+) bacteria]

185

Alexander-Strete-Niles:

V. Medical Microbiology

25. Assessing Antibiotic

©The McGraw-Hill

Lab Exercises in
Organismal and Molecular
Microbiology

Effectiveness: The
Kirby-Bauer Method

Companies, 2003

186 Section V Medical Microbiology

Evaluating Effectiveness

When a disease-causing bacterium is isolated from a
patient, the physician must determine which antibiotic
to administer for treatment. The most widely used method
to evaluate the effectiveness of antibiotics against specific
bacteria is the Kirby-Bauer method. In this method, out-
lined in figure 25.1, Mueller-Hinton agar (table 25.2)
is inoculated with a culture of a bacterial isolate. After
inoculation, antibiotic disks are placed on the agar surface.
Plates are incubated to allow for bacterial growth and then
inspected for zones of inhibition around antibiotic disks.
Zones of inhibition are measured in millimeters and com-
pared to an interpretive standard to determine if the iso-
late is susceptible or resistant to the antibiotic. Antibiotics
that the organism is susceptible to are candidates for use
in treating the patient.

Table 25.2

Composition
Agar

of Mueller-Hinton

Beef infusion

300 g

Cas amino acids

17.5 g

Starch

1.5 g

Agar

17 g

Distilled water

1 ,000 ml

Final pH

7.3

Source: The Difco Manual. Eleventh Edition. Difco Laboratories

Cultures (24 hours in tryptic soy broth)
Bacillus cereus, Gram-positive rod
Escherichia coli, Gram-negative rod
Pseudomonas aeruginosa, Gram-negative rod
Staphylococcus aureus, Gram-positive coccus

All agents in red are BSL2 bacteria.

Media

Mueller-Hinton agar plates, 4 mm thick
(25 ml/plate)

Chemicals and reagents

Antibiotic disks (loose in sterile petri dish)

Ampicillin

B acitracin

Chloramphenicol

Erythromycin

Gentamicin

Penicillin G

Polymyxin B

Streptomycin

Tetracycline

Vancomycin
Ethanol, 70%

Equipment

Incubator (35 °C)

Miscellaneous supplies
Beaker, 250 ml
Bunsen burner and striker
Cotton-tipped swabs, sterile
Disposable gloves
Forceps

Ruler, millimeter
Test tube rack
Wax pencil

Alexander-Strete-Niles:

V. Medical Microbiology

25. Assessing Antibiotic

©The McGraw-Hill

Lab Exercises in
Organismal and Molecular
Microbiology

Effectiveness: The
Kirby-Bauer Method

Companies, 2003

Assessing Antibiotic Effectiveness: The Kirby-Bauer Method EXERCISE 25 187

(a) Dip a cotton-tipped swab into a broth culture.

(b) Spread the culture over the entire plate. Dip and spread
two more times, as depicted in figure 25.2.

(d) Pick up antibiotic disk, and place on inoculated plate
Repeat for four other antibiotics.

Zones of inhibition

(e) After incubation, examine plates for zones of
inhibition, indicative of antibiotic effectiveness.

ONVTO

(f) Measure zones of inhibition to the nearest millimeter
(mm), and compare to the interpretive standard. This zone
measures 24 mm.

Figure 25.1 An outline of the Kirby-Bauer method.

Alexander-Strete-Niles:

V. Medical Microbiology

25. Assessing Antibiotic

©The McGraw-Hill

Lab Exercises in
Organismal and Molecular
Microbiology

Effectiveness: The
Kirby-Bauer Method

Companies, 2003

188 Section V Medical Microbiology

Procedure

First Session: Preparation of Plates

1 . Dip a sterile cotton-tipped swab into one of the
broth cultures, and use it to inoculate a Mueller-
Hinton agar plate using the procedure depicted in
figure 25.2. Inoculation of the plate in this way
ensures a lawn of bacterial growth after incubation.
Repeat this inoculation procedure for a second
plate using the same organism. Label both plates.

2. Repeat step 1 for the other three cultures. You
should now have a total of eight inoculated and
labeled plates, two for each culture. After
inoculation, allow all plates to dry for 15 minutes
before proceeding to the next step.

3. Pour some 70% ethanol into a 250 ml beaker.

CAUTION

Caution: Keep this beaker away
from your flame.

a. Dip your forceps into the alcohol, and then
pass the forceps over the Bunsen burner
flame to sterilize them.

b. Now pick up an antibiotic disk from one of
the petri dishes, and place it on one of your
inoculated plates.

c. After placement on the agar, tap it once to
make sure it is secure.

Repeat steps a-c until you have placed this
disk on a plate for each culture. Proceed to the
next disk until five disks have been placed on a
plate for each culture. Place the other five
disks on the second plate, for a total of 10
disks per culture.

4. When all disks are in place, put your eight
plates into a 35 °C incubator.

Second Session: Examination
of Plates

1. Your plates must be examined after 16-18
hours of incubation. If you cannot examine
them then, place them in a refrigerator
until examination.

2. Examine your plates for zones of inhibition.
Measure these with a millimeter ruler across
the disk as shown in figure 25 . If. Record the

# ♦♦>>>♦♦>> ♦ ♦
♦ ♦ ♦ ♦ ♦ ♦„> ♦ ♦ ♦ ♦ ♦

-♦♦♦♦♦♦♦♦ ♦ ♦ ♦ ♦ -

(a) Area of initial swab

(b) Area of second swab

Figure 25.2 Inoculation of a Mueller-Hinton agar plate,
(a) Dip a cotton swab in the culture, and swab across the
surface of the agar without leaving any gaps, (b) Using the
same swab, dip in the culture again, and swab the agar in a
direction perpendicular to the first inoculum, (c) Dip and
swab a third time at a 45 ° angle to the first inoculum.

diameter of the zone to the nearest whole
millimeter in the laboratory report. If only one
side of the zone can be measured, multiply the
number obtained by 2 to obtain a full zone of
inhibition. If there is no zone (i.e., if growth
occurs up to the edge of the disk), record a zero.
Note: You might see colonies within the zone
of inhibition. These colonies consist of cells that
are resistant to the antibiotic. Continue recording
the zones of inhibition until you have all
40 measurements.

Now compare the zone of inhibition you obtained
to the interpretive standards for these antibiotics
in table 25.3. Record whether each organism
is resistant, susceptible, or intermediate to
the antibiotic.

Complete this exercise by recording for each
type of bacteria the antibiotics the organism is
susceptible to. These represent possible drugs of
choice to treat infections by these bacteria.

Alexander-Strete-Niles:

V. Medical Microbiology

25. Assessing Antibiotic

©The McGraw-Hill

Lab Exercises in
Organismal and Molecular
Microbiology

Effectiveness: The
Kirby-Bauer Method

Companies, 2003

Assessing Antibiotic Effectiveness: The Kirby-Bauer Method EXERCISE 25 189

Table 25.3 Interpretive

Standards for Antibiotics Selected for This Exerc

ise

Antimicrobial agent

Abbreviation

Concentration

Diameter of zone of inhibition (mm)
Resistant Intermediate Susceptible

Ampicillin

10ng

Gram-negative

12-13

Staphylococci

21-28

Bacitracin

10 units

9-12

Chloramphenicol

30 (ig

13-17

Erythromycin

15 Hg

14-22

Gentamicin

10 Hg

13-14

Penicillin G

1 units

Staphylococci

21-28

Other organisms

12-21

Polymyxin B

300 units

9-11

Streptomycin

lOng

12-14

Tetracycline

30 ng

15-18

Vancomycin

30 ng

10-11

Source: Antimicrobial Susceptibility Test Discs. Technical information published by Becton Dickinson Microbiology Systems,
Cockeysville, Maryland.

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

V. Medical Microbiology

25. Assessing Antibiotic
Effectiveness: The
Kirby-Bauer Method

Alexander-Strete-Niles:

V. Medical Microbiology

25. Assessing Antibiotic

©The McGraw-Hill

Lab Exercises in
Organismal and Molecular
Microbiology

Effectiveness: The
Kirby-Bauer Method

Companies, 2003

Name

Lab Section

EXERCISE

Laboratory Report

Date

Assessing Antibiotic Effectiveness: The Kirby-Bauer Method

1 Record the diameter of the zones of inhibition (in mm).

Bacteria

Antibiotic

Disk code

Bacillus
cereus

Escherichia
coli

Pseudomonas
aeruginosa

Staphylococcus
aureus

Ampicillin

AM10

Bacitracin

BIO

Chloramphenicol

C30

Erythromycin

E15

Gentamicin

GM10

Penicillin G

P10

Polymyxin B

PB300

Streptomycin

S10

Tetracycline

TE30

Vancomycin

VA30

2. Evaluate the bacteria based on the interpretive standards in table 25.3. Record whether each type
is R = resistant, S = susceptible, or I = intermediate based on the standard.

Bacteria

Antibiotic

Disk code

Bacillus
cereus

Escherichia
coli

Pseudomonas
aeruginosa

Staphylococcus
aureus

Ampicillin

AM10

Bacitracin

BIO

Chloramphenicol

C30

Erythromycin

E15

Gentamicin

GM10

Penicillin G

P10

Polymyxin B

PB300

Streptomycin

S10

Tetracycline

TE30

Vancomycin

VA30

191

Alexander-Strete-Niles:

V. Medical Microbiology

25. Assessing Antibiotic

©The McGraw-Hill

Lab Exercises in
Organismal and Molecular
Microbiology

Effectiveness: The
Kirby-Bauer Method

Companies, 2003

192 Section V Medical Microbiology

3. Which antibiotics are the test organisms susceptible to, and hence, candidates for treating infections
caused by these organisms?

Bacteria

Antibiotics susceptible to (candidates for treatment)

Bacillus cereus

Escherichia coli

Pseudomonas aeruginosa

Staphylococcus aureus

4. In your results, did you find evidence of broad- spectrum antibiotics (i.e., ones effective against both
Gram-positive and Gram- negative bacteria)? Which antibiotics were broad-spectrum? Were these
results as you expected?

5. In your results, did you find evidence of narrow- spectrum antibiotics (i.e., ones effective against only
either Gram-positive or Gram-negative bacteria)? Which antibiotics were narrow-spectrum? Were these
results as you expected?

6. Answer the following questions based on these photographs:

a. Name the clear area around some of these
antibiotic disks

b. Explain these two colonies within the clear
area around this TE 30 disk.

What do they indicate?

Alexander-Strete-Niles:

V. Medical Microbiology

26. Identification of a

©The McGraw-Hill

Lab Exercises in
Organismal and Molecular
Microbiology

Clinical Bacterial
Unknown

Companies, 2003

Identification of a Clinical Bacterial Unknown

Previous exercises have covered aspects of clinical iso-
lates from various regions of the body, such as the skin,
throat, intestinal tract, and urinary tract. In this exercise,
you will apply what you have learned about clinical iso-
late diagnosis to the identification of a clinical bacter-
ial unknown.

Materials

Cultures (24-48-hour broth)
Enterobacter aerogenes
Enterococcus faecalis
Escherichia coli
Klebsiella pneumoniae
Proteus vulgaris
Pseudomonas aeruginosa
Salmonella typhimurium
Shigella flexneri
Staphylococcus aureus
Staphylococcus epidermidis
Streptococcus pneumoniae
Streptococcus pyogenes

AH agents in red are BSL2 bacteria.

Stains

Gram- stain reagents

Media

Bile esculin agar (BE A) tubes
Blood agar plates
Lactose broth tubes
Mannitol salt agar (MSA) plates
Motility test agar tubes
MR-VP medium tubes
OF glucose tubes
SIM medium tubes
Simmons citrate agar tubes
Tryptic soy agar (TSA) plates
Urea broth tubes

Chemicals and reagents
Bacitracin (A) disks
Coagulase test (rabbit plasma or rapid latex

agglutination test kit)
Hydrogen peroxide (catalase test)
Methyl red pH indicator
Optochin disks
Oxidase reagent

Equipment

Incubator (35 °C)

Miscellaneous supplies
Bottle with tap water
Bunsen burner and striker
Disposable gloves
Glass slides
Immersion oil
Inoculating loop and needle
Lens paper
Mineral oil (sterile)
Pasteur pipette with bulb
Staining tray
Test tube rack
Wax pencil

1 . You will select an unknown culture, or one will
be assigned to you. In either case, record the
number of your unknown in the laboratory report.

2. Begin with a streak-plate on TSA. After
incubation, examine the streak-plate to make sure
you have good growth and a pure culture. If
growth is slow or poor, try a streak-plate on a
blood agar plate.

3. After recording culture characteristics, do a Gram
stain (see Exercise 1 1) on your streak-plate
culture to determine cell shape, cell arrangement,
and Gram reaction. Record your results.

4. Examine the identification scheme in figure 26.1
to determine the test to be done next. If a catalase
test is required, see Exercise 19; for coagulase

193

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26. Identification of a

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Clinical Bacterial
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194 Section V Medical Microbiology

Gram stain

Coccus shape

Gram-positive

Catalase

Bile esculin hydrolysis

Coagulase and
mannitol fermentation

Rod shape

Gram-negative

O-F glucose
and oxidase

Facultatively
anaerobic and
oxidase (-)

Aerobic and
oxidase (+)

Lactose

Straight rods

(Pseudomonas
aeruginosa)

(Staphylococcus (Staphylococcus
epidermidis) aureus) ^~'

Methyl red

Hemolysis
and susceptibility

Gamma-hemolysis

(Enterococcus
faecalis)

Alpha-hemolysis
and optochin
susceptibility

(Streptococcus
pneumoniae)

Beta-hemolysis
and bacitracin
susceptibility

(Streptococcus
pyogenes)

Motile, swarming

on agar (Proteus

vulgaris)

Motile (Escherichia

coli)

Nonmotile,
citrate (-)
(Shigella
flexneri)

Motile,

citrate (+)

(Salmonella

typhimurium)

(Klebsiella
pneumoniae)

(Enterobacter
aerogenes)

Figure 26.1 Identification scheme for 12 clinical bacterial unknowns.

and mannitol fermentation tests, see Exercise 21;
for bile esculin hydrolysis, see Exercise 24; for
hemolysis and susceptibility tests, see Exercise
22; for O-F glucose, oxidase, lactose, methyl red,
and H 2 S tests, see Exercise 19; for motility, see
Exercise 18.

5. A urea test is required to separate rapid urea
utilizers, such as Proteus vulgaris, from other
bacteria. The test employs urea broth (table
26.1) and is read 18-24 hours after inoculation
Urea utilization turns the medium pink;
nonutilization displays no color change.

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26. Identification of a

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Clinical Bacterial
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Identification of a Clinical Bacterial Unknown EXERCISE 26 195

Table 26.1

Composition

of Urea Broth

Yeast extract

0.1 g

Potassium phosphate

18.6 g

Urea

20 g

Phenol red

0.01 g

Distilled water

1 ,000 ml

Final pH

6.8

Source: The Difco Manual. Eleventh Edition. Difco Laboratories

6. If citrate utilization is required, inoculate a tube
of Simmons citrate agar (table 26.2) and
examine for color change. A blue color after
24-48 hours incubation indicates a citrate-
positive test; no color change is a negative test.

7. Continue with your tests until your unknown has
been identified. Be sure to record the results of
all tests and the identity of your unknown in the
laboratory report.

Table 26.2 Composition

of Si

immons

Citrate

Agar

Magnesium sulfate

0.2 g

Ammonium dihydrogen

phosphate

Dipotassium phosphate

Sodium citrate

Sodium chloride

Agar

15 g

Bromthymol blue

0.08 g

Distilled water

1 ,000 ml

Final pH

6.8

Source: The Difco Manual. Eleventh Edition. Difco Laboratories

8 . Your laboratory instructor may wish to see all
results when you are finished. Therefore, keep all
slides, plates, and tubes until examined by your
laboratory instructor.

Alexander-Strete-Niles:

V. Medical Microbiology

26. Identification of a

©The McGraw-Hill

Lab Exercises in
Organismal and Molecular
Microbiology

Clinical Bacterial
Unknown

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Alexander-Strete-Niles:

V. Medical Microbiology

26. Identification of a

©The McGraw-Hill

Lab Exercises in
Organismal and Molecular
Microbiology

Clinical Bacterial
Unknown

Companies, 2003

Name

Lab Section

EXERCISE

Laboratory Report

Date

Identification of a Clinical Bacterial Unknown

Unknown no.

1. Follow the identification scheme in figure 26.1 to identify your clinical bacterial unknown. Be sure to
perform only the tests required to identify your unknown.

2. Record your results for the required tests.

Procedure

Observations

Results

Culture characteristics
Broth

Agar

Staining characteristics
Cell shape

Cell arrangement

Gram reaction

Biochemical/other characteristics
Bacitracin susceptibility

Bile esculin hydrolysis

Catalase test

Citrate utilization

Coagulase

Hemolysis

Hydrogen sulfide (H 2 S)
production

Lactose utilization

Mannitol fermentation

Methyl red test

Motility

O-F glucose test

Optochin susceptibility

Oxidase test

Urea utilization

197

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26. Identification of a

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Lab Exercises in
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Clinical Bacterial
Unknown

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198 Section V Medical Microbiology

3. After following the scheme in figure 26.1 and recording the results for the required tests, I conclude that
my unknown is

4. Answer the following questions based on these photographs

a. This culture is growing in urea broth

Is the culture urea-positive?

b. These two cultures are growing on citrate
agar. Does the culture on the right utilize

citrate?

How do you know?

Alexander-Strete-Niles:
Lab Exercises in
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Microbiology

VI. Controlling the Risk and
Spread of Bacterial
Infections

27. Killing Bacteria with
High Temperature

Killing Bacteria with

High Temperature

Dry and Wet (Moist) Heat

Heat is one of the most effective methods used to kill bac-
teria. Heat is generally divided into dry and wet (moist)
heat (table 27.1). Dry heat, which includes incineration
and the hot-air oven, kills bacteria by oxidizing compo-
nents of the cell. Wet (moist) heat, which includes boiling
water, autoclave/pressure cooker, pasteurization, and frac-
tional sterilization, kills bacteria by coagulating proteins
in the cell, including essential enzymes and cell structures.

Using Dry Heat in the Kitchen

Dry heat is used for grilling on the stovetop or baking
in the oven. When properly used, dry heat in the kitchen
can effectively eliminate the risk of contracting certain
types of bacterial diseases.

Pathogenic strains of Escherichia coli, such as the
0157:H7 strain, cause diarrhea, and can be contracted by
eating undercooked hamburger. Cooking hamburger meat
to a temperature of 80°C or above should kill all vegeta-

tive cells of E. coli, if present. Likewise, species of Sal-
monella, such as S. enteritidis and S. typhimurium, are
associated with eating undercooked chicken and eggs,
causing salmonellosis. The thorough grilling or baking of
chicken and eggs to a temperature of 80°C or above
should kill all vegetative cells of Salmonella, if present.

Using Wet Heat in the Kitchen

Boiling water has been used for a long time around the
home in cooking and disinfecting items, such as baby
bottles and canning jars. Drinking water may also
require boiling on occasion. For example, whenever
water flow is interrupted in water lines by a rupture or
drop in pressure, there is a chance of bacterial con-
taminants entering the water supply. In these cases, city
officials may advise people to boil their water prior to
use. This eliminates the risk of contracting a water-
borne infection until normal service is restored.

In summary, when properly used, heat is an effec-
tive household tool to eliminate the risk of bacterial
infection. This exercise will demonstrate the killing
power of wet heat.

Table 27.1

Types of Heat Used to Kill Bacteria

Type

of heat

Examples

Effect on cells

Uses

Dry

Incineration

Oxidizes cell components

Used to sterilize laboratory loops and
needles; used to destroy waste and
infectious materials

Hot-air oven

Oxidizes cell components

Used to sterilize laboratory glassware;
used in home cooking

Wet

Boiling water

Coagulates cell proteins

Used in home disinfection and cooking

Autoclave/pressure

Coagulates cell proteins

Autoclave used to sterilize laboratory

cooker

media; pressure cooker used in
home cooking/canning

Pasteurization

Coagulates cell proteins

Used to disinfect liquids (e.g., milk) to
increase shelf life and kill pathogens

Fractional sterilization

Coagulates cell proteins

Used to sterilize heat- sensitive
instruments and chemicals

200

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Lab Exercises in
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Microbiology

VI. Controlling the Risk and
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Infections

27. Killing Bacteria with
High Temperature

Killing Bacteria with High Temperature EXERCISE 27 201

Materials

Culture (24-hour in tryptic soy broth)
Escherichia coli

Media

Tryptic soy broth tubes (18): 16 x 150 mm
tubes containing 5 ml broth per tube, capped

Equipment

Incubator (35 °C)

Miscellaneous supplies
Beaker (1 liter)
Bunsen burner and striker
Pipette (1 ml, sterile); pipette bulb
Test tube rack
Thermometer (°C)

Tripod with ceramic-lined wire mesh
Wax pencil

First Session: Inoculation
and Heating of Broth Tubes

1 . Place a pipette bulb onto a 1 ml sterile pipette and
fill the pipette with the broth culture of E. coli.

Caution: Do not pipette by
mouth.

This should be sufficient culture to inoculate 17
of the 18 broth tubes.

Aseptically transfer 1 drop of culture to each of
17 broth tubes. Note: Insert the pipette into the
tube close to the surface of the liquid, and aim
the drop directly into the liquid. A drop deposited
on the side of the glass may not reach the broth,
resulting in a false negative.

Thoroughly mix the drop into the broth. Place one
of the inoculated tubes in a test tube rack. Label this
tube the control. Place the remaining 1 6 inoculated
tubes in the 1 liter beaker, and fill the beaker with
tap water to a level above the broth. Now carefully
insert the thermometer in the uninoculated broth
tube, and place the tube in the water.

4 . Place the beaker on the wire mesh platform
mounted on the tripod. Move a lighted Bunsen

Figure 27.1 Experimental setup for heating broth tubes
inoculated with Escherichia coli.

burner to a position beneath the tripod to heat the
water. Examine figure 27.1 to see this experimental
setup without the 16 inoculated tubes.

During heating, remove one tube at every 5°C
interval, beginning at 25 °C. Label each tube with
the temperature at which it was removed, and
place it in the test tube rack with the control tube.
When the water reaches 100°C, remove the last
tube, and turn off the Bunsen burner.

Caution: Use care when
disposing of the hot water!

6. Place the test tube rack with the 17 tubes in a
35 °C incubator.

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VI. Controlling the Risk and
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27. Killing Bacteria with
High Temperature

202 SECTION VI Controlling the Risk and Spread of Bacterial Infections

Second Session: Examination
of Broth Tubes

1 . After 48 hours, examine each tube for growth. If
viable cells remained after heating, they will have
multiplied into millions of cells, turning the broth
cloudy or turbid. In this case, you will not be able
to see through the liquid. Score these tubes as (+)
for growth, indicating that the temperature wasn't
sufficient to kill all vegetative cells. If all
vegetative cells were killed after heating, none

will have been left to multiply, leaving the broth
clear. In this case, you will be able to see through
the liquid. Score these tubes as (-) for growth,
indicating that the temperature was sufficient to
kill all vegetative cells. Record your score for
each tube in the laboratory report.

Continue scoring tubes as (+) or (-) using the
criteria in step 1 until all tubes have been scored.
Evaluate the results of your experiment as related
to the use of heat in your home.

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Spread of Bacterial
Infections

27. Killing Bacteria with
High Temperature

EXERCISE

Laboratory Report

Name

Date

Lab Section

Killing Bacteria with High Temperature

1. In the following table, record your scores for each tube; use a (+) for tubes with cloudy, turbid growth;
use a (-) for tubes with clear broth.

Temperature (°C)

Broth turbid (T)
or clear (C)?

Growth (+) or (-)?

Heat killed all
vegetative cells?

100

2. According to your results in this experiment, what is the minimum temperature required to kill all vege-
tative cells of E. colil What application might this have for cooking your hamburger meat at home?

3. If you received a notice from city officials to boil your water before use, would boiling kill E. coll and
other vegetative bacterial cells if they were present? Explain.

203

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VI. Controlling the Risk and
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Infections

28. Skin Disinfection:
Evaluating Antiseptics and
Hand Sanitizers

Skin Disinfection: Evaluating Antiseptics
and Hand Sanitizers

A variety of chemical agents display antimicrobial activ-
ity against bacteria. One category of antimicrobial chem-
ical agents, the antibiotics, was examined in Exercise 25.
Two other categories of chemical agents commonly used
in the household are antiseptics and disinfectants. Anti-
septics are chemicals safe enough to be applied to the
skin; they are used to prevent wound infections and to dis-
infect skin. Some commonly used antiseptics and their
effects on bacterial cells are presented in table 28.1.

The effectiveness of these skin-applied chemical
agents will be examined in this exercise. Disinfectants
are chemicals considered too harsh to be applied to the
skin, and are only used on inanimate surfaces. Disin-
fectants will be evaluated in Exercise 29.

Evaluating Antiseptics: The Filter
Paper Method

Antiseptics are commonly used on the skin to prevent
wound infections. One of the ways to determine the
effectiveness of antiseptics is to use the filter paper

method, outlined in figure 28.1. In this method, filter
paper disks are dipped into an antiseptic and then placed
on an agar plate that has been inoculated with a bacte-
rial culture. The plate is then incubated to allow bac-
terial growth. After growth, plates are examined for
zones of inhibition around the chemical- soaked disks,
indicating chemical effectiveness. In this exercise, you
will use the filter paper method to examine the effec-
tiveness of antiseptics commonly applied to the skin.

Evaluating Hand Sanitizers

Bacteria are numerous on the hands, and represent both
members of the normal flora and transients picked up
from the environment. While the normal flora is typi-
cally not harmful, transients can be disease-causing
agents. One of the simplest and most effective ways
to eliminate these transient disease-causing agents is to
wash your hands. Hungarian physician Ignaz Semmel-
weis advocated hand washing as a means of preventing
disease transmission in the mid- 1800s. This simple task
is still recommended today by health-care specialists as
one of the most effective means of preventing infection.

Table 28.1 Commonly Used Antiseptics

Chemical agent

Effect on cells

Commercial uses

Alcohol (ethyl or isopropyl)

Dehydrates the cell; alters cell

Skin cleansing and degerming

membrane; denatures cell proteins

agent; skin antiseptic

Benzalkonium chloride

Alters cell membrane

Skin antiseptics

Cetylpyridinium chloride

Alters cell membrane

Mouthwashes

Hexachlorophene

Alters cell membrane; denatures
cell proteins

Soaps and skin antiseptics

Hydrogen peroxide

Oxidizes cell components

Skin antiseptic

Mercurochrome or

Denatures cell proteins

Skin antiseptic

Merthiolate

Tincture of iodine

Denatures cell proteins

Skin antiseptic

Triclosan

Alters cell membrane; denatures
cell proteins

Antibacterial soaps

205

Alexander-Strete-Niles:
Lab Exercises in
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Microbiology

VI. Controlling the Risk and
Spread of Bacterial
Infections

28. Skin Disinfection:
Evaluating Antiseptics and
Hand Sanitizers

206 SECTION VI Controlling the Risk and Spread of Bacterial Infections

(a) Obtain a sterile disk using sterile forceps, and dip the
disk halfway into antiseptic to allow the disk to soak up
the chemical.

(b) Place the chemical- soaked disk on an inoculated plate
Repeat for three other antiseptics.

Zones of inhibition

Figure 28.1 The filter paper method for evaluating antiseptics.

Today, using a hand sanitizer is a popular way to
clean the hands. These products are popular because they
can be used to disinfect the hands while away from home
or when soap, water, or towels are not available. These gel
products are dispensed from plastic bottles onto the hands.
The hands are then rubbed together until dry. The active
ingredient in these products is 62% ethyl alcohol.

This exercise will also evaluate the effectiveness
of hand sanitizers in removing bacteria from the hands.

Cultures (24-hour in tryptic soy broth)
Bacillus cere us
Escherichia coli

Pseudomonas aeruginosa
Staphylococcus aureus

All agents in red are BSL2 bacteria

Media

Tryptic soy agar (TSA) plates
Tryptic soy broth tubes

Chemicals and reagents
Antiseptics

Alcohol, ethyl or isopropyl
Benzalkonium chloride (found in
skin antiseptics)

Alexander-Strete-Niles:
Lab Exercises in
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Microbiology

VI. Controlling the Risk and
Spread of Bacterial
Infections

28. Skin Disinfection:
Evaluating Antiseptics and
Hand Sanitizers

Skin Disinfection: Evaluating Antiseptics and Hand Sanitizers EXERCISE 28 207

Cetylpyridinium chloride (found

in mouthwashes)
Hexachlorophene (found in soaps

and skin antiseptics)
Hydrogen peroxide
Mercurochrome or Merthiolate
Tincture of iodine

Triclosan (found in antibacterial hand soaps)
Ethanol, 70%

Hand sanitizer (active ingredient,
62% ethyl alcohol)

Equipment

Incubator (35 °C)

Miscellaneous supplies
Beaker, 250 ml
Bunsen burner and striker
Cotton-tipped swabs, sterile
Filter paper disks, sterile, in a petri dish
Forceps
Wax pencil

Procedure

First Session

Evaluating Antiseptics: The Filter
Paper Method

1 . Dip a cotton-tipped swab into one of the four
cultures, and use it to inoculate a tryptic soy agar
plate using the procedure outlined in Exercise 25
(see figure 25.2). Note: A lawn of bacterial
growth is necessary for this method, as it was for
antibiotic testing in Exercise 25. Repeat this
inoculation procedure for a second plate using the
same culture. Label each plate with a wax pencil.

2. Repeat step 1 for the remaining three cultures.
You should now have a total of eight plates, two
for each culture. After inoculation, allow all
plates to dry for 1 5 minutes before proceeding to
the next step.

3. Pour some 70% ethanol into a 250 ml beaker.

Caution: Keep the alcohol away
from the flame!

b. Now pick up a sterile disk with the forceps,
and insert it halfway into a drop of the
antiseptic poured into a beaker or a petri dish.
Let the disk soak up the chemical; when
thoroughly soaked, lift the disk and place it on
an inoculated plate.

c. After placement, tap the disk lightly to make
sure it is secure.

Repeat steps a-c until you have placed
this antiseptic on a plate for each culture.
Proceed to the next antiseptic until you have
placed four disks on a plate for each culture.
Place the remaining four antiseptics on the
second plate, for a total of eight antiseptics
per culture. Note: Place the disks as far apart
as possible, and mark the antiseptic on the
bottom of the plate.

4. When all disks are in place, put your plates into
a 35 °C incubator.

Evaluating Hand Sanitizers

1 . Dip a cotton-tipped swab into a tube of tryptic
soy broth to wet the cotton. Rub lightly on the
inside of the tube to remove excess liquid.

2. Swab the left hand as follows: Begin at the top of
the first finger (nearest the thumb) and swab
down to the base of the thumb; roll the swab, and
come back up to the fingertip; repeat this two
more times to cover this area of the finger and
palm (figure 28.2). Use this swab to inoculate a
tryptic soy agar plate. Swab the entire surface of
the plate, turn 90°, and swab the entire surface
again. Be sure to rotate the swab as you go to
deposit all the bacteria lifted from the hand.
Label this plate "Before, Replicate 1."

3. Repeat step 2 for the third finger of the left hand,
swabbing the finger and palm as before with a
fresh swab, and then transferring the bacteria
lifted to a second tryptic soy agar plate. Label
this plate "Before, Replicate 2."

4. Take the hand sanitizer, and place a thumbnail-
sized amount in the palm of the left hand. Rub
the palms of both hands together, covering all
inside surfaces of the hands with sanitizer.
Continue rubbing until the gel has disappeared
and the hands are dry.

a. Dip your forceps into the alcohol, and pass them
over a Bunsen burner flame to sterilize them.

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

VI. Controlling the Risk and
Spread of Bacterial
Infections

28. Skin Disinfection:
Evaluating Antiseptics and
Hand Sanitizers

208 SECTION VI Controlling the Risk and Spread of Bacterial Infections

Before,
Replicate 1

(a)

Before,
Replicate 2

(b)

Washing with hand sanitizer

(c)

After,
Replicate 1

(d)

After,
Replicate 2

(e)

5. After sanitizer treatment, take a fresh swab, and
wet it in broth as before. Swab the second finger,
starting at the tip and moving downward to the
base of the palm. Rotate the swab, and move
upward to the fingertip. Repeat this down-and-up
process two more times as before (figure 28.2).
Inoculate a third tryptic soy agar plate as before.
Label this plate "After, Replicate 1."

6. Using a fresh swab, repeat the swabbing
procedure in step 5 for the fourth finger
(smallest). Inoculate a fourth tryptic soy agar
plate as before, and label it 'After, Replicate 2."

7. Place these four plates in a 35 °C incubator with
the antiseptic plates.

Figure 28.2 Testing the effectiveness of hand sanitizers.

Second Session

Examining Antiseptic Plates

1 . After 48-72 hours, examine the culture plates
containing antiseptic disks. Examine the growth
around the disks.

2. For each disk, look for a zone of inhibition. As
for antibiotics, these areas indicate the
effectiveness of a chemical agent in preventing
growth. However, in this case, the diameter of the
zone may not equate to a degree of effectiveness,
since chemicals vary in their volatility and
diffusion through the agar. Therefore, record only
a (+) for a zone of inhibition around a disk
indicating susceptibility. Record a (-) for no zone
of inhibition, indicating resistance.

3. Complete your observation of all disks for the
four cultures, recording a (+) or (-) in the
laboratory report.

Examining Hand Sanitizer Plates

1 . After 48-72 hours, examine the plates inoculated
with the swabs of your left hand. Separate these
into "before" and "after" plates.

2 . Count the total number of colonies on the two
replicate "before" plates and the total number of
colonies on the two replicate "after" plates. Record
these numbers in your laboratory report. Calculate
a "before" average and an "after" average.

3. Record the percentage of bacteria killed by the
hand sanitizer.

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

VI. Controlling the Risk and
Spread of Bacterial
Infections

28. Skin Disinfection:
Evaluating Antiseptics and
Hand Sanitizers

Name

Lab Section

EXERCISE

Laboratory Report

Date

Skin Disinfection: Evaluating Antiseptics and Hand Sanitizers

Antiseptics

1. In the following table, record your results for antiseptic plates. Record a (+) for the presence of a zone of
inhibition around the disk. Record a (-) for no zone of inhibition.

Culture

Antiseptic

Bacillus
cereus

Escherichia
coli

Pseudomonas
aeruginosa

Staphylococcus
aureus

Benzalkonium chloride

Cetylpyridinium chloride

Ethanol (70%)

Hexachlorophene

Hydrogen peroxide

Isopropyl alcohol

Mercurochrome or Merthiolate

Tincture of iodine

Triclosan

2. Which antiseptic(s), if any, had the widest spectrum of activity? How would this trait make this a useful
antiseptic? Explain.

209

Alexander-Strete-Niles:
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VI. Controlling the Risk and
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28. Skin Disinfection:
Evaluating Antiseptics and
Hand Sanitizers

210 SECTION VI Controlling the Risk and Spread of Bacterial Infections

Hand Sanitizer

1 . In the following table, record your results for the hand sanitizer. Record the total number of colonies on
the two "before" plates and the total number of colonies on the two "after" plates.

Total number of colonies

Replicate

Before hand sanitizer

After hand sanitizer

Average

2. Calculate the average percent reduction of bacteria on the hand:

°7t

3. Did the hand sanitizer remove the large majority of bacteria from your hand? Based on these results,
would you buy this product for use when away from home? When would it be useful?

Alexander-Strete-Niles:
Lab Exercises in
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Microbiology

VI. Controlling the Risk and
Spread of Bacterial
Infections

29. Cleaning Countertops
with Disinfectants

Cleaning Countertops with Disinfectants

Antimicrobial chemical agents are important in the con-
trol of microorganisms. Exercise 25 examined the effec-
tiveness of antibiotics, while Exercise 28 evaluated the
effectiveness of antiseptics. A third category of chem-
ical agents, disinfectants, are considered too harsh for
use on or in the human body; however, they are useful
on inanimate surfaces. Some of the chemical agents
commonly used in disinfectants are listed in table 29.1.

Disinfectants are widely used around the house to
remove bacteria from surfaces. Surfaces that require
disinfection at home include the kitchen sink and coun-
tertops, bathroom sink and countertops, toilet, shower,
and bathtub. Similar surfaces that require periodic dis-
infection are also found in public facilities and at work.
Keeping these surfaces clean and low in bacterial num-
bers is one of the most effective means of controlling
the occurrence and spread of infectious agents.

In this exercise, you will evaluate the effectiveness
of several commercially available disinfectants con-
taining the chemical compounds listed in table 29.1.

Media

Tryptic soy agar plates
Tryptic soy broth tubes

Chemicals and reagents

Disinfectants, commercially available (those
listed in table 29.1 or others that contain the
same chemicals)

Equipment

Incubator (35 °C)

Miscellaneous supplies
Adhesive tape

Bottles, spray-dispenser type
Cotton-tipped swabs, sterile
Paper towels
Ruler, metric
Wax pencil

Table 29.1 Chemical

Agents Commonly Used in Disinfectants

Chemical agent

Effect on cells

Commercial uses

Sodium hypochlorite

Oxidizes cell components

Surface disinfectants and bleach

Orthophenylphenol

Denatures cell proteins

Surface disinfectants, such as
Lysol

Alkyldimethylbenzyl

Alters cell membrane

Surface disinfectants, such as

ammonium chloride

Formula 409

Pine oil

Alters cell membrane;

Surface disinfectants, such as

denatures cell proteins

Pine-Sol

211

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

VI. Controlling the Risk and
Spread of Bacterial
Infections

29. Cleaning Countertops
with Disinfectants

212 SECTION VI Controlling the Risk and Spread of Bacterial Infections

Procedure

First Session: Inoculation of Plates

1 . Select two surfaces to be cleaned. A laboratory
countertop and a bathroom or kitchen
countertop are recommended. If a bathroom
or kitchen is unavailable, select a second
laboratory countertop.

2. Mark off a 3,600 cm 2 area of the first surface to
be cleaned. Use four 60 cm pieces of adhesive
tape to mark the edges of this area. Also place a
piece of adhesive tape in the center of this area.
The center piece of tape will help delineate four
areas within the 3,600 cm 2 area: an upper left
area, upper right area, lower left area, and lower
right area. Designate these four areas as test areas
for disinfectants 1, 2, 3, and 4, respectively
(figure 29.1).

3 . In each test area, use pieces of adhesive tape
10 cm long to mark the edges of two adjacent
100 cm 2 areas, one designated A, before cleaning
with disinfectant, and the other designated B,
after cleaning with disinfectant (figure 29.1).

Countertop area (3,600 cm 2 )

60 cm

10 cm

Disinfectant 1

10 cm

Disinfectant 2

10 cm

Disinfectant 3

Disinfectant 4

60 cm

A: Before cleaning, swab each A area with a wet, cotton-tipped
swab. Inoculate a tryptic soy agar plate.

B: After cleaning with disinfectant, swab each B area with
another swab. Inoculate a second tryptic soy agar plate.

4. Dip a sterile, cotton-tipped swab into tryptic soy
broth. Use it to swab the 100 cm 2 area denoted as
A, Disinfectant 1. Swab the entire 100 cm 2 area
twice, the second time at a 90° angle to the first.
Use the swab to inoculate a tryptic soy agar plate.
Rub the swab over the entire surface of the plate,
rolling the swab as you do so. Rotate the plate
90° and swab again. Label this plate 'A,
Disinfectant 1."

5. Take disinfectant 1, and clean the entire
disinfectant 1 test area. Do not spray into any
of the other areas. Prepare the disinfectant per
the directions on the container, mixing the
disinfectant with water in a spray-type dispenser.
In this way, the disinfectant can be thoroughly
sprayed over the entire surface before wiping
with a paper towel. Be sure to wipe the surface
dry. Do not wipe into any of the other areas.

6. Dip a fresh cotton- tipped swab in sterile broth,
and use it to swab the 1 00 cm 2 area denoted as B ,
Disinfectant 1 . Again, be sure to swab the entire
100 cm 2 area twice. Use this swab to inoculate a
second tryptic soy agar plate as before. Label this
plate "B, Disinfectant 1."

7. Repeat steps 4-6 to complete the sampling of
each A and B area for disinfectants 2, 3, and 4.
When finished, you should have inoculated a total
of eight tryptic soy agar plates.

8. After completing your sampling of the first
surface, repeat steps 2-7 for the second surface.
You should have inoculated another eight tryptic
soy agar plates for this surface, giving you a total
of 16 plates for the two surfaces.

9. Place all plates into a 35 °C incubator.

Second Session: Examination of Plates

1 . After 48-72 hours, examine your plates. Sort
the plates by surface cleaned, disinfectant used,
and before cleaning (A) and after cleaning
(B). Count the total number of bacterial
colonies on each plate, and fill in your results
in the laboratory report.

2. Calculate the percent decrease in the bacteria on
each cleaned surface for each disinfectant.

Figure 29.1 Procedure for testing the effectiveness
of disinfectants.

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

VI. Controlling the Risk and
Spread of Bacterial
Infections

29. Cleaning Countertops
with Disinfectants

Name

Lab Section

EXERCISE

Laboratory Report

Date

Cleaning Countertops with Disinfectants

1 . Record the number of colonies on your plates,
a. Laboratory countertop (first surface)

Disinfectant

Before (A)

After (B)

Percent Decrease

1 =

2 =

3 =

4 =

b. Second surface

Disinfectant

Before (A)

After (B)

Percent Decrease

1 =

2 =

3 =

4 =

2. Explain the difference between disinfection and sterilization. Which of these terms applies to the action
of the chemicals used in this exercise?

3. Do these chemical agents work effectively to remove bacteria from surfaces? Were there any that seemed
to work best?

4. Based on your results, do you think the use of these chemicals around the home is justified? If so, when
and where would you use these products?

213

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VI. Controlling the Risk and
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Infections

30. Bacteriological
Examination of Drinking
Water Using the MPN
Method

Bacteriological Examination of Drinking Water
Using the MPN Method

Coliforms, Indicators
of Fecal Contamination

Water is routinely tested to ensure that it is safe for drink-
ing. A widely used indicator of the suitability of drink-
ing water is coliform bacteria. Coliforms are
Gram-negative, non-endospore-forming rods that are fac-
ultatively anaerobic and produce acid and gas from lac-
tose within 48 hours at 35 °C. The key indicator organism
in this group is Escherichia coli, which is not normally
present in soil and water, but present in large numbers
in the intestines and feces, and capable of long-term sur-
vival in the environment. Therefore, the presence of
E. coli is indicative of human or animal fecal waste. Water
contaminated with fecal material, as determined by the
presence of coliforms, is considered nonpotable, mean-
ing unsuitable for drinking. Water that is coliform-free
is considered potable and safe for drinking.

Human fecal waste may also carry intestinal
pathogens, such as Salmonella typhi, the cause of
typhoid fever; Salmonella typhimurium, the cause of
salmonellosis; Vibrio cholerae, the cause of cholera;
and Shigella sonnei, the cause of shigellosis. Each of
these intestinal pathogens is transmitted by fecal con-
tamination of drinking water. However, their presence
is difficult to detect since they do not typically occur
in large numbers and do not survive long in soil and
water. As a consequence, coliforms, especially E. coli,
are used as the indicator of fecal contamination.

Testing Water for Coliforms

One of the methods used to detect coliforms in drinking
water is the most probable number (MPN) method.
This method, outlined in figure 30. 1 , consists of three
parts: (1) a presumptive test; (2) a confirmed test; and
(3) a completed test.

In the presumptive test, three series of five tubes
each, or 15 tubes total, are inoculated with a water sam-
ple. Each tube contains 10 ml of lactose broth and a
durham tube. Each tube in the first series of five tubes

Presumptive test:

Inoculate lactose broth;
incubate 24-48 hours.

Acid and gas produced:
Positive presumptive test

Acid and gas not produced
Negative presumptive
test — water potable

Confirmed test:

Streak from lactose broth

onto eosin methylene blue

(EMB) plates; incubate

24 hours.

Typical coliform colonies:

dark centers, metallic sheen

Positive confirmed test

Colonies not coliform:
Negative confirmed test-
water potable

Completed test:

Select typical coliform

colonies; inoculate lactose

broth and agar slant;

incubate 24 hours.

Lactose
broth

Agar
slant

Acid and gas not produced
Negative completed test-
original isolate not
coliform; water potable

Acid and

gas
produced

Gram-negative

rods present;

no spores

present

Coliform group present:
Positive completed test-
water nonpotable

Figure 30.1 The MPN method used to detect coliforms
in drinking water.

215

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

VI. Controlling the Risk and
Spread of Bacterial
Infections

30. Bacteriological
Examination of Drinking
Water Using the MPN
Method

216 SECTION VI Controlling the Risk and Spread of Bacterial Infections

receives 10 ml of sample; each tube in the second series
of five tubes receives 1 ml of sample; and each tube in
the third series of five tubes receives 0.1 ml of sample.
After 24 hours incubation at 35 °C, tubes are examined
for the presence of acid and gas, products of lactose fer-
mentation. A positive tube, which has turned yellow and
has a gas bubble in the durham tube, is depicted in
figure 30.2a. Also depicted is a negative tube, which is
unchanged in color and has no gas bubble in the durham
tube (figure 30.2b). After 48 hours of incubation, nega-
tive tubes are examined again for a delayed positive reac-
tion. All tubes after 48 hours are denoted as either (+)
or (-), and a most probable number is assigned accord-
ing to the index shown in table 30.1. If only one tube
scores positive, this is considered a positive presump-
tive test-that is, it presumes that coliforms are present.
However, their presence must be confirmed in the next
part. If all tubes score negative, this is considered a neg-
ative presumptive test. In this case, the water is consid-
ered free of coliforms and, therefore, potable.

In the confirmed test, all positive tubes from the
highest dilution of sample are streaked onto eosin
methylene blue (EMB) agar (table 30.2). This agar
selects for and differentiates coliform bacteria. E. coli
is especially easy to differentiate since it produces a dis-
tinctive green, metallic sheen on this agar. The presence
of colonies on EMB with this characteristic is consid-
ered a positive confirmed test — that is, it confirms the
presence of coliforms. However, their presence must be
further substantiated by the completed test described
next. The absence of colonies on EMB with this char-
acteristic is considered a negative confirmed test, and
the water is considered absent of coliforms and potable.

Yellow

Gas bubble
in durham tube

Red

No gas bubble
in durham tube

(a)

(b)

Figure 30.2 Lactose broth, (a) Positive tube,
(b) Negative tube.

In the completed test, colonies from EMB with a
green, metallic sheen are transferred to a lactose broth
tube and a nutrient agar slant. If acid and gas are produced
in the lactose broth tube within 24 hours and a Gram stain
detects a Gram-negative rod, this is considered a posi-
tive completed test, meaning that the confirmation of col-
iforms in the water is complete. The water is considered
contaminated with coliforms and unsafe to drink.

In this exercise, you will use the MPN method to
examine the bacteriological quality of three water sam-
ples: sewage, surface water, and tap water.

Materials

Water samples
Sewage

Sewage may contain pathogens

Surface water (from pond, lake, or stream)
Tap water

Media

Eosin methylene blue (EMB) plates
Lactose broth tubes: each with 10 ml broth

and a durham tube, both double- strength

and single- strength
Nutrient agar slant

Chemicals and reagents
Gram- stain reagents

Equipment

Incubator (35 °C)
Light microscope

Miscellaneous supplies
Bunsen burner and striker
Inoculating loop
Immersion oil
Lens paper
Microscope slides

Pipettes, 10 ml and 1 ml, sterile; pipette bulb
Test tube racks
Wax pencil

Procedure

First Session: Inoculation
of Lactose Broth Tubes

1. Take 15 lactose tubes, five double- strength and 10
single-strength, and align into three rows of five in
a test tube rack. Place the five double- strength

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

VI. Controlling the Risk and
Spread of Bacterial
Infections

30. Bacteriological
Examination of Drinking
Water Using the MPN
Method

Bacteriological Examination of Drinking Water Using the MPN Method EXERCISE 30 217

tubes in the front row. In a similar manner, arrange
15 tubes for each of the other two samples, for a
total of 45 tubes. Number the tubes in each row
1 to 5 ; also designate the sample type and sample
amount added: 10 ml (front row), 1 ml (middle
row), or 0.1 ml (back row).

2. Place a pipette bulb onto a 10 ml pipette

CAUTION:

Caution: Do not pipette
by mouth!

Table 30.1

MPN Index and 95% Confidence Limits for Various Combinations of Positive Results
When Five Tubes Are Used per Dilution (10 ml, 1 .0 ml, 0.1 ml)

Combination of MPN index/
positives 100 ml

95% confidence
limits

Combination of
positives

MPN index/
100 ml

95% confidence
limits

Lower

Upper

Lower

Upper

000

4-3-0

0-0-1

1.0

4-3-1

0-1-0

1.0

4-4-0

0-2-0

1.0

5-0-0

9.0

1-0-0

1.0

5-0-1

110

1-0-1

1.0

5-0-2

140

1-1-0

1.0

5-1-0

120

1-1-1

2.0

5-1-1

150

1-2-0

2.0

5-1-2

180

2-0-0

1.0

5-2-0

170

2-0-1

2.0

5-2-1

210

2-1-0

2.0

5-2-2

250

2-1-1

3.0

5-3-0

250

2-2-0

3.0

5-3-1

110

300

2-3-0

5.0

5-3-2

140

360

3-0-0

3.0

5-3-3

170

410

3-0-1

4.0

5-4-0

130

390

3-1-0

4.0

5-4-1

170

480

3-1-1

6.0

5-4-2

220

100

580

3-2-0

6.0

5-4-3

280

120

690

3-2-3

7.0

5-4-4

350

160

820

4-0-0

5.0

5-5-0

240

100

940

4-0-1

7.0

5-5-1

300

100

1,300

4-1-0

7.0

5-5-2

500

200

2,000

4-1-1

9.0

5-5-3

900

300

2,900

4-1-2

5-5-4

1,600

600

5,300

4-2-0

9.0

5-5-5

> 1,600

4-2-1

Source: Standard Methods for the Examination of Water and Wastewater. 18th edition. Copyright 1992 by the American Public Health Associ
ation, the American Waterworks Association, and the Water Environment Federation. Reprinted with permission.

Alexander-Strete-Niles:
Lab Exercises in
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VI. Controlling the Risk and
Spread of Bacterial
Infections

30. Bacteriological
Examination of Drinking
Water Using the MPN
Method

218 SECTION VI Controlling the Risk and Spread of Bacterial Infections

Table 30.2

Composition

of Eosin

Methylene Blue

(EMD) Agar

Peptone

10 g

Lactose

Sucrose

Dipotassium phosphate

Eosin Y

0.4 g

Methylene blue

0.065 g

Agar

13.5 g

Distilled water

1 ,000 ml

Final pH

7.2

Source: The Difco Manual. Eleventh Edition. Difco Laboratories

Add 10 ml of the first sample to each of the five
tubes in the front row. Do the same for the
second and third samples. Use a fresh 10 ml
pipette for each sample.

3 . After all the tubes in the front row have been
inoculated, use a 1 ml pipette with bulb to
inoculate the second and third row of tubes
for each sample.

#• *

Caution: Do not pipette
by mouth!

The tubes in the second row each receive 1 ml
of sample, while those in the third row each
receive 0.1 ml. Be sure to change pipettes
between each sample.

Place all pipettes that were used on the
sewage sample in a disinfectant solution or
in some other waste container designated by
your laboratory instructor.

4. After completing the inoculation of all tubes,
place the test tube racks in a 35°C incubator.

Second Session: Examination of
Lactose Broth Tubes (Presumptive Test)

1 . After 24-48 hours, examine each tube for the
presence of acid and gas. Record tubes with a
yellow color and gas as (+) in the laboratory
report. Record tubes without a color change or
gas as (-). Use the (+) and (-) results to calculate
an MPN for each sample (table 30.1).

2. For samples with a positive presumptive test (i.e.,
one or more tubes with a yellow color and gas),
continue to the confirmed test by streak-plating
positive tubes of the highest dilution onto EMB
agar plates. Place these plates in a 35 °C incubator.

Third Session: Examination of EMB
Agar Plates (Confirmed Test)

1. After 24-48 hours, examine each EMB plate for
the presence of colonies with a green, metallic
sheen. The presence of these colonies represents
a positive confirmed test, while their absence
represents a negative confirmed test.

2. If one or more samples have coliform colonies,
continue to the completed test by selecting a green,
metallic sheen colony from an EMB plate and
using it to inoculate a lactose broth tube and a
nutrient agar slant. Place these in a 35 °C incubator.

Fourth Session: Examination
of Lactose Broth Tube and
Gram Stain (Completed Test)

1 . After 24 hours, examine the lactose broth tube for
acid and gas. If positive, do a Gram stain from
the nutrient agar slant to determine if the culture
is a Gram-negative rod. If lactose-positive and a
Gram-negative rod, the confirmation of coliforms
in the sample is complete.

2. Based on your results, determine the potability of
each water sample.

Alexander-Strete-Niles:
Lab Exercises in
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VI. Controlling the Risk and
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Infections

30. Bacteriological
Examination of Drinking
Water Using the MPN
Method

Name

Lab Section

EXERCISE

Laboratory Report

Date

Bacteriological Examination of Drinking Water
Using the MPN Method

1. Results for water sample #1
a. Presumptive test

(+) or (-)

Sample
added

Tubel

Tube 2

Tube 3

Tube 4

Tube 5

Number of
positive tubes

10 ml

1 ml

0.1ml

Combination of positives =
MPN index/ 100 ml =

Presumptive test: positive or negative?
b. Confirmed test

Number of tubes of highest dilution streaked onto EMB plates
Number of these plates with green, metallic- sheen colonies

Confirmed test: positive or negative?

c. Completed test

Number of green, metallic- sheen colonies selected from EMB plates

Number of these colonies that produced acid and gas from lactose and were Gram-negative rods

Completed test: positive or negative?

d. Conclusion: Water potable or nonpotable?

219

Alexander-Strete-Niles:
Lab Exercises in
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VI. Controlling the Risk and
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30. Bacteriological
Examination of Drinking
Water Using the MPN
Method

220 SECTION VI Controlling the Risk and Spread of Bacterial Infections

2. Results for water sample #2
a. Presumptive test

(+) or (-)

Sample
added

Tubel

Tube 2

Tube 3

Tube 4

Tube 5

Number of
positive tubes

10 ml

1 ml

0.1ml

Combination of positives =
MPN index/100 ml =

Presumptive test: positive or negative?
b. Confirmed test

Number of tubes of highest dilution streaked onto EMB plates
Number of these plates with green, metallic- sheen colonies

Confirmed test: positive or negative?

c. Completed test

Number of green, metallic-sheen colonies selected from EMB plates

Number of these colonies that produced acid and gas from lactose and were Gram-negative rods

Completed test: positive or negative?

d. Conclusion: Water potable or nonpotable?

3. Results for water sample #3:
a. Presumptive test

(+) or (-)

Sample
added

Tubel

Tube 2

Tube 3

Tube 4

Tube 5

Number of
positive tubes

10 ml

1 ml

0.1ml

Combination of positives =
MPN index/100 ml =

Presumptive test: positive or negative?

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

VI. Controlling the Risk and
Spread of Bacterial
Infections

30. Bacteriological
Examination of Drinking
Water Using the MPN
Method

Bacteriological Examination of Drinking Water Using the MPN Method EXERCISE 30 221

b. Confirmed test

Number of tubes of highest dilution streaked onto EMB plates
Number of these plates with green, metallic- sheen colonies

Confirmed test: positive or negative?

c. Completed test

Number of green, metallic- sheen colonies selected from EMB plates

Number of these colonies that produced acid and gas from lactose and were Gram-negative rods

Completed test: positive or negative?

d. Conclusion: Water potable or nonpotable?

4. What are coliforms? Why is their presence in drinking water routinely monitored?

5. What action should be taken if coliforms are detected in drinking water?

6. Answer the following questions based on these photographs:

A water sample yielded these results for the presumptive test (left) and the confirmed test (right)
Collectively, what do these results indicate?

What would be the next step?

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VII. Bacterial Genetics

31. Bacterial DNA Isolation

©The McGraw-Hill

Lab Exercises in
Organismal and Molecular
Microbiology

and Southern Analysis

Companies, 2003

Bacterial DNA Isolation

and Southern Analysis

The sequence of the genome of one strain of Escherchia
coli, K12, was completed in 1997 by researchers at the
University of Wisconsin, Madison. The genome, con-

sisting of a single, circular, double- stranded DNA chro-
mosome, is 4,639,221 base pairs long and contains
4,403 genes. A partial genetic map of the E. coli K12
chromosome is shown in figure 31.1.

O °Q

* fe 5 a

CO <i>

malA

9 %

sef°
pyrD

■purB

att<t>80

tr ^Ac AE

rt\

Figure 31.1 Genetic map of E. coli K12 with the locations of selected genes. E. coli K12 strains
are used for fundamental work in biochemistry, genetics, and biotechnology, acting as carriers of
genes encoding therapeutic proteins.

224

Alexander-Strete-Niles:

VII. Bacterial Genetics

31. Bacterial DNA Isolation

©The McGraw-Hill

Lab Exercises in
Organismal and Molecular
Microbiology

and Southern Analysis

Companies, 2003

Bacterial DNA Isolation and Southern Analysis EXERCISE 31 225

In preparation for analysis, the DNA must be iso-
lated from a pure culture of the bacteria. The isolation
involves lysing the cells, degrading cellular RNA and
protein with enzymes, and separating cellular debris
from the DNA through extraction with an organic sol-
vent. The DNA is then cut into fragments with a restric-
tion endonuclease, an enzyme that cuts through
double-stranded DNA at a particular recognition
sequence, (see also Exercise 33 and table 33.1). The
restriction enzyme EcoRI, for example, cuts DNA
wherever it contains the sequence,

-GAATTC-
-CTTAAG-

Therefore, cutting a series of DNA samples from
the same source with EcoRI will always generate the
same set of restriction fragments. These fragments can
be separated by size using gel electrophoresis.

However, cellular DNAs are so long (here, over
4 million base pairs) that when they are cut with a

restriction enzyme and the fragments are separated on
a typical electrophoresis gel, no clear restriction pattern
can be seen. Only a smear of DNA representing frag-
ments of just about every possible size is visible (figure
31.2). Think of this DNA smear as a ladder that has so
many rungs so close together that you cannot distin-
guish one rung from the next, or as a barcode that is
solid black — there is no information there. Southern
blotting allows the detection of a discrete region of
the DNA, revealing a restriction pattern of just that part
of the genome (figure 31.3). Southern blotting is also
often employed to generate DNA fingerprints (see
Exercise 36).

In this exercise, you will isolate DNA from bacte-
ria for restriction analysis (figure 31.3 a-c). If time per-
mits, you may proceed with a Southern blot over the
next few lab sessions (figure 31.3 d-i) in order to iden-
tify the restriction pattern of the bacterial gene lacZ. The
lacZ gene encodes the enzyme p-galactosidase.

Size marker
(base pairs)

23,130

9,416

6,557

4,361

2,322
2,027

(a)

Size marker

(base pairs)

23,130^

9,416-

6,557-

■ ^Bf'

4,361-

2,322-
2,027 -

L|^mmAHH

(b)

Figure 31.2 Agarose gel electrophoresis of DNA isolated from E. coli. The 0.8%
agarose gels have been stained with (a) methylene blue or (b) ethidium bromide.
Both gels contain the following samples: bacteriophage lambda DNA cut with the
restriction enzyme Hindlll (size marker, lane 1), E. coli DNA cut with the restriction
enzyme EcoRI (lane 2), and E. coli DNA that has not been cut with a restriction
enzyme (lane 3). The fragments (bands) in lane 1 are distinct because the lambda
genome is only about 49,000 base pairs long, and the enzyme cut the DNA into
discernible fragments. The E. coli DNA restriction fragment lengths in lane 2 are
indistinguishable from one another by this method, and appear as a smear.

Alexander-Strete-Niles:

VII. Bacterial Genetics

31. Bacterial DNA Isolation

©The McGraw-Hill

Lab Exercises in
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Microbiology

and Southern Analysis

Companies, 2003

226 Section VII Bacterial Genetics

(a) Isolation of DNA from tissues, cells, or viruses:

The DNA is mechanically sheared during this
procedure, generating large fragments.

(b) Restriction enzyme digestion: The large fragments
of DNA are cut at specific sites with a restriction
enzyme, generating restriction fragments
characteristic of the organism.

(c) Agarose gel electrophoresis: The restriction
fragments are separated by size; the distance
migrated by a fragment during electrophoresis is
inversely proportional to its size.

Longer
fragments

Shorter
fragments

(d) DNA denaturation: The DNA fragments in the
gel are made single-stranded.

NaOH

dsDNA

ssDNA

(e) DNA transfer (blotting): DNA is transferred
from the gel to the surface of a membrane, such
as nitrocellulose. The method of transfer shown
here is called capillary blotting.

Weight

Sponge

Dry paper

Membrane

Salt solution

(f) DNA immobilization: The membrane is baked to
irreversibly bind the DNA to the membrane.

Bake

80°

(g) Hybridization: The membrane is submerged in a
solution containing many molecules of a specific
single-stranded DNA "probe," labeled in some
way for later detection. The probe DNA forms
base pairs with target DNA molecules on the
membrane.

Membrane

Hybridization
solution containing
labeled probe
molecules

(h) Washing: Probe that is not extensively base-
paired to the immobilized DNA is washed away;
probe that is nonspecifically bound is removed.

^ — M

(i) Development /detection: Restriction fragments
that have hybridized with probe appear as a
pattern on the membrane (or on the film if the
label was a radioisotope).

Alexander-Strete-Niles:

VII. Bacterial Genetics

31. Bacterial DNA Isolation

©The McGraw-Hill

Lab Exercises in
Organismal and Molecular
Microbiology

and Southern Analysis

Companies, 2003

Bacterial DNA Isolation and Southern Analysis EXERCISE 31 227

Figure 31.3 (opposite page) Overview of Southern blotting and hybridization. With the com-
pletion of the Southern technique, what was once visible only as a smear of DNA fragments on
a gel now becomes a distinct pattern of specific restriction fragments on a membrane.

First Session: Bacterial DNA Isolation
and Restriction Digestion

Cultures

E. coli B and S. marcescens, each grown
overnight in 2 ml LB broth and
then inoculated into 50 ml fresh LB
for log growth

Media

LB broth: 10 g bacto-tryptone, 5 g yeast
extract, 1 g NaCl per liter

Reagents

TNE (10 mM Tris, pH 8.0, 10 mM NaCl,

0.1 mM EDTA), autoclaved
TE (10 mM Tris, pH 8.0, 0.1 mM EDTA),

autoclaved
HTE (50 mM Tris, pH 8.0, 20 mM EDTA),

autoclaved
2% sarcosyl (N-lauroyl sarcosine) in HTE
RNase on ice (pancreatic RNase A, 1 mg/ml,

in TE, preheated to 80°C for 10 minutes to

inactivate DNases)
Pronase on ice (10 mg/ml, in TNE, preheated

to 37°C for 15 minutes to inactivate DNases)

Phenol, equilibrated with 0.5 mM Tris, pH 8.0
Chloroform (chloroform:isoamyl

alcohol, 24:1)
3.0 M sodium acetate
Isopropanol
70% ethanol

Distilled water, autoclaved
Restriction enzyme and control reaction mixes

(table 31.1)

Equipment

37°C bacterial incubator with shaker platform

Microwave oven

Water bath or heat block at 37°C

Water bath or heat block at 50°C

Miscellaneous supplies
Laboratory marker
Latex gloves (when handling DNA; to protect

DNA from deoxyribonucleases on hands)
Ice

Microfuge tubes
Pasteur pipettes/bulb
1 .0 ml serological pipette/pipettor
Micropipettors/tips (1-10 jll, 10-100 jll,
100-1,000 jil)

Control Mix. Ad

Table 31.1 Components of the Restriction Enzyme Mix

mix to the corresponding reaction and control tubes. Store mixes on ice.

Restriction

mix
components

EcoRI

No enzyme
control

Use 10 nl
restriction mix,

Use 10 ^il
no enzyme control mix,

lOx restriction buffer

3ul

3 |il

Sterile distilled water

6ul

7 |il

EcoRI (10-20 units/ul)

1 ul

Oul

Total mix volume

10 ul

10 |xl

Total reaction volume

with 20 ul bacterial DNA

30 |il

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31. Bacterial DNA Isolation

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Lab Exercises in
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and Southern Analysis

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228

Section VII Bacterial Genetics

Second Session: Agarose Gel
Electrophoresis, Staining,
and Southern Transfer

Reagents

0.8 % agarose gel prepared with TBE: Tris-

Borate-EDTA (108 g Tris-base, 55 g boric

acid, 40 ml 0.5 M EDTA, pH 8.0, per liter)
DNA standard, lambda-Hindlll, 1 jig per 30 jll

TBE; one per gel
DNA sample loading buffer (tracking dyes):

0.25% bromphenol blue, 0.25% xylene

cyanol, 30% glycerol in distilled water
DNA Blue InstaStain™
Denaturing solution (0.5 N NaOH, 1.5 M NaCl)
Neutralization solution (0.5 M Tris, pH 7.5,

1.5 M NaCl)
20x SSC (3 M NaCl, 0.3 M sodium citrate),

diluted to 1 Ox SSC

Equipment

Horizontal gel electrophoresis system and

power source
Kitchen sponge (one per gel, for Southern

transfer)

Miscellaneous supplies

Micropipettors/tips (1-10 Jill, 10-100 jll)

1 25 ml Erlenmeyer flask

Laboratory marker

Latex gloves (when handling DNA samples)

1 .0 ml microcentrifuge tubes

Weigh boat or shallow dish (for staining)

Optitran BA-S supported nitrocellulose

membranes
3MM chromatography paper

Third Session: Probe Preparation and
Southern Hybridization

Reagents

DIG-High Prime DNA Labeling and Detection

Starter Kit I (table 31.2)
Probe DNA: pBLU digested with Hindlll

(1 Jig in 16 (il distilled, autoclaved water).

One probe for every 2 membranes.
20x SSC (3 M NaCl, 0.3 M sodium citrate),

diluted to 2x SSC

Equipment

Oven set at 80°C

Oven set at 42°C with a rocker platform

covered with bench-coat absorbent paper
Water bath set at 42°C
Boiling water bath or heat block set at 100°C

Miscellaneous supplies

Micropipettors/tips (1-10 Jill, 10-100 jll)
50 ml conical tubes

Fourth Session: Washing
and Blot Development

Reagents

20x SSC (3 M NaCl, 0.3 M sodium citrate),

diluted to 2x SSC
2xSSC, 0.1%SDS
0.5xSSC, 0.1%SDS

Equipment

Oven set at 42°C with a rocker platform

covered with bench-coat absorbent paper
Water bath set at 42°C
Water bath or oven at 68°C
Bench top rocker or shaker platform

Miscellaneous supplies

3MM chromatography paper
Large weigh dishes

Procedure

First Session: Bacterial DNA Isolation
and Restriction Digestion

Yesterday, each E. coli strain was inoculated into 2 ml
of LB for overnight growth at 37°C with shaking. Ear-
lier today, each 2 ml culture was transferred into
50 ml of fresh broth in 125 ml flasks and incubated at
37°C with shaking.

1 . Remove a flask of bacteria from the 37°C
incubator (the culture is expected to be in the log
phase of growth), and pipette 1 ml of it into a
microfuge tube. Centrifuge the sample in a
microfuge at full speed (14,000 RPM) for

15 seconds. Decant the supernatant into a waste
receptacle, and let the liquid drain off onto a
tissue. Dispose of the tissue in a biohazard bag.

2. Resuspend the cell pellet in 0.3 ml HTE, mixing
until there are no remaining cell clumps.

3. Add 0.35 ml 2% sarcosyl in HTE. Mix well by
capping and inverting the tube. Note that the
liquid is quite cloudy. Once lysis is complete
(after step 4), the liquid will be less cloudy.

CAUTION

Note: Wear glo
point on.

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VII. Bacterial Genetics

31. Bacterial DNA Isolation

©The McGraw-Hill

Lab Exercises in
Organismal and Molecular
Microbiology

and Southern Analysis

Companies, 2003

Bacterial DNA Isolation and Southern Analysis EXERCISE 31 229

Table 31.2 DIG-H

igh Prime DNA Detection

Starter Kit 1 Reagent Descriptions

and

Buffer

Preparations

Amount required

Reagent

Purpose

Preparation

(approximate)

Hybridization solution

For prehybridization

Add 64 ml of autoclaved, cooled

20 ml per blot

and hybridization

dH 2 0, in two portions, stirring, at
37 °C for 5 minutes

Posthybridization: blot treatment and development

Buffer 1 (maleic

For preparation of

0. 1 M maleic acid

2 liters

acid buffer)

wash buffer and
buffer 2 (blocking

0.15MNaCl

buffer)

pH to 7.5 with solid NaOH

Buffer 1 + Tween-20

For washing the blot

0.3% Tween-20 (v/v) in buffer 1

1.5 liters

(wash buffer)

before blocking and

after antibody

Tween-20 (polyoxyethlenesorbitan

incubation

mono laureate, Sigma # P 1379)

Buffer 2

Coats the membrane

Dilute lOx blocking solution

75 ml per blot

(blocking solution)

with proteins to

(provided in kit) 1:10 in

prevent antibodies

buffer 1

from binding directly

to the membrane in the

fourth session, step 6;

also used to make the

antibody solution

Buffer 3

For equlibration of

100 mM TrisCl

40 ml per blot

(detection buffer)

the blot prior to

100 mM NaCl

development, and

50 mM MgCl 2

for preparation of

ph9.5

substrate solution

Antibody solution

Antibodies, covalently

Anti-DIG-AP (provided in kit)

10 ml per blot

linked to the enzyme

diluted 1:5,000 in buffer 2

AP and specific for

the digoxigenin groups

along the probe DNA

Substrate solution

Colorless substrate will

200 ul NBT/BCIP (provided in

10 ml per blot

(blocking solution)

be converted to
colored product in the
presence of AP

kit) in 10 ml buffer 3

Buffer 4 (TE)

For stopping the

10 mM TrisCl

20 ml per blot

development reaction

1 mM EDTA, pH 8.0

4. Add 5 jil RNase, and incubate at 37°C for 15
minutes. Add 35 jil of pronase, and heat at 50°C
until lysis is complete, about 30 minutes.

5. Cap the tube securely, and vortex the sample for
2 minutes at the highest setting (figure 31.4).

6. Phenol and chloroform extractions: Add an

equal volume (700 jil) of phenol, shake well, and
centrifuge at full speed for 3 minutes to separate
the phases. Pipette the upper phase into a fresh
microfuge tube, being careful to avoid the

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31. Bacterial DNA Isolation

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230

Section VII Bacterial Genetics

Figure 31.4 Vortex the sample for 2 minutes at the high-
est setting. This mechanically shears the DNA, generating
fragments that are about 20 kilobases (kb). Later, you will
further fragment the DNA with the restriction enzyme
EcoRI.

Figure 31.5 Pipette the upper phase into a fresh
microfuge tube, being careful to avoid the interface. The
interface contains amphipathic substances such as proteins
associating with both the aqueous phase above and the
organic phase below. The DNA is dissolved in the upper,
aqueous phase.

flocculent interface (figure 31.5). Dispose of the
phenol waste in an approved receptacle. Extract
the sample again with an equal volume of
chloroform, centrifuging briefly to separate the
phases. Always retain the upper phase and avoid
the interface.

7. DNA precipitation: Pipette 70 jlxI of 3M sodium
acetate into the sample and mix well. To the mix
sample, add an equal volume of isopropanol
(700 jil). Mix well by shaking.

8. Centrifuge for 5 minutes at full speed. Look for
the pellet as you remove the tube from the
centrifuge (figure 31.6). Even if your pellet is not

Figure 31.6 The pellet of DNA should be visible as a
tiny white clump. Even if you do not see a pellet, the DNA
is likely present at the bottom back wall of the tube.

visible at this point, DNA is likely present.
Remove as much of the liquid as you can with a
Pasteur pipette, being careful not to disturb the
DNA pellet. If you do not see a pellet, avoid the
back bottom wall of the tube as you pipette.

9. Wash the DNA pellet by adding about 1 ml of
70% ethanol to the tube. Then remove the ethanol
without disturbing the pellet. If the pellet comes
loose, centrifuge it as in step 8.

10. After removing as much liquid as possible, allow
the pellet to air-dry. The pellet will be difficult to
see once it is dry, but it is there!

1 1 . Suspend the pellet in 50 (il autoclaved distilled
water. Label the tube with your name, the date,
and the name of the bacterial strain you used.
Store the samples in the freezer, or proceed to the
next step.

12. Label two microfuge tubes with your initials.
Then label one tube "EcoRI." EcoRI is the name
of the enzyme you will be using to digest the
DNA. Label the other tube "control."

13. Transfer 20 jil of your DNA sample into
each tube. Add 10 jil of restriction mix to
the tube labeled "EcoRI" and 10 pi of the
no- enzyme control mix to the tube labeled
"control" (table 31.1).

14. Mix each sample well by gently pipetting up and
down, and place both tubes in a 37°C heat block
or water bath for one hour. Samples can also be
left overnight at 37°C.

15. Store the samples in the freezer until it is time for
the electrophoresis step.

Alexander-Strete-Niles:

VII. Bacterial Genetics

31. Bacterial DNA Isolation

©The McGraw-Hill

Lab Exercises in
Organismal and Molecular
Microbiology

and Southern Analysis

Companies, 2003

Bacterial DNA Isolation and Southern Analysis EXERCISE 31 231

Second Session: Agarose Gel
Electrophoresis, Staining,
and Southern Transfer

Working with one or two other groups, prepare
one gel. Weigh out 0.4 g of agarose, and place it
into a 1 25 ml Erlenmeyer flask. Add 50 ml of
TBE to the flask, and swirl it gently. Using a lab
marker, draw a line on the side of the flask
indicating the level of fluid.

Microwave the mixture for about 1 minute,
checking to make sure it does not boil over. Using
a hot glove, gently swirl the flask, and return it to
the microwave. Heat for 15 seconds, repeating
this until no more flecks of agarose are visible in
the flask. If there has been obvious loss of volume
through evaporation, add hot distilled water to the
flask using the line you drew as a marker. Let the
molten agarose cool until the flask is comfortable
to handle, but still quite warm.

The sample will be hot after
boiling.

While you are waiting for the molten agarose to
cool slightly, prepare the horizontal
electrophoresis chamber according to the
manufacturer's instructions. An example of a
horizontal minigel system is shown in figure 31.7

When the agarose has cooled as described in step
2, pour the molten agarose, and position the
comb. With the long side of the electrophoresis
chamber parallel to the edge of the lab bench, the
comb should be positioned far to the left. It is
important to keep in mind that the samples will
run from the black lead end (the negatively
charged cathode) toward the red lead end (the
positively charged anode).

Figure 31.7 Assembly of a horizontal minigel system
(VWR #CBMGU-202). (a) Place dams securely, (b) With
the electrode connections toward the back, place the comb
so that the comb bar touches the left side dam. Be sure that
the teeth of the comb are about 2 mm above the floor of
the gel platform, and that the comb is level, (c) When the
flask is cool enough to handle, pour the gel.

(a)

(b)

(c)

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31. Bacterial DNA Isolation

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Lab Exercises in
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and Southern Analysis

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232 Section VII Bacterial Genetics

Figure 31.8 Load the agarose gel throught the TBE run-
ning buffer. Insert the micropipette tip just inside the well,
and gently release the sample. Do not release your thumb
from the pipette plunger until you have lifted the
micropipettor out of the running buffer.

5. While the agarose is solidifying, prepare the
"EcoRI" and "control" samples for loading by
adding 6 (il of DNA sample loading buffer. In
addition, obtain a DNA standard sample (one per
gel) such as lambda-Hindlll. Add 6 jil of sample
loading buffer to it.

6. When the gel is solid, gently remove the comb
and the dams, and pour about 250 ml of TBE
into the electrophoresis chamber until the gel is
fully submerged.

7. Set a micropipettor at 35 jil. Pipette 35 jil of
each sample into its designated well as shown
in figure 31.8, changing the micropipette

tip between samples.

8 . Place the lid on the
electrophoresis chamber, and
connect the leads to the power
source. Remember that the DNA
will migrate from the black lead
end toward the red lead end.

9. Set the power source at 90 volts
(constant voltage), and allow the
electrophoresis to proceed for 1
hour. As the gel begins to run,
you will see that the tracking dye
is moving toward the red lead
end. The dye front allows you to
check the progress of the
electrophoresis; it does not stain
the DNA.

10. Wearing gloves and using a spatula, gently
remove the gel from the electrophoresis chamber.
Place the gel into a weigh boat or small dish, and
stain the gel using the DNA Blue Instastain
method. Place a staining sheet over the gel,
firmly running your fingers over the surface
several times. Then place a glass or plastic plate
on top with an empty beaker as a weight, and let
the gel and staining sheet set for 1 5 minutes
(figure 31.9).

1 1 . Remove the staining sheet, and place the gel into
a shallow dish. Add distilled water heated to
37°C, changing the warm water every 10 minutes
until the bands become visible.

12. Examine the banding patterns, comparing the
EcoRI-digested and the uncut samples. Diagram
your results in your laboratory report. Store the
gel wrapped in plastic wrap in the refrigerator, or
proceed to the next step.

1 3 . Cut the gel off above the wells (slice through the
wells), and notch the gel at its lower left-hand
corner (figure 31.10). Measure and record the
dimensions of the gel (length and width).

14. Transfer the gel to a small dish containing
denaturing solution. Be sure that the entire gel is
submerged. Incubate the gel at room temperature
for 15 minutes with occasional agitation.

1 5 . Holding the gel in place with a gloved hand,
pour the denaturing solution into a beaker,
and pour fresh denaturing solution over the
gel, submerging it once again. Incubate the
gel at room temperature for 1 5 minutes with
occasional agitation.

(a)

(b)

Figure 31.9 Stain the agarose gel after electrophoresis with a methylene blue
staining sheet. Make sure there is even contact between the gel and the sheet by
(a) running your fingers over the surface several times and (b) placing a plate
on top with an empty beaker as a weight.

Alexander-Strete-Niles:

VII. Bacterial Genetics

31. Bacterial DNA Isolation

©The McGraw-Hill

Lab Exercises in
Organismal and Molecular
Microbiology

and Southern Analysis

Companies, 2003

Bacterial DNA Isolation and Southern Analysis EXERCISE 31 233

Figure 31.10 Preparation of the gel for capillary
transfer. Cut the gel off above the wells (slice through
the wells), and notch the gel at its lower left-hand corner.
Then measure the length and width of the gel.

16. Holding the gel in place with a gloved hand, pour
the denaturing solution into a beaker, and rinse
the gel briefly with distilled water (collect it from
a carboy). Holding the gel in place with a gloved
hand, pour the distilled water into the sink.

17. Pour neutralization solution into the dish. Be sure
that the entire gel is submerged. Incubate the gel
at room temperature for 1 5 minutes with
occasional agitation.

1 8 . Holding the gel in place with a gloved hand,
pour the neutralization solution into the sink,
and pour fresh neutralization solution over
the gel, submerging it. Incubate the gel at
room temperature for 1 5 minutes with
occasional agitation.

19. During the incubation steps, 14-18, prepare
materials for transfer:

a. Wearing clean gloves, cut a piece of

nitrocellulose the same size as the gel. Use a
razor blade on a cardboard surface. Keep your
cut membrane on a clean surface. Notch the
membrane at the same position that you
notched the gel (lower left-hand corner). Write
your initials and the date on the bottom edge
with a ballpoint pen.

Note: The nitrocellulose
membrane should be handled
with clean gloves throughout the
Southern procedure.

b. Using scissors, cut two pieces of Whatman
3MM chromatography paper that are the same
size as the gel, and two pieces of paper that are
1 cm larger than the gel in each dimension.

c. Cut several paper towels the same size as the
gel (a 2-inch stack when compressed).

20. Wet the nitrocellulose membrane by flotation
in a small dish containing lOx SSC. Once it
is wet, submerge it.

21. When the gel has been neutralized (after step 18),
set up the transfer as shown in figure 31.11.
Allow capillary transfer to proceed overnight.

(a)

Weight

Stack of dry paper towels

2 pieces of 3MM paper,
same size as gel

Nitrocellulose, same
size as gel; align notch

Notched gel, placed facedown

2 pieces of 3MM paper,
larger than gel

Sponge saturated with 10 x SSC

Dish containing 10 x SSC

(b)

Figure 31.11 Southern transfer by capillary blotting, (a) Diagram and (b) photograph of the transfer apparatus. The denatured
DNA will migrate from the gel onto the nitrocellulose membrane as the salt solution is taken up by capillary action.

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VII. Bacterial Genetics

31. Bacterial DNA Isolation

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Lab Exercises in
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Microbiology

and Southern Analysis

Companies, 2003

234 Section VII Bacterial Genetics

Preparation for the Third Session:
Disassembly of the Capillary Transfer
Apparatus and Membrane Baking

1. Disassemble the transfer apparatus: Throw away
the wet paper and gel, and air-dry the
nitrocellulose membrane by leaving it on a clean
piece of Whatman paper for about 20 minutes.

2. Bake the membrane at 80° C for 1 hour,
sandwiched between two pieces of clean
Whatman paper with a glass weight on top. Store
the membrane, now called the blot, the same way
at room temperature.

Third Session: Probe Preparation and
Southern Hybridization

A summary of the following steps is presented in
table 31.3.

1 . DNA probe labeling

a. The probe is pBLU cut with Hindlll. Note:
Make one probe for every two blots. Obtain

1 jig of Hindlll-cut pBLU DNA suspended in
16 (il of dH 2 0. Boil this sample for 10 minutes
(or use heat block at 100°C) to denature the
DNA. The pBLU DNA molecules must be
denatured so they are free to anneal to the
random primers and to act as a template for
DNA synthesis.

b. After the 10-minute denaturation step, give
the tube a quick spin, and immediately place
it on ice.

c. Add 4 jil of DIG-High Prime (labeling mix) to
the denatured DNA, and mix well by gently
pipetting up and down. Incubate the sample

1 hour at 37°C.

2. Prehybridization

a. While the probe labeling reaction is going on,
wet the nitrocellulose membrane containing
DNA by floating it on 2x SSC. Once it is
completely wet, submerge it in the 2x SSC.

b. Transfer 10 ml of hybridization solution (table
31.2) into a 50 ml conical tube, and place into
a 42°C water bath.

Table 31 .3 The Steps in Southern Hybridization and Development (in Brief)

Step

Description

DNA probe labeling

Prehybridization

Hybridization

Washing

Antibody incubation

Development

Single-stranded (denatured) DNA is used as template for the synthesis of
labeled DNA. The primers for synthesis are random hexanucleotides,
expected to anneal at random sites along the DNA

During synthesis, dGTP, dATP, dTTP, and dCTP are incorporated along with
Digoxigenin-dUTP (the label).

The probe DNA must be denatured by boiling prior to hybridization.

The membrane with denatured DNA bound to it is submerged in hybridization
solution without the labeled probe. This step helps block the membrane to
prevent nonspecific binding of the DNA probe directly to the membrane.

The digoxigenin-labeled DNA probe is added to the membrane in

hybridization solution. During hybridization, which typically proceeds
overnight, the single- stranded DNA probe binds with complementary
sequences of DNA bound to the membrane.

Washing the membrane removes nonspecifically bound probe.

Antibodies specific for the digoxigenin group bind to digoxigenins along the
DNA probe. The antibodies are covalently linked to an enzyme, alkaline
phosphatase (AP).

The membrane, now containing labeled probe hybridized at specific sites, is
placed into a colorless substrate, BCIP/NBT, which is converted to a
colored product by the enzyme AP Color appears only at sites where AP-
antibody is located, and the AP-antibody is located wherever digoxigenin
(probe) is hybridized.

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31. Bacterial DNA Isolation

©The McGraw-Hill

Lab Exercises in
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Microbiology

and Southern Analysis

Companies, 2003

Bacterial DNA Isolation and Southern Analysis EXERCISE 31 235

Figure 31.12 Place the blot into a 50 ml conical tube
containing warm DIG-Easy hybridization solution with the
DNA side toward the center of the tube.

c. Place the blot into the 50 ml conical tube
containing warm DIG-Easy hybridization
solution with the DNA side toward the center
of the tube (figure 31.12).

d. Place the securely capped conical tube on a
rocker platform covered with bench-coat
absorbent paper. Incubate with rocking at 42°C
until the probe is ready (30 minutes).

3. Hybridization

a. Heat the labeled pBLU probe for 10 minutes in
a boiling water bath.

b. Give the tube a quick spin, and add 10 (il to
the 50 ml conical tube containing your blot
and hybridization buffer. Return the conical
tube to the oven, and incubate at 42°C with
rocking until the next session.

Fourth Session: Washing
and Blot Development

Steps 1-3 are designed to remove nonspecifically
bound probe. Steps 4-10 are designed for membrane
development

1 . Remove the hybridized blot from the oven, and
turn the oven temperature up to 68 °C.

2. Decant the hybridization solution into a waste
receptacle, and wash the blot by adding 40 ml of
2x SSC, 0.1% SDS wash to the conical tube.
Keeping the tube at room temperature, mix it
occasionally over the course of 5 minutes.
Decant the solution, and repeat the wash with
fresh 2x SSC, 0.1% SDS.

3. Decant the 2x SSC, 0.1% SDS wash, and add 40
ml of warmed 0.5x SSC, 0.1% SDS to the
conical tube. Return the tube to the oven, now at
68°C, for 15 minutes with rocking. Decant the
solution, and repeat the wash with fresh 0.5x
SSC, 0.1% SDS.

4. Place the blot into a weigh dish with the DNA
side up. Wash the membrane with 20 ml of buffer
1 containing 0.3% Tween-20 for 1 minute at
room temperature with rocking.

5. Holding the blot in place with a gloved hand,
decant buffer 1 /Tween-20. Transfer 50 ml of
buffer 2 into the dish, covering the blot
completely. Incubate the blot for 30 minutes at
room temperature with rocking.

6. Decant buffer 2, and transfer 20 ml of prepared
antibody (alkaline phosphatase-conjugated anti-
digoxigenin antibody diluted 1:5,000 in buffer 2
into the dish, covering the blot. Incubate at room
temperature for 15 minutes with rocking.

7. Decant the antibody, and wash the blot with 50
ml of buffer 1 + Tween-20 for 15 minutes at
room temperature with rocking. Repeat with fresh
buffer 1.

8. Decant buffer 1 + Tween-20, and add 20 ml of
buffer 3. Gently swirl the dish for 2 minutes.

9. Decant buffer 3 and transfer 10 ml of freshly
prepared substrate solution (200 jil NBT/BCIP
stock in 10 ml buffer 3). Place the dish in a dark
place such as a drawer. No rocking is necessary.

10. Within 3 to 10 minutes, purple-gray bands should
appear on the blot. When bands have developed,
but before the membrane itself begins to discolor,
stop the reaction by adding 50 ml of buffer 4 to
the dish. After 5 minutes, decant the solution, and
add distilled water to the dish. Pick up the blot,
and place it on a clean piece of Whatman paper,
allowing it to air- dry. Store the membrane flat,
sandwiched between two pieces of Whatman
paper, with a weight on top.

1 1 . Record your results in your laboratory report.

Alexander-Strete-Niles:

VII. Bacterial Genetics

31. Bacterial DNA Isolation

©The McGraw-Hill

Lab Exercises in
Organismal and Molecular
Microbiology

and Southern Analysis

Companies, 2003

Alexander-Strete-Niles:

VII. Bacterial Genetics

31. Bacterial DNA Isolation

©The McGraw-Hill

Lab Exercises in
Organismal and Molecular
Microbiology

and Southern Analysis

Companies, 2003

Name

Lab Section

EXERCISE

Laboratory Report

Date

Bacterial DNA Isolation and Southern Analysis

1. Diagram the banding pattern of your stained gel (or place a photograph of your gel here). Number
each lane of the gel. Below the gel diagram or photo, list the lane numbers and what you loaded
into each lane.

2. Describe any differences you see in the restriction enzyme-digested sample compared with the
control sample.

237

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VII. Bacterial Genetics

31. Bacterial DNA Isolation

©The McGraw-Hill

Lab Exercises in and Southern Analysis Companies, 2003

Organismal and Molecular

Microbiology

238 Section VII Bacterial Genetics

3. If you completed the Southern portion of the lab, diagram your results in the blank space in question 1,
right, and indicate the contents of each lane. Can you distinguish /^cZ-specific restriction fragments? If
so, how many fragments do you see? Do you think that the probe hybridized to other regions of DNA in
the genome or to the bacteriophage lambda DNA fragments? If so, this is known as nonspecific
hybridization.

4. The rate at which a DNA fragment migrates on a gel during electrophoresis is inversely proportional to
the log of its molecular weight. Given this fact, where on the gel are the largest fragments, and where are
the smallest fragments?

5. If DNA from a cell is cut with a restriction enzyme and loaded onto a typical agarose gel, only a smear
of DNA is seen on a stained gel. How does using the Southern technique overcome this limitation?

6. In a Southern blot, the consequences of not denaturing the DNA in the gel are the same as the conse-
quences of not boiling the probe before adding it to the hybridization solution. Please explain.

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

VII. Bacterial Genetics

32. Mutagenesis in
Bacteria: The Ames Test

Mutagenesis in Bacteria: The Ames Test

An animal or plant cell becomes cancerous when it
accumulates mutations that lead to unregulated cell
division, chromosomal instability, and/or the inability
to undergo normal cell death (apoptosis). Therefore,
any natural or synthetic agent that damages DNA is a
potential carcinogen. In 1971, Dr. Bruce Ames devel-
oped a rapid method for identifying mutagens — and so,
potential carcinogens — using a special strain of Sal-
monella enterica (formerly S. typhimurium). The strain
has two features that make it ideal as a sensor for muta-
gens. First, it lacks DNA repair enzymes so that mis-
takes in DNA synthesis are not corrected. Second, it
carries a point mutation that renders it a histidine aux-
otroph (his~)\ it is unable to synthesize this amino acid
from ingredients in its culture medium. In the presence
of a mutagen, reversions or back mutations to the his
phenotype occur at a high rate, and the revertants are
easily identified.

In the Ames test, the auxotrophic strain is exposed
to a test chemical and cultured on a nutrient medium
containing only a small amount of histidine. The his~
cells can survive until their histidine is used up. Cells
that have reverted to the his + phenotype continue to
grow even in the absence of exogenous histidine. The
number of colonies on the test plate is therefore pro-
portional to the efficiency of the mutagen. For example,
as shown in figure 32.1, substance A produced a higher
frequency of reversion than the control, while substance
B did not. The results suggest that substance A is a
mutagen but substance B is not.

This bacteria-based mutagenesis test provides a
fast, inexpensive way to identify potential carcinogens.
It is important to note, however, that some substances
that cause cancer in laboratory animals are not muta-

Figure 32.1 An example of Ames test results. The
concentration of the amino acid histidine is limiting in
each plate, so only his + revertants grow. The control plate
is at the center. Substance A produced a higher frequency
of reversion than the control, while substance B did not.
The results suggest that substance A is mutagenic and
substance B is not.

genie in the Ames test, and some substances identi-
fied as mutagens in the Ames test do not appear to
cause cancer. Some chemicals (called pro-mutagens)
are not mutagenic unless they are converted to more
active derivatives by liver enzymes. For example,
benzo[a]pyrene is not mutagenic, but it is converted
by liver enzymes to diolepoxides, which are potent
mutagens and carcinogens. Therefore, to test for
pro-mutagens, an extract of rat liver enzymes is usually
included in the Ames test.

Since Salmonella is pathogenic in humans, we will
be using a harmless strain of E. coli that is auxotrophic
with respect to histidine (and thiamine) as our mutagen-
sensor strain. Although this strain is not optimized for
mutagenesis (it is capable of DNA repair), the princi-
ple of the test is the same. In addition, we will not
include liver enzymes in the test, so we will not be test-
ing for pro-mutagens.

239

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

VII. Bacterial Genetics

32. Mutagenesis in
Bacteria: The Ames Test

240

Section VII Bacterial Genetics

Materials

Cultures

Overnight culture of E. coll strain AB 3612

in nutrient broth
or 5. typhimurium, Ames test strain

All agents In red are BSL2 bacteria

Media

1 minimal medium agar plates (per group)
40 plates:

Sodium phosphate dibasic, 6 g
Potassium phosphate monobasic, 3 g
Sodium chloride, 0.5 g
Ammonium chloride, 1 g
15 g agar

1 liter distilled H 2

After autoclaving, add 50 ml warmed, sterile
40% glucose, and swirl gently to mix.
Reagents

lOx thiamine solution (20 mg/ml)
Sterile distilled water (for water-soluble solids
to be tested)

Chloroform (for water-insoluble solids
to be tested)

70% ethanol in a shallow dish

Test substances provided in the
laboratory (such as diethyl sulfate,
4-nitro-o-phenylenediamine or sodium
nitrite) and those supplied by students (such
as household products) . The effects of UV
radiation can also be tested if a UV lamp is
available, along with UV-safe goggles
and gloves.

Equipment

37°C incubator with shaker platform
Bunsen burner

Miscellaneous supplies

Sterile Pasteur pipettes/bulb or

transfer pipettes
Microfuge tubes (~8)
1 .0 ml serological pipette/pipettor
Micropipettors/tips (100-1,000 jil)
Spreader
Sterile forceps

Sterile filter paper disks (0.75 cm diameter)
Laboratory marker

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

VII. Bacterial Genetics

32. Mutagenesis in
Bacteria: The Ames Test

Mutagenesis in Bacteria: The Ames Test EXERCISE 32 241

Procedure

Obtain ten minimal medium agar plates : four
plates for each substance you are testing
(2 substances) and two control plates. Pipette
0.1 ml of lOx thiamine solution onto each of the
minimal medium agar plates. Distribute the liquid
as evenly as possible with a sterilized spreader
(figure 32.2).

Once the plates have dried, pipette 0.1 ml of the
overnight culture of E. coli strain AB 3612 onto
each plate with a sterilized spreader. Spread the
cells as evenly as you can. Label the plate
bottoms with your name(s) and the date.

While the plates are drying, prepare two
substances that you wish to test for mutagenicity.
If the material is a solid, weigh out 1 mg using an
analytical balance, place it into a microfuge tube,
and dissolve it in 1 ml of sterile, distilled water.
Note: If the substance does not dissolve in water,
weigh out another milligram, and dissolve it in
1 ml chloroform. If the substance is a liquid,
record its concentration, if known.

Prepare dilutions of both liquids: For each
substance to be tested, label three microfuge
tubes with the name of the test substance, and
number them 2, 3, and 4 (tube 1 is the original,
undiluted sample). Pipette 1.0 ml of the
appropriate diluent (chloroform or sterile water)
into the tubes numbered 2, 3, and 4. If you use
chloroform, keep the tubes capped. Then, using
a micropipettor, transfer 1 \i\ of the undiluted

liquid sample into tube 2, and mix well. Using
the same tip, transfer 1 jil of sample from tube
2 into tube 3, and mix well. Using the same tip,
transfer 1 \\1 of sample from tube 3 into tube 4,
and mix well. Repeat this series of dilutions on
the second liquid substance.

Label the plates the same way you labeled the
microfuge tubes (1-4 and substance name). Label
the two remaining plates "dry disk control" and
"solvent control."

6. Using sterile forceps, place a sterile filter paper
disk at the center of each plate (figure 32.3).

7 . Using a sterile Pasteur pipette or transfer pipette,
add 1 drop of a liquid sample to the center of the
filter paper disk on the corresponding plate. The
filter paper should be saturated but not dripping
wet. If needed, add additional sample, drop by
drop, until the paper is saturated. Count the
number of drops you use.

8. To the "dry disk control" plate, add no liquid. To
the "solvent control" plate, add either sterile
water or chloroform, drop wise, as in step 7. If
you used both solvents, choose just one, but be
sure that someone else in the class performs the
other solvent control.

9. Place the plates into the 37°C incubator, inverted.
Be sure that the disk continues to adhere to the
agar. Incubate the plates for 2 days (the plates
will then be stored in the refrigerator).

10. Examine your plates, and record the results in
your laboratory report.

Figure 32.2 Spread 0.1 ml (lOOul) lOx thiamine solu
tion onto a minimal medium agar plate.

Figure 32.3 Place a sterile filter paper disk at the center
of the agar plate.

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

VII. Bacterial Genetics

32. Mutagenesis in
Bacteria: The Ames Test

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

VII. Bacterial Genetics

32. Mutagenesis in
Bacteria: The Ames Test

EXERCISE

Laboratory Report

Name

Date

Lab Section

Mutagenesis in Bacteria: The Ames Test

1. Complete the following data tables.

Test substance A:

Substance description:

Concentration
(if known)
in mg/ml:

Sample
number

Sample
dilution

Total

material

tested (mg)

Description of results
(number and distribution of any colonies)

Mutagenic at
this level?

Test substance B:

Substance description:

Concentration
(if known)
in mg/ml:

Sample
number

Sample
dilution

Total

material

tested (mg)

Description of results
(number and distribution of any colonies)

Mutagenic at
this level?

243

Alexander-Strete-Niles:

VII. Bacterial Genetics

32. Mutagenesis in

Lab Exercises in Bacteria: The Ames Test Companies, 2003

Organismal and Molecular
Microbiology

244 Section VII Bacterial Genetics

2. Briefly discuss the results of the Ames test for each of the substances you tested in light of the data you
have gathered, comparing these results with the controls.

3. An overnight culture is expected to be at the stationary phase of growth and at a density of about
10 9 cells/ml. Given this, approximately how many cells did you plate initially?

4. If you detected revertant colonies on any of the plates, select one plate, and do the following
a. Calculate the approximate surface area (cm 2 ) of the region where the colonies appear.

b. What percentage of the total plate surface area does the affected area (determined in 4a) represent?

c. Given your answer to question 3, calculate the number of cells that you plated in the affected area.

d. What is the "mutagenesis efficiency" of this substance, expressed as the number of revertant colonies
per total number of cells plated?

5. What conclusion would you reach if you observed the following: 20 scattered colonies around the disk
of a test plate and 18 scattered colonies around the disk of the "solvent control" plate.

Alexander-Strete-Niles:

VII. Bacterial Genetics

33. Plasmid Isolation and

©The McGraw-Hill

Lab Exercises in
Organismal and Molecular
Microbiology

Restriction Mapping

Companies, 2003

Plasmid Isolation and Restriction Mapping

The capacity of a bacterium to respond to environmen-
tal conditions, to reproduce, or to cause disease depends
on the expression of its genes. Most of the genes are
located on a single, circular, double- stranded DNA
(deoxyribonucleic acid) molecule — the bacterial chro-
mosome (see Exercise 31). However, some bacteria
also harbor several copies of a much smaller circular,
double-stranded DNA molecule called a plasmid, or
episome (figure 33.1). Plasmids contain genes that are
not necessary for day-to-day metabolic processes, but
that confer specialized functions, such as the ability to
transfer DNA to another bacterium (in the case of a
plasmid called a fertility factor, or F factor) or to pro-
duce toxins or antibiotic resistance factors.

Plasmids are particularly valuable to a bacterium
because they can be present in multiple copies. While a
single bacterium has just one chromosome (or two, if it
is about to undergo binary fission), it can have as many as
200 copies of a plasmid. Thus, plasmids can offer as many

as 200 copies of a gene encoding an antibiotic resistance
factor, for example. Plasmids can be replicated because
they have a site for DNA polymerase binding, called the
origin of replication (ORI or rep). They are replicated
more rapidly than the chromosome because they are so
much smaller. Figure 33.2 presents a comparison of the
features of plasmid and bacterial chromosomal DNA.

Figure 33.1 Electron micrograph of a plasmid.

E. coli bacterial chromosome

Plasmid

Double-stranded, circular

One origin of replication

Length: 4,669,221 base pairs

Antibiotic resistance gene(s)

Restriction sites throughout

Engineered restriction sites

Figure 33.2 A comparison of
plasmid and bacterial chromosome
features. The highlighted boxes are
features common to both.

245

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VII. Bacterial Genetics

33. Plasmid Isolation and

©The McGraw-Hill

Lab Exercises in
Organismal and Molecular
Microbiology

Restriction Mapping

Companies, 2003

246

Section VII Bacterial Genetics

While plasmids occur naturally, they have also been
modified for use in gene cloning and gene transfer. Such
plasmids have useful marker genes (including antibiotic
resistance genes) and restriction sites for the insertion
of foreign DNA. A particular restriction site is recog-
nized by a particular restriction endonuclease, a type of
enzyme that occurs naturally in bacteria, which protects
bacteria from foreign DNA, typically bacteriophage
(virus) DNA. Thus, the term restriction endonuclease
makes sense; the enzyme restricts the growth of viruses
in bacteria by cutting double- stranded DNA (nuclease)
within a DNA molecule (endo). There are now hundreds

of restriction enzymes available for research. Just a few
of them, along with their recognition sites, are listed
in table 33.1.

In this exercise, you will isolate the plasmid
pBR322 from E. coli, cut the plasmid with two differ-
ent restriction enzymes, electrophorese the restriction
fragments on an agarose gel, analyze the gel to deter-
mine the fragment sizes (lengths in base pairs), and for-
mulate a restriction map, showing the relative
positions of the restriction sites and the distances
between each site. A genetic map of pBR322 is pre-
sented in figure 33.3

Table 33.1

Examples of Restriction Endonucleases and Their Recogn

ition Sites*

Restriction

Bacterial source

Recognition site

DNA ends resulting

enzyme

from restriction

BamHI

Bacillus amyloliquefaciens H

— GGATCC—

— G GATCC—

— CCTAGG—

— CCTAG G—

EcoRI

Escherichia coli

— GAATTC—

— G AATTC—

— CTTAAG—

— CTTAA G—

Hindlll

Haemophilus influenzae Rd

— AAGCTT—

—A AGCTT—

— TTCGAA—

— TTCGA A—

Nml

Nocardia rubrai

— TCGCGA—

— TCG CGA—

— AGCGCT—

— AGC GCT—

PstI

Providencia stuartii

— CTGCAG—

— CTGCA G—

— GACGTC—

— G ACGTC—

*Each restriction enzyme was isolated from bacteria, and each recognition site is composed of a molecular palindrome; it reads the same on
the upper strand left to right as on the lower strand right to left. The positions at which the enzyme cuts the DNA are indicated by an arrow (T).
Notice that the restriction enzyme cuts at equivalent positions on the two strands.

Alexander-Strete-Niles:

VII. Bacterial Genetics

33. Plasmid Isolation and

©The McGraw-Hill

Lab Exercises in
Organismal and Molecular
Microbiology

Restriction Mapping

Companies, 2003

Plasmid Isolation and Restriction Mapping EXERCISE 33 247

pBR322

4,361 base pairs

Figure 33.3 A genetic map of the plasmid pBR322
which confers resistance to both tetracycline and ampi-
cillin. The region called rep contains the origin of
replication The gene rop encodes a protein that helps
regulate replication.

First Session: Plasmid Isolation
("Plasmid Miniprep")
and Restriction Digestion

Cultures

E. coli RR1 (wild- type strain, transformed with
pBR322), grown on an ampicillin-
containing LB agar plate (1 plate per pair)

Media

LB agar: 10 g bacto-tryptone, 5 g yeast
extract, 10 g NaCl, 12 g agar per liter,
ampicillin 100 (ig/ml

Reagents

Solution I: 25 mM Tris-Cl, pH 8.0, 50 mM

glucose, 10 mM EDTA
Solution I with lysozyme, 4 mg/ml
Solution II: 0.2 N NaOH, 1.0% SDS
Solution III: 3 M potassium acetate (120 ml
5M potassium acetate plus 23 ml
glacial acetic acid, bring volume to 200 ml)

Isopropanol

TE/RNase: Tris-EDTA (10 mM Tris, pH 8.0,

1 mM EDTA), 50 jag/ml RNase, heat-treated

to denature DNAses
Restriction endonuclease EcoRI and restriction

endonuclease PstI, prepared as restriction

mixes E, P, and E+P (table 33.2)

Equipment

Microcentrifuge

Vortexer

37°C heat block or water bath

Miscellaneous supplies
Laboratory marker
Latex gloves (to protect DNA from

deoxyribonucleases on hands)
Ice

1.5 ml microfuge tubes
Pasteur pipettes/bulb
1 .0 ml serological pipette/pipettor
Micropipettors/tips (1-10 jil, 10-100 jil,

100-1,000 |il)
Plastic ruler

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VII. Bacterial Genetics

33. Plasmid Isolation and

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Lab Exercises in
Organismal and Molecular
Microbiology

Restriction Mapping

Companies, 2003

248

Section VII Bacterial Genetics

Second Session: Agarose
Gel Electrophoresis

Reagents
Agarose
TBE:Tris-Borate-EDTA (108 g Tris-base, 55 g

boric acid, 40 ml 0.5 M EDTA, pH 8.0,

bring volume to 1 liter)
DNA sample loading buffer (tracking dyes):

0.25% bromphenol blue, 0.25% xylene

cyanol, 30% glycerol in distilled water
DNA standard, lambda- Hindlll, 1 jag per 25 |il

TBE; one per gel
DNA Blue InstaStain™

Equipment

Microwave oven

Horizontal gel electrophoresis system and
power source

Miscellaneous supplies
Latex gloves

Micropipettors/tips (1-10 jal, 10-100 jal)
1 25 ml Erlenmeyer flask
Bacterial waste beaker
Semilog graph paper

First Session: Plasmid Isolation
and Restriction Digestion

1 . Pipette 200 jil of solution I into a 1 .5 ml
microfuge tube. Scrape a large loopful of E. coli
RR1 (wild- type) cells from the plate, and mix
into the solution, tapping the loop in order to
release the cells. Mix the sample until there are
no clumps by pipetting up and down or by
vortexing the capped tube.

2. Centrifuge the sample for 15 seconds in a
microcentrifuge to pellet cells.

3 . Pipette off the liquid supernatant above the pellet.
Remove as much of the liquid as you can, and
discard it into a bacterial waste beaker.

4. Add 200 (ll of solution I containing lysozyme (4
mg/ml) to the cell pellet. Pipette up and down to
resuspend the cells well.

5. Place the sample on ice for 1 minute.

6. Add 400 (ll of solution II. Mix gently by inverting
several times, and place on ice for 1 minute.

7. Add 300 jal of solution III (cold). Cap the tube
securely, and vortex the sample for 1 seconds at
the highest setting. You will see a white

Figure 33.4 A flocculent precipitate forms after the
addition of solution III and vortexing.

precipitate forming. Place the sample on ice for 5
minutes. This is bacterial chromosomal DNA,
RNA, proteins, and bacterial debris (figure 33.4).

8. Centrifuge the sample at 14,000 RPM for 5
minutes at 4°C.

9. Using a Pasteur pipette, transfer the supernatant
(which contains the plasmid) into a fresh 1.5 ml
centrifuge tube, avoiding bacterial debris. If you
transfer debris, centrifuge the sample a second
time in the fresh tube. In the end, transfer the
precipitate- free supernatant into a fresh tube.

10. Add 700 jil of isopropanol (a volume equal to the
sample volume). Shake vigorously. After about 1
minute, centrifuge the sample for 5 minutes.

1 1 . Look for a tiny white pellet as you remove the
tube from the centrifuge. It will be located on the
back side of the tube bottom. Using a Pasteur
pipette, remove and discard the supernatant,
avoiding the plasmid pellet.

12. Wash the pellet with a little squirt of 70% ethanol,
then pipette the ethanol back off and discard it,
being careful not to discard the pellet. If the pellet
begins to float, centrifuge the sample again.

1 3 . Remove as much liquid (ethanol) as possible, and
air- dry the pellet.

14. Suspend the DNA pellet in 50 jil TE containing
RNAse (50 |ig/ml).

15. Label tube with your name, the date, and the
contents (pBR322).

1 6 . Pipette 7 jil of plasmid DNA into each of three
fresh microfuge tubes, and place them on ice.
Label the tubes E (for EcoRI), P (for PstI) and
E+P (for both). Write your initials on the tubes as
well. The remaining DNA can be stored in the
refrigerator or freezer.

17. Add 23 jil of restriction enzyme mix to each
plasmid sample as shown in table 33.2.

Alexander-Strete-Niles:

VII. Bacterial Genetics

33. Plasmid Isolation and

©The McGraw-Hill

Lab Exercises in
Organismal and Molecular
Microbiology

Restriction Mapping

Companies, 2003

Plasmid Isolation and Restriction Mapping EXERCISE 33 249

Table 33.2 Components of the Restriction Mix. Add 23 jal of each restriction mix to the

corresponding reaction tube.

Restriction

mix
components

TubeE

TubeP

Tube E + P

23 ^1E
restriction mix

23 |Lil P
restriction mix

+23 |nl E + P
restriction mix

lOx restriction buffer

3 pi

3 Ml

Sterile distilled water

19 pi

19 Ml

18 Ml

EcoRI

1 pi

1 Ml

PstI

1 Ml

Total restriction mix

23 |Lil

23 Ml

Total reaction volume

with 7 jil plasmid

30 Ml

1 8 . Mix the sample well by pipetting up and down
gently a few times. If needed, centrifuge for a
moment to bring the liquid to the bottom of the tube

19. Incubate the samples at 37°C for at least 1 hour.
They can be left longer, but should not be left
overnight. After incubation, store the digested
DNA in the refrigerator or freezer.

Second Session: Agarose
Gel Electrophoresis

1 . Weigh out 0.4 g of agarose, and place it into a
125 ml Erlenmeyer flask. Add 50 ml of TBE to the
flask, and swirl it gently. Using a lab marker, draw
a line on the side of the flask indicating the level
of fluid. Microwave it about 1 minute, checking to
make sure it does not boil over. Return the flask to
the microwave, and heat again as needed until
there are no more flecks of agarose in the flask. If
there has been obvious loss of volume through
evaporation, add hot distilled water to the flask
using the line you drew as a marker. Let the
molten agarose cool until the flask is comfortable
to handle, but still quite warm.

2. While the agarose is cooling, prepare the
horizontal electrophoresis chamber according to
the manufacturer's instructions (see figure 31.7).

3 . When the molten agarose has cooled slightly,
pour the gel and position the comb. With the long
side of the electrophoresis chamber parallel to the
edge of the lab bench, the comb should be
positioned far to the left. It is important to keep
in mind that the samples will run from the black

lead end (the negatively charged cathode) toward
the red lead end (the positively charged anode).

4. The agarose will solidify as it cools, within about
15 minutes. While the gel is solidifying, prepare
your samples for loading. You have three digests,
labeled E, P, and E + P. To each of these tubes,
add 6 M^l of sample loading buffer. In addition,
prepare a sample of undigested pBR322 by
pipetting 7 (J.1 into a fresh microfuge tube, adding
23 ^il TBE and 6 p.1 of sample loading buffer.

5 . Each gel must also contain a size marker, or
DNA standard. The standard (here, lambda [X]
DNA cut with Hindlll) is a set of fragments of
known lengths (figure 33.5). Later you will use
the standard to deduce the lengths of your
restriction fragments. Add 6 ^1 of sample loading
buffer to a 24 ^1 sample of standard.

6. When the gel is solid, gently remove the comb
and the dams, and pour about 250 ml of TBE
into the electrophoresis chamber until the gel
is fully submerged.

7. Set a micropipettor at 35 (il. Load 35 ^il of
each sample into its designated well, changing
the micropipette tip between samples. Load

in this order:

pBR322

Lane:

Sample:

DNA

standard

X-Hindlll

E
EcoRI
digest

E+P
EcoRI and
PstI double

digest

P
PstI

digest

Undigested
plasmid

Alexander-Strete-Niles:

VII. Bacterial Genetics

33. Plasmid Isolation and

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Lab Exercises in
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Microbiology

Restriction Mapping

Companies, 2003

250

Section VII Bacterial Genetics

X-Hindlll fragments

23,130

9,416

6,557

4,361

12.

tawf

■

2,322
2,027

Figure 33.5 Bacteriophage lambda ( X ) DNA digested
with Hindlll yields eight restriction fragments. Because
the lengths of the fragments are known, they can be used
as a size standard. Here, the two smallest fragments are
not visible.

8. Place the lid on the electrophoresis chamber, and
connect the leads to the power source. Remember
that the DNA will migrate from the black lead
end toward the red lead end.

9. Set the power source at 80 volts (constant
voltage), and allow the electrophoresis to proceed
for about 1 hour. As the gel runs, you will see that
the tracking dyes are moving toward the red lead
end as well. The dye fronts allow you to check
the progress of the electrophoresis. The dye does
not indicate the position of DNA fragments.

10. After 1 hour, turn off the power. Wearing gloves
and using a spatula, gently remove the gel from
the electrophoresis chamber. Place the gel onto a
piece of plastic wrap, and stain the gel using the
DNA Blue InstaStain method. Place a staining
sheet over the gel, firmly running your fingers
over the surface several times. Then place a glass
or plastic plate on top of the gel with an empty
beaker as a weight, and let the gel and staining
sheet set for 15 minutes.

1 1 . Remove the staining sheet, and place the gel into
a shallow dish. Add distilled water heated to
37°C, changing the warm water every 10 minutes
until the bands become visible. Gels can be left to
destain overnight.

Using a plastic ruler, measure and record the
distance migrated (cm) by each of the standard
fragments (in the lamba-Hindlll lane). Be sure to
use the same start point for each measurement,
such as the top end of the gel or the bottom of
the well. Record each value in your laboratory
report. Then measure and record the distances
migrated by your restriction digest fragments in
each lane: E, E + P, and P.

Using a piece of semilog paper, graph the
standard. Plot the distance migrated by each
standard fragment on the x (linear) axis versus the
log of its length (in base pairs) on the y (log)
axis. When you use log paper, you do not need to
calculate log. Alternatively, you may use a
graphing program to plot the data.

Draw the best straight line. Do not include the
data points from the largest two standard
fragments (23,130 and 9,416). An example of a
semilog plot is shown in figure 33.6.

Using the distances you recorded for each of the
restriction fragment bands, determine their
lengths using the standard graph. Include this
graph in your laboratory report.

■ -n fcn&of tf1,'_/^w* R:-Ju,.-t. (TjrJ ttf**Qm0it'i*/iidL{t)

—4-_ . . . M_

-i.i - J K m L-i h-n

J*BjtffAttrt <ftW*ff*C fmt)

Figure 33.6 Graph of migration distances (cm) versus
length in base pairs for the DNA size standard, lambda-
Hindlll. The red line shows that the length of an unknown
fragment can be deduced from its migration distance. Here,
a fragment that migrates 1.5 cm is deduced to be approxi-
mately 2,500 base pairs long.

Alexander-Strete-Niles:

VII. Bacterial Genetics

33. Plasmid Isolation and

©The McGraw-Hill

Lab Exercises in
Organismal and Molecular
Microbiology

Restriction Mapping

Companies, 2003

EXERCISE

Laboratory Report

Name

Date

Lab Section

Plasmid Isolation and Restriction Mapping

1 . Complete the following table of DNA standard fragment lengths and migration distances based
on your measurements.

Lambdaphage DNA Hindlll
standard fragment lengths

(base pairs)

Migration distance (cm)

2. Using the semilog paper provided, graph the standard fragment lengths versus migration distances as
described in the second session, step 13.

3. List the migration distances of the band or bands you measured in each of the pBR322 digest lanes.

EcoRI
digest

EcoRI and PstI

double-digest

PstI

digest

Note: Your gel includes an undigested sample of pBR322 in lane 5. Be sure to compare the bands you see in the digested
lanes with those in the undigested lane. If a band in a digested lane matches a strong band in the undigested lane, it may
be incompletely digested plasmid and should be ignored.

251

Alexander-Strete-Niles:

VII. Bacterial Genetics

33. Plasmid Isolation and

Lab Exercises in
Organismal and Molecular
Microbiology

Restriction Mapping

Companies, 2003

252 Section VII Bacterial Genetics

4. You can make some predictions about the restriction map of pBR322. Draw a circle
representing a plasmid.

a. If you cut a circular DNA at one position, how many fragments will be generated? _

b. If you cut a circular DNA at two positions, how many fragments will be generated?

5. Consider each of the pBR322 digest results.

EcoRI

How many fragments or bands do you see?

Based on this, how many EcoRI sites are there in pBR322?
EcoRI + PstI

How many fragments or bands do you see?

PstI

How many fragments or bands do you see?

Based on this, how many PstI sites are there in pBR322?

6. Briefly state what you know about the restriction map of pBR322 at this point

7. Determine the lengths of each of the pBR322 digest fragments using the standard graph you have already
prepared. In the space provided, list the fragment lengths for each digest. For each, total the fragment
lengths to obtain the total length of the plasmid. Each of the three totals should agree.

EcoRI fragment(s)

EcoRI + PstI fragment(s)

PstI fragment(s)

Sum of fragment
lengths in each
digest (bp)

8. Using the circle you drew in number 4, draw a restriction map of pBR322 providing:

• the total length of the plasmid (in base pairs)

• the relative positions of the EcoRI and PstI sites

• the distance between these sites (in base pairs)

You can also include the origin of replication and the two antibiotic resistance genes in your map as
shown in figure 33.3. Here are some hints on the placement of these sequences: The ampicillin resistance
gene is about 1,000 base pairs long, and the PstI site is located within this gene. The tetracycline
resistance gene is about 1,200 base pairs long and is located about 300 base pairs to one side of the
EcoRI site.

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34. Acquiring Antibiotic
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Bacterial Transformation

Acquiring Antibiotic Resistance Through
Bacterial Transformation

By the 1970s, it appeared that many once-devastating
infectious diseases had been all but defeated by antibi-
otics and highly effective, preventive vaccines. Viral
diseases such as poliomyelitis and smallpox were
well under control owing to intensive immunization
programs, and bacterial diseases such as tuberculosis
(TB) were effectively treated with antimicrobial drugs.
Since then, these victories, including the eventual erad-
ication of smallpox by 1980, have been greatly tem-
pered by the recent, sharp rise in the rate of infectious
diseases worldwide.

Many factors have contributed to the emergence
and spread of old, new, and more virulent infectious
agents, including climate change, environmental degra-
dation, mass movement of displaced people, interna-
tional travel, poverty, and the lack of public health
measures and surveillance. At the same time, of course,
microorganisms have adapted and thrived in new hosts
and environments. The adaptability of microbes can be
seen in the alarming rise in antibiotic-resistant strains
of bacteria, a trend that has been fueled by the misuse
of antibiotics in recent decades, and the general sense
that bacterial infections are treatable and are therefore
of little consequence. In fact, we commonly encounter
bacteria that are resistant to more than one antibiotic,
the so-called multidrug-resistant, or MDR strains.

A bacterium can acquire resistance to an antibiotic
by random, spontaneous mutation within its genome, or
by taking in whole antibiotic resistance factor-encoding
genes from other microbes. Bacteria can take up foreign
genes in one of three ways: transduction, conjugation,
or transformation. In transduction, a piece of bacter-
ial DNA is transported from one bacterium to another
by a bacteriophage, a virus that infects bacteria. Con-
jugation involves the direct transfer of DNA from one
bacterium to another through an appendage called the sex
pilus. This mode of natural gene transfer is described in
more detail in Exercise 35. Both conjugation and trans-
duction can result in changes in the recipient cell because
they involve the transfer of genes. You could say that
the recipient cell can be "transformed" from one phe-
notype to another — for example, from being antibiotic-
sensitive to being antibiotic-resistant.

The term transformation, however, is reserved for
the third mode of bacterial gene transfer: the uptake of
free DNA from the surrounding environment. In the late
1920s, the English biochemist Frederick Griffith dis-
covered what came to be known as transformation by
chance while working to develop a pneumonia vaccine.
Griffith found that a nonvirulent form of Streptococ-
cus pneumoniae became virulent by taking up mater-
ial (later shown to be DNA) from dead, virulent
streptococci (figure 34.1).

253

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34. Acquiring Antibiotic
Resistance Through
Bacterial Transformation

254 Section VII Bacterial Genetics

Death
(a)

■

Survival

Heat

Heat-killed S

^j<o

Live R

"transforming factor"
from S

+ Live R

Survival
(b)

11 ss

Live S

Figure 34.1 The transforming principle. Streptococcus pneumoniae exists in two
forms: the S, or smooth, form is highly virulent because it bears a capsule that resists
phagocytosis. On the other hand, the R, or rough, form does not cause disease
because it has no capsule and is readily eliminated by phagocytic cells, (a) When
Griffith inoculated mice with the S strain alone, they succumbed to the infection,
while those infected with the R strain alone remained alive and healthy, (b) He then
inoculated mice with the heat-killed S strain, and as expected, the dead cells could
not establish an infection, and the mice lived, (c) However, when heat-killed S cells
were mixed with live, nonpathogenic R cells and introduced together, the mice suc-
cumbed to the infection, suggesting that the nonvirulent R cells had been transformed
into virulent S cells by taking in genetic material released from the dead S cells.
Twenty years later (1944), Avery McCarty, and McLeod extended these studies and
provided evidence that this genetic material, or "transforming factor," is DNA.

The DNA that enters the cell can remain as a plas-
mid, independent of the chromosome, or it may be
incorporated into the bacterial chromosome. Not all
bacteria can take up free DNA this way. Those that can,
such as Streptococcus pneumoniae, are said to be nat-
urally competent. Other bacteria, including E. coli,
must be treated to become competent for transforma-
tion. E. coli can be made competent by first suspending

the cells in a solution of calcium chloride. The bacter-
ial cell membrane is permeable to chloride ions, but
nonpermeable to calcium ions. As chloride ions enter
the cells, so do water molecules, causing the cells to
swell slightly and become porous. When the cells are
then "heat- shocked" (42°C, 2 minutes), free DNA mol-
ecules such as plasmids are swept through the transient
pores into the cell (figure 34.2).

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34. Acquiring Antibiotic
Resistance Through
Bacterial Transformation

Acquiring Antibiotic Resistance Through Bacterial Transformation EXERCISE 34

H 2 Cl

HoO

Cytosol

Inner
membrane

Outer
membrane

Transient
pore

42C°

e Calcium
ions

Plasmid DNA

Extracellular fluid

Figure 34.2 The transformation of competent cells. The negatively charged
plasmid DNA is associated with calcium ions, while chloride ions and water enter
the cell, causing it to swell slightly. During the heat-shock step, the plasmic DNA
is swept into the cell. Under the conditions of transformation, typically one plasmid
molecule enters a cell.

In this exercise, you will transform antibiotic- sensi-
tive E. coli with the plasmid you isolated in Exercise 33,
pBR322, yielding transformants resistant to ampicillin
and tetracycline (figure 34.3; see figure 33.3 for a map
of the plasmid) . Here, you will select for transformants by
growing the cells on nutrient agar containing ampicillin.
The mechanism of ampicillin action as well as the mech-
anism of resistance to it is shown in figure 34.4.

Materials

Figure 34.3 pBR322 confers resistance to both ampi-
cillin and tetracycline. E. coli transformed with pBR322
grows on an ampicillin- containing plate (left). E. coli
carrying no plasmid does not.

Cultures

E. coli RR1 (host strain) mid-log culture
(10 ml per group)

Media

LB broth: 10 g bacto-tryptone, 5 g yeast

extract, 1 g NaCl per liter
LB agar (12 g/L) plates (100 x 15 mm)

containing ampicillin at 100 jig/ml

(2 per group)
LB agar plates ( 1 00 x 15 mm) with no

antibiotic (2 per group)

Reagents

50 mM calcium chloride (sterile, cold)

TE (Tris-EDTA: 10 mM Tris, pH 8.0, 1 mM

EDTA)
70% ethanol in a shallow dish

Equipment

37°C incubator with shaker platform
Heat block or water bath at 42°C
Buns en burner

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34. Acquiring Antibiotic
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Bacterial Transformation

256 Section VII Bacterial Genetics

(a) Peptidoglycan synthesis

Polysaccharide
chain

Peptide bridge <

Pentapeptide

(b) Ampicillin
inactivates
transpeptidase

Transpeptidase

cleaves alanine
from the pentapeptide,
forming a tetrapeptide
that becomes linked
to a peptide bridge,
joining two adjacent
polysaccharide chains.

Ampicillin

Figure 34.4 The action of ampicillin and ampicillin resistance, (a) Peptidoglycan synthesis, (b) Ampicillin blocks
peptidoglycan synthesis. It contains a beta-lactam ring that binds irreversibly to the bacterial enzyme, transpeptidase,
blocking a key step in peptodoglycan synthesis. Note that ampicillin does not damage already existing peptidoglycan.
(c) The ampicillin resistance factor inactivates ampicillin. It is a beta-lactamase that inactivates ampicillin by breaking
its beta-lactam ring.

Miscellaneous supplies
Ice

Laboratory marker
Latex gloves (to protect DNA from

deoxyribonucleases on hands)
5 ml pipettes/pipettor
Micropipettor/tips (10-100 Jill)
1 .0 ml serological pipettes/pipettor
Spreader

Prior to today's lab, a 2 ml sample of nutrient broth was
inoculated with E. coli RR1 (host strain) for overnight
growth at 37°C with shaking. Earlier today, 100 ml of
nutrient broth was inoculated with the overnight culture
and incubated at 37°C with shaking.

Preparation of Competent Cells (yields
enough for five transformations)

1. Obtain 10 ml of mid-log E. coli RR1 (host strain)
cells in a 15 ml conical tube; place it on ice.

2. Pellet the cells by centrifugation at 2,000 RPM
(1,000 x g) for 10 minutes at 4°C.

3 . Decant the supernatant into a waste receptacle,
being careful not to discard the pellet. Leave a
small volume of broth over the pellet.

4. Tap the tube vigorously to disperse the pellet
in the residual broth. Place the cells on

ice immediately.

5. Add 5 ml of sterile, ice cold 50 mM CaCl 2 to the
cells, resuspending them gently with the pipette.

6. Immediately place the cells on ice for 20 minutes.

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34. Acquiring Antibiotic
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Bacterial Transformation

Acquiring Antibiotic Resistance Through Bacterial Transformation EXERCISE 34 257

7. Centrifuge as in step 2, but for 5 minutes.

8. Decant the supernatant as in step 3. This time, the
cell pellet is softer and more diffuse. Be sure it
doesn't pour out with the supernatant.

9. Resuspend the cell pellet with 1 ml of ice-cold
50 mM CaCl 2 , by pipetting the cells up and down
very gently. Immediately place these cells back
on ice. These are your competent cells.

Transformation of Competent E. coli
with pBR322

1. Label two microfuge tubes, "pBR322" and
no plasmid."

2. Place the two labeled tubes on ice, and transfer
200 jil of competent cells into each of the tubes.
Be sure that the cells are well suspended
before you transfer them!

3. Obtain a sample of the plasmid you isolated in
Exercise 33. Keep it on ice.

4. Transfer 20 jil of plasmid (~1 jig) into your
tube labeled "pBR322." Mix gently, keeping
the cells on ice.

5. Using a fresh micropipette tip, transfer 20 jil of
TE into your "no plasmid" tube. Mix gently,
keeping the cells on ice.

6. Incubate the cells on ice for 20 minutes.

7. While your transformation reactions set on ice,
label two ampicillin plates and two antibiotic-free
plates with your name and the date. Label one
ampicillin plate and one antibiotic- free plate
"pBR322." Label the other ampicillin plate

and the other antibiotic-free plate "no
plasmid control."

8. After the transformation reactions have been on
ice for at least 20 minutes, heat- shock each by
placing them in a 42°C water bath for 2 minutes.
Return them to ice.

9. Add 1 ml of fresh, sterile nutrient broth to each
tube, cap tightly, and tape the tubes, side down,
onto the shaker platform in the 37°C incubator.
Incubate for 40 to 60 minutes with shaking. This
allows the cells to recover from the calcium
chloride and heat- shock treatment before you
plate them. It also allows the antibiotic resistance
gene(s) on the plasmid to begin to be expressed,
before the cells are exposed to ampicillin.

10. When the recovery period is completed, plate the
bacteria by spreading with a sterile spreader:

100 jil of pBR322 Tf reaction onto the ampicillin
plate labeled "pBR322"

100 |il of pBR322 Tf reaction onto the "no
antibiotic" plate labeled "pBR322"

100 jil of "no plasmid" control reaction onto the
ampicillin plate labeled "no plasmid control"

100 jil of "no plasmid" control reaction onto the
"no antibiotic" plate labeled "no plasmid control"

11. Place the inverted plates in the 37°C
incubator overnight.

12. Examine the plates and address the questions in
your laboratory report.

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34. Acquiring Antibiotic
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Bacterial Transformation

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

VII. Bacterial Genetics

34. Acquiring Antibiotic
Resistance Through
Bacterial Transformation

EXERCISE

Laboratory Report

Name

Date

Lab Section

Acquiring Antibiotic Resistance Through Bacterial Transformation

1. Examine each plate. Describe and discuss the results of each with respect to the presence or absence of
bacterial growth, and whether or not the plates with growth contain isolated (individual) colonies. Are the
results what you expected?

2. Count the total number of colonies on each plate that has individual colonies. Note: If the plate is very
crowded, it may be easier to count if you divide the plate into quarters or eighths and then multiply the
count by 4 or 8, respectively. Record these counts here.

3. Transformation efficiency is a measure of the success of transformation. It is expressed as the number of
antibiotic-resistant colonies per \ig of DNA transformed. A typical transformation efficiency is about 10 6
colonies/jig. Using the number of colonies on the ampicillin plate labeled "pBR322," determine the
transformation efficiency. Keep in mind that you transformed 1 \ig of DNA, and that you plated about
one-twelfth of the total transformation reaction (100 jil from 1,200 jil total).

4. Do you think the cells that grew on the ampicillin plate are also resistant to tetracycline?
Why or why not?

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260

Section VII Bacterial Genetics

5. Why are most antibiotics safe for humans and other animals (other than side effects) even though they
can be very harmful to bacteria?

6. How does ampicillin kill ampicillin- sensitive bacteria? How do ampicillin-resistant bacteria avoid being
killed by ampicillin?

7. Why is having an antibiotic resistance gene on a plasmid more beneficial to the bacterium than having
the gene on the bacterial chromosome?

8. An isolated colony represents one cell that landed at that spot on the agar when you spread the bacteria
Why is the colony considered a clone?

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35. Acquiring Antibiotic
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Acquiring Antibiotic Resistance Through
Bacterial Conjugation

Conjugation involves the direct transfer of plasmid or
chromosomal DNA from one bacterium to another via
an extended appendage called the sex pilus, or conju-
gation pilus. The donor cell, also called the male, pos-
sesses a plasmid (fertility factor, or F factor) that
allows the cell to synthesize the sex pilus and to repli-
cate and transport the F factor itself. The recipient cell,
or female, is a closely related strain or species (usually
Gram- negative) that has a recognition site on its surface.

F + (donor) F (recipient)

minutes

2 minutes
Pilus formation

10 minutes

DNA replication with
continued pilus formation

15 minutes
DNA transfer

20 minutes
Conjugates separate

Specialized conjugation plasmids known as resistance
factors, or R factors, not only carry genes that control con-
jugation, but can also carry genes that confer resistance to
antimicrobial drugs. Thus in a single conjugation event, a
recipient cell can receive a "shield" of one or more drug
resistance genes. Later, the recipient can spread that resis-
tance to other cells, again through conjugation. Indeed,
this type of horizontal gene transfer contributes to the
emergence of multidrug-resistant bacteria. Penicillin- and
tetracycline-resistant Neisseria gonorrhoeae is thought to
result from the conjugative transfer of an R factor.

In this exercise, the donor (F + ) strain is Escherichia
coli BB4. Its conjugative plasmid carries a tetracycline
resistance gene, so the plasmid could be called an R fac-
tor. The recipient (F") strain is E. coli SCS 1 . Its chro-
mosome carries the gene for ampicillin resistance.
When the cells are mixed and conjugation occurs, a
copy of the R factor of E. coli BB4 moves to E. coli
SCSI, conferring tetracycline resistance on the recipi-
ent cell (figure 35.1). The success of conjugation can be
measured by the appearance of colonies resistant to
both ampicillin and tetracycline.

Figure 35.1 Bacterial conjugation. In this example, conju-
gation is conferred by a fertility factor that carries a tetracy-
cline resistance gene, (a) During conjugation, a copy of the
R factor of E. coli BB4 moves to E. coli SCSI. The SCSI cell
gains tetracycline resistance, (b) An electron micrograph of
two E. coli cells during conjugation. The F + cell to the right is
covered with small pili, and a sex pilus connects the two cells.

(a)

(b)

261

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262 Section VII Bacterial Genetics

Materials

Cultures

Overnight culture of E. coli BB4 (tetracycline-

resistant donor strain)
Overnight culture of E. coli SCSI (ampicillin-

resistant recipient strain)

Media

Tryptic soy broth (tryptone 1 5 g, soytone 5 g,
sodium chloride 5 g, in 1 liter distilled water)

Tryptic soy agar plates (tryptic soy broth, agar
1 5 g/liter )

with ampicillin at 1 00 jig/ml (one per group)
with tetracycline at 1 5 (ig/ml (one per group)
with ampicillin at 100 jig/ml and

tetracycline at 1 5 (ig/ml (2-4 per group)

Reagents

70% ethanol in a shallow dish

Equipment

37 °C incubator with shaker platform
Bunsen burner

Miscellaneous supplies

1 .0 ml serological pipettes/pipettors

Pasteur pipettes/bulb

1 ml culture tube (one per group)

Spreader

Inoculating loop (for spreading bacteria on a

plate half)
Laboratory marker

1. Using a 1.0 ml serological pipette, transfer 0.1 ml
of the overnight culture of E. coli BB4 (donor
cells) into a culture tube. With a fresh pipette, add
0.9 ml of an overnight culture of E. coli SCSI
(recipient cells) to the donor cells. Add 5 ml of
sterile nutrient broth. Incubate the conjugation
mix at 37°C with shaking. Record the time.

2. Obtain one ampicillin plate and one tetracycline
plate. Using a lab marker, divide each plate in
half by drawing a line on the bottom plate. Label
one half of each plate "donor," and the other half
of each plate "recipient."

3 . Using a sterile Pasteur pipette or a 1.0 ml
serological pipette, transfer one drop of the

E. coli BB4 overnight (be sure that the cells are
well suspended first) onto the "donor" half of the
ampicillin plate and then onto the donor half of

the tetracycline plate. Use a sterile spreader or
sterile inoculating loop to spread the bacteria on
each plate, being careful not to go beyond your
drawn line (figure 35.2).

4. Repeat step 3 using a fresh pipette and
transferring a drop of the E. coli SCSI overnight
on the "recipient" half of each plate.

5 . Near the end of the lab period, * remove the
conjugation mix (from step 1) from the incubator,
and transfer one drop of the culture onto an
ampicillin/tetracycline plate. Use a sterile spreader
to distribute the cells evenly over the plate surface.
Record the time, and return the conjugation mix to
the incubator shaker platform for overnight
growth. Label the plate "conjugation mix," and
write the time on the plate.

*Note: You may be asked to plate the conjugation
mix at earlier time points as well. Label a fresh
ampicillin/tetracycline plate for each time point.

Label all plates with your name(s), the date, and
the time of mating period (on the bottom side,
along the plate edge). Place all the plate cultures
in the 37 °C incubator, inverted, overnight.

6. If possible, repeat step 5 the next day.

7. Examine the plate and record your observations
in your laboratory report. In addition, count and
record the number of colonies on the ampicillin/
tetracycline plate(s).

Figure 35.2 Spread the "donor" half of an ampicillin
plate with E. coli BB4 and the "recipient" half of the plate
with E. coli SCSI. Do the same with a tetracycline plate.

Alexander-Strete-Niles:
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35. Acquiring Antibiotic
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Bacterial Transformation

Name

Lab Section

EXERCISE

Laboratory Report

Date

Acquiring Antibiotic Resistance Through Bacterial Conjugation

1. What was the purpose of plating E. coli BB4 and E. coll SCSI separately on plates containing ampicillin
alone and tetracycline alone? What are the expected results?

2. Diagram and briefly describe the results of the plating addressed in question 1.

Ampicillin

Tetracycline

3. Formulate a table showing

• the number of conjugation mixtures you plated

• the length of the mating period for each

• the number of colonies you counted on each plate

263

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Organismal and Molecular Bacterial Transformation

Microbiology

264 Section VII Bacterial Genetics

4. Discuss the results presented in question 3

5. Approximately what volume of conjugation mixture did you plate at each time point (about how many
microliters are there in a drop of fluid)?

For each time point, what is the conjugation efficiency as expressed in conjugation events per ml?
Keep in mind that each colony represents a single cell.

6. Why was it important to use a recipient cell that contained a stable marker such as ampicillin resistance
in this experiment?

7. If you were to mix the doubly resistant SCSI cells with an appropriate, antibiotic-sensitive recipient
E. coli strain, do you think the recipient strain might become tetracycline resistant? Why or why not?
Do you think the recipient strain might become ampicillin resistant? Why or why not?

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Microbiology

Acquiring Antibiotic Resistance Through Bacterial Conjugation EXERCISE 35 265

8. The cells that survive on the ampicillin/tetracycline plates are E. coli SCSI, not E. coli BB4.
How do you know this?

9. Two E. coli strains, mating pairs X and Z, are mixed. X is F + , harboring an R factor that confers

resistance to penicillin. Z is F~ and is sensitive to all antibiotics. However, Z has been engineered with a
gene that makes its colonies appear blue. After 10 minutes, the cells are placed into a blender at high
speed to disrupt the conjugates.

One drop of the mating mixture is spread on a plate containing no antibiotic, and another drop is
spread on a plate containing penicillin. Describe the growth you expect on each plate.
Briefly explain your answer.

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VIM. Viruses

36. Viral DNA

Lab Exercises in
Organismal and Molecular
Microbiology

Fingerprinting

Companies, 2003

DNA Fingerprinting

Recall that during the second half of the nineteenth cen-
tury, Robert Koch, Louis Pasteur, and others began to
identify the microorganisms that cause a number of
infectious diseases. The researchers relied on the fact
that each microbe could be collected in a bacteria-proof
filter, grown in nutrient medium, and observed by light
microscopy. However, the causes of a number of trans-
missible diseases, such as foot-and-mouth disease,
rabies, and smallpox, remained a mystery. By the dawn
of the twentieth century, it had become clear that dis-
eases such as these are associated with a fundamentally
different kind of infectious agent — one that is smaller
than bacteria (it could not be retained in filters of that
time, and it cannot be seen by light microscopy) and
is incapable of reproduction outside of cells (cannot
be grown in laboratory media).

A virus is an obligate intracellular parasite con-
sisting of nucleic acid (an RNA or DNA genome) con-
tained within a coat of proteins called a capsid (figure
36.1). More complex viruses contain additional struc-

tures, sometimes including a membranous envelope
studded with protein spikes, or peplomers (figure 36.2).
The power of a virus, an inert, nonliving agent, is in
its capacity to enter and be replicated within its host
cell. As a result of one virus entering a single cell, hun-
dreds of newly formed viruses may be released as the
cell dies. Each virus, or virion, is then capable of infect-
ing a nearby cell, effectively extending the cellular
injury and leading to symptoms that may arise from the
infection or from the immune response to it. In humans,
viruses cause a number of diseases, including smallpox
(eradicated as of 1980), the common cold, chickenpox,
influenza, poliomyelitis, rabies, ebola hemorrhagic
fever and AIDS. A few viruses have even been linked to
the development of cancer.

In recent years, a number of methods have been
developed for the rapid identification of microbes
through DNA fingerprinting, or DNA typing. A DNA
fingerprint is a ladder of fragmented or newly synthe-
sized DNA molecules that form a barcode-like pattern
unique to an organism. The key to a DNA fingerprint,
then, is having an identifiable pattern of bands on a

s -I- ■• ■.-.-. - -.

■if- 2 ':":■••• ..-■ ".<

*ra-^gfrifigra

r.- w- ..■<* S i WMT3 X. vt i ?

(a)

Capsid

(b)

Figure 36.1 An adenovirus consists of a DNA genome surrounded by a capsid. Adenoviruses
cause upper respiratory infections, such as the common cold, and other infections, such as pink-
eye, (a) Electron micrograph of an adenovirus, (b) Computer- simulated model of an adenovirus.

268

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36. Viral DNA

Lab Exercises in
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Microbiology

Fingerprinting

Companies, 2003

Viral DNA Fingerprinting EXERCISE 36 269

Peplomers

Lipid envelope

50 nm

(a)

(b)

Figure 36.2 An influenza virus consists of an RNA genome surrounded by a capsid enclosed in a membranous
envelope. The envelope contains viral proteins called peplomers, or spikes, (a) Electron micrograph of influenza virus
particles, (b) Diagram of an influenza virus.

gel. As you learned in Exercise 31, gel electrophoresis
of cellular DNA that has been cut with a restriction
endonuclease generates a smear of restriction fragments
on a gel. In Exercise 33, however, you saw how the elec-
trophoresis of smaller DNAs, such as plasmids or bac-
teriophage DNAs, cut with restriction enzymes, can
result in a discernible pattern. Among the methods used
to generate DNA fingerprints of cellular DNAs are
pulsed-field gel electrophoresis (PFGE), the poly-
merase chain reaction (PCR), and Southern blotting
and hybridization. In Exercise 31, you learned how a

Southern blot is used to detect particular DNA
sequences within a complex genome, such as that of
E. coll. PFGE and PCR are outlined in figure 36.3.

In this exercise, we will take advantage of the
smallness of viral genomes, and generate DNA finger-
prints simply through restriction enzyme digestion fol-
lowed by agarose gel electrophoresis and staining. It
will be possible to identify a simulated, unknown "clin-
ical sample" by comparing its restriction pattern with
those of known viral DNA samples.

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270

Section VIII Viruses

(a) Pulsed-field gel electrophoresis (PFGE)

Isolation of DNA from tissues, cells, or viruses: The DNA is

mechanically sheared during this procedure, generating large
fragments.

Restriction enzyme digestion: The large fragments of DNA
are cut at specific sites with a restriction enzyme, generating
restriction fragments characteristic of the organism.

Agarose gel electrophoresis: Very long fragments of DNA
(from 40 kb to 5 Mb) are separated by size using alternating
electric fields.

Field A on, field B off: DNA migrates on end, parallel to field A,
toward the anode (DNA has a net negative charge).

Field B on, field A off: DNA migrates on end, parallel to field B,
toward the anode.

Alternate fields several times.

As in standard gel electrophoresis, the distance migrated by a
fragment during electrophoresis is inversely proportional to its size
However, PFGE conditions allow for very long fragments to
separate by size so they can be distinguished as bands on a stained
gel. The restriction fragment lengths, unique to a particular
microbe, provide the organism's DNA fingerprint.

Field A
cathode (-)

Field B
anode (+)

Field B
cathode (-)

Field A
anode (+)

Stained gel

Longer
fragments

Shorter
fragments

Figure 36.3 Pulsed-field gel electrophoresis and the polymerase chain reaction can be used to generate DNA fingerprints
without Southern blotting, (a) PFGE: During typical gel electrophoresis, DNAs longer than 30 kb migrate with the same
mobility regardless of size. However, if the DNA is made to change direction during electrophoresis, as in PFGE, these larger
fragments can separate from each other. Through PFGE, it is possible to resolve fragments as long as 40,000-5,000,000 base
pairs, (b) PCR: Using DNA encoding 16S rRNA as a template, 16S rDNA primers can be used to generate a set of synthe-
sized products that is unique to a bacterial species, for example.

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Viral DNA Fingerprinting EXERCISE 36 271

(b) The polymerase chain reaction (PCR)

Isolation of DNA from tissues, cells, or viruses: The DNA is

mechanically sheared during this procedure, generating large
fragments.

DNA denaturation: The DNA is subjected to a high temperature
so that it becomes single-stranded. Once it is single-stranded, it
can act as a template for DNA synthesis.

95°C

Primer annealing: In order for DNA synthesis to begin, a primer
(a short strand of DNA or RNA), must be base-paired to the
template, and provide a 3 ' hydroxyl at the end of its sequence.
DNA polymerase can then link a series of nucleotides to the
primer, one after another, all complementary to the template strand

Since the primer must base-pair to the template, the particular
sequence of the primer dictates where synthesis begins. The two
primers depicted here base-pair at repetitive sequences on both
strands. Repetitive sequences are short, highly conserved stretches
of DNA that are present throughout the genomes of all bacteria
tested so far. However, the distances between repetitive sequences
differ from strain to strain.

50°C

= Primer, base-paired to
rRNA gene sequences,
providing a 3 ' OH group
for DNA polymerization

DNA synthesis (primer extension): DNA polymerase adds
nucleotides, complementary to the template.

Repeat denaturation, annealing, and DNA synthesis about
30 times.

Agarose gel electrophoresis: The PCR products are separated by
size, generating a fingerprint that can be used to identify and
compare bacterial strains.

70°C

After multiple cycles, rRNA
gene sequences are amplified

Many copies
of each product

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272 Section VIII Viruses

Materials

First Session: Restriction Digestion

Reagents

pBR322 containing viral genomes (prepared
as knowns and as "clinical samples").
(These DNAs should be transformed into
E. coli for storage and propagation, and
isolated from the cells in preparation for
this exercise.)
pBRSV SV40 virus DNA
pAM6 hepatitis B virus (HBV) DNA
pHPV-18 human papilloma virus

(HPV) DNA
Restriction endonuclease, EcoRI prepared

as a restriction mix (see table 33.2)

All agents in red are BSL2 bacteria.

Equipment

Microcentrifuge

Vortexer

37°C heat block or water bath

Miscellaneous supplies
Laboratory marker

Latex gloves (when handling DNA samples)
Ice

1.5 ml microfuge tubes
Pasteur pipettes/bulb
1 .0 ml serological pipette/pipettor
Micropipettors/tips (1-10 jil, 10-100 jll,
100-1,000)11)

Second Session: Agarose Gel
Electrophoresis and Staining

Reagents

Agarose

TBE: Tris-Borate-EDTA (108 g Tris-base,
55 g boric acid, 40 ml 0.5 M EDTA,
pH 8.0, bring volume to 1 liter)

DNA sample loading buffer (tracking dyes)
0.25% bromphenol blue, 0.25% xylene
cyanol, 30% glycerol in distilled water

DNA standard, lambda- Hindlll, ljig per
30 \x\ TBE; one per gel

DNA Blue InstaStain™

Equipment

Microwave oven

Horizontal gel electrophoresis system and
power source

Miscellaneous supplies

Latex gloves (to protect DNA from

deoxyribonucleases on hands)
Micropipettors/tips (1-10 jal, 10-100 jll)
1 25 ml Erlenmeyer flask
Bacterial waste beaker
Plastic ruler
Semilog paper

First Session: Restriction Digestion

1 . Pipette 7 jll of S V40 DNA into a microfuge tube,
and place it on ice. Do the same for the hepatitis
B DNA and the human papilloma virus DNA. In
addition, obtain 7 jll of an unknown, "clinical
sample," and place it on ice.

2. Add 23 |il of restriction enzyme mix to each of
the four viral DNA tubes.

3 . Using a different micropipette tip for each
sample, mix well by gently pipetting up and
down. If needed, centrifuge for a moment to
bring the liquid to the bottom of the tube.

4. Incubate the samples at 37°C for at least 1 hour.
They can be left longer, but should not be left
overnight. After incubation, store the digested
DNA in the refrigerator or freezer, or proceed to
the next step.

Second Session: Agarose Gel
Electrophoresis and Staining

1 . Weigh out 0.4 g of agarose, and place it into a
1 25 ml Erlenmeyer flask. Add 50 ml of TBE to the
flask, and swirl it gently. Using a lab marker, draw
a line on the side of the flask indicating the level of
fluid. Microwave it about 1 minute, checking to
make sure it does not boil over. Return the flask to
the microwave, and heat again as needed until there
are no more flecks of agarose in the flask. If there
has been obvious loss of volume through
evaporation, add hot distilled water to the flask
using the line you drew as a marker. Let the molten
agarose cool until the flask is comfortable to
handle, but still quite warm.

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Viral DNA Fingerprinting EXERCISE 36 273

2. While the agarose is cooling, prepare the
horizontal electrophoresis chamber according to
the manufacturer's instructions (see figure 31.7).

4. The agarose will solidify as it cools, within about
15 minutes. While the gel is solidifying, prepare
your samples for loading. To each of your four
samples, add 6 jil of sample loading buffer.

5 . Each gel must also contain a DNA standard (see
figure 33.5). Later you will use the standard to
deduce the lengths of your restriction fragments.
Add 6 (il of sample loading buffer to a 30 \i\
sample of standard.

6. When the gel is solid, gently remove the comb
and the dams, and pour about 250 ml of TBE
into the electrophoresis chamber until the gel is
fully submerged.

7. Set a micropipettor at 36 jil. Load 36 jil of
each sample into its designated well,
changing the micropipette tip between
samples. Load in this order:

EcoRI digests

~~\

Lane:

Sample: DNA standard

X-Hindlll

SV40 HBV HPV Clinical

DNA DNA DNA DNA sample

8. Place the lid on the electrophoresis chamber, and
connect the leads to the power source. Remember
that the DNA will migrate from the black lead
end toward the red lead end.

9. Set the power source at 80 volts (constant
voltage), and allow the electrophoresis to proceed
for 2 hours. As the gel runs, you will see that the
tracking dyes are moving toward the red lead end
as well. The dye fronts allow you to check the

progress of the electrophoresis. The dye does not
indicate the position of DNA fragments.

10. After 2 hours, turn off the power. Wearing gloves
and using a spatula, gently remove the gel from
the electrophoresis chamber. Place the gel onto a
piece of plastic wrap, and stain the gel using the
DNA Blue InstaStain method. Place a staining
sheet over the gel, firmly running your fingers
over the surface several times. Then place a glass
or plastic plate on top of the gel with an empty
beaker as a weight, and let the gel and staining
sheet set for 15 minutes (see figure 31.9).

12. Using a plastic ruler, measure and record the
distance migrated (cm) by each of the standard
fragments (in the lamba-Hindlll lane). Be sure to
use the same start point for each measurement,
such as the top end of the gel or the bottom of
the well. Then measure and record in your
laboratory report the distances migrated by your
restriction digest fragments in each of the other
lanes: SV40 DNA, HBV DNA, HPV DNA, and
the "clinical sample."

1 3 . Using a piece of semilog paper, graph the
standard. Plot the distance migrated by each
standard fragment on the x (linear) axis versus the
log of its length (in base pairs) on the y (log)
axis. When you use log paper, you do not need to
calculate log. Alternatively, you may use a
graphing program to plot the data.

14. Draw the best straight line. Do not include the
data points from the largest two standard
fragments (23,130 and 9,416). For an example of
a semilog plot, see figure 33.6.

15. Using the distances you recorded for each of the
restriction fragment bands, determine their
lengths using the standard graph.

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36. Viral DNA

Lab Exercises in
Organismal and Molecular
Microbiology

Fingerprinting

Companies, 2003

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36. Viral DNA

Lab Exercises in
Organismal and Molecular
Microbiology

Fingerprinting

Companies, 2003

EXERCISE

Laboratory Report

Name

Date

Lab Section

Viral DNA Fingerprinting

1 . Examine the restriction patterns of the three known samples and the single "clinical sample"
and compare them. Can you identify the source of the unknown sample DNA? If so, what is it?

2. Complete the following table of DNA standard fragment lengths and migration distances based
on your measurements.

Lambda-Hindlll
standard fragment lengths

(base pairs)

Migration distance (cm)

3. Graph the standard fragment lengths versus migration distances using semilog paper or a
graphing program.

4. List the migration distances of the band or bands you measured in each of the EcoRI digest lanes
Using the standard graph, deduce the size of each.

SV40 DNA

HBV DNA

HPV DNA

Clinical DNA sample

Migration

distance

(cm)

Deduced
length

(bp)

Migration

distance

(cm)

Deduced
length

(bp)

Migration

distance

(cm)

Deduced
length

(bp)

Migration

distance

(cm)

Deduced
length

(bp)

275

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276 Section VIII Viruses

5. Choose one of the DNAs from the table in question 4, and draw a restriction map of it. The map is
circular, as in a plasmid restriction map. Include the following in the restriction map:

• the total length of the plasmid (in base pairs)

• the relative positions of the EcoRI

• the distance between these sites (in base pairs)

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37. Isolation of

Lab Exercises in
Organismal and Molecular
Microbiology

Bacteriophage from
Sewage and Determination
of Phage Titer

Companies, 2003

solation of Bacteriophage from Sewage and
Determination of Phage Titer

Virtually any type of cell is susceptible to virus infec-
tion; viruses cause disease in plants and animals, and
can also infect procaryotes and unicellular eucaryotes.
Viruses that infect procaryotes are known as bacterio-
phages, or phages, because when they were first dis-
covered, they appeared to eat bacterial cells, generating
a clearing, or plaque, on a lawn of susceptible bacte-
ria. In reality, the bacteria are killed by lysis as newly
produced phages are released from the damaged cells.

Like all viruses, bacteriophages consist of nucleic
acid (RNA or DNA) surrounded by a protein coat, or
capsid. Unlike some plant and animal viruses, bacte-
riophages are not enveloped. Some phages have elab-
orate structures for attaching to the bacterial surface and
injecting nucleic acid into the cytoplasm. A diagram
of one such bacteriophage, T4, is shown in figure 37.1.

Most bacteriophages are lytic; that is, each infec-
tion event leads to the production of new virions and the
death of the cell by lysis. Some bacteriophages — most
notably bacteriophage lambda (A) — are categorized as
temperate. Sometimes X DNA is integrated into the bac-
terial chromosome, with its genes largely silent. The
infected cell survives as a lysogen. In some X infections,
the DNA remains independent of the host chromosome,
and is replicated many times over; its genes are
expressed at high levels, and newly assembled phages
are released. The "choice" between a lysogenic, non-
productive infection and a lytic, productive infection
depends on environmental conditions. For example, UV
exposure can cause a X infection to switch from lyso-
genic to lytic. A typical lytic bacteriophage infection
cycle is depicted in figure 37.2.

110 nm

ds linear DNA

Neck

Collar
Whiskers

Sheath

Internal tail tube

Tail fiber

Base plate
with pins

(a)

(b)

Figure 37.1 Bacteriophage or "phage" T4, a DNA virus
of E. coll. (a) Diagram of phage T4. (b) Electron micro-
graph of phage T4 particles.

277

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37. Isolation of

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Microbiology

Bacteriophage from
Sewage and Determination
of Phage Titer

Companies, 2003

278 Section VIII Viruses

(T) Attachment
(adsorption)

(2) Entry

(3) High-level gene
expression
(protein synthesis)

(4J Genome
replication

(5) Assembly

(V) Release

Transcription
Translation

dt>9

Virus proteins

Linear DNA
enters and
circularizes

Synthesis of
multiple copies
of circular DNA
and further
gene expression

Shift to synthesis
of linear DNA

(a)

(b)

Figure 37.2 The infection cycle of bacteriophage T4. (a) This kind of infection is "productive" because new viruses are
produced. The steps listed on the left are generally applicable to any productive virus infection, (b) Electron micrograph of
E. coli infected with phage T4 (36,500x).

In this exercise, we will focus on the bacteriophages
of coliform bacteria. Coliform bacteria are relatively
harmless microorganisms that live in large numbers in
the intestines of mammals, where they aid in the diges-
tion of food. Escherichia coli is a common fecal col-
iform bacterium. The presence of fecal coliform
bacteria in water indicates that it has been contaminated
with human or other animal feces, and that a potential
health risk exists for those who use the water. Raw,
untreated sewage contains large numbers of E. coli.
Therefore, we will use raw sewage as a source of bac-
teriophages that infect E. coli.

In this exercise, you have the opportunity to:
(1) amplify (increase the numbers of) phages in the

sewage sample by allowing them to infect and reproduce
within fresh E. coli, (2) collect the phages from the cul-
ture by centrifugation and filtration, and (3) detect and
titer the amplified, isolated phages using a plaque assay.
The assay is based on the fact that each plaque on a lawn
of bacteria, although it contains 10 6 to 10 7 virions along
with bacterial debris, represents a single infecting phage
that entered one cell at the start of the culture. The infec-
tion then "spread" as the viruses reproduced and cells
lysed, eventually forming a visible plaque (figure 37.3).
The titer of a phage suspension, therefore, is determined
by counting the number of plaques that form from a given
volume of suspension. Phage titer is expressed as plaque-
forming units (PFU) per milliliter (ml) .

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Microbiology

Bacteriophage from
Sewage and Determination
of Phage Titer

Companies, 2003

Isolation of Bacteriophage from Sewage and Determination of Phage Titer EXERCISE 37 279

Bacteriophage

E.coli

(a)

(b)

Figure 37.3 Phage plaques, (a) A lawn of E. coli B containing plaques, (b) Each
clearing or plaque contains 10 6 to 10 7 bacteriophages and bacterial debris, but rep-
resents a single phage that infected one cell at the approximate center of that site.

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Microbiology

Bacteriophage from
Sewage and Determination
of Phage Titer

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280

Section VIII Viruses

Materials

Cultures

Overnight culture of E. coli B
1 (125 ml) Erlenmeyer flask containing 40 ml
of raw sewage

Media

Nutrient broth (1 g Peptone, 0.5 g yeast

extract, 0.25 g NaCl, 0.8 g potassium

phosphate, dibasic in 100 ml

distilled water)
1 Ox strength nutrient broth (Peptone 20 g,

yeast extract 10 g, NaCl 5 g, potassium

phosphate-dibasic 16 g, in 200 ml

distilled water)
Warmed nutrient agar plates (6, 100 x 15 mm

plates) (12-15 g agar/liter nutrient medium)
Tubes containing 3 ml each of warm, top

agarose (one per plate) (7.5 g agarose/liter

nutrient broth, molten, cooled to 45 °C)

Reagents

Phosphate-buffered saline (PBS) (sodium
chloride 1.6 g, potassium chloride
0.04 g, sodium phosphate-dibasic 0.22 g,
potassium phosphate-monobasic 0.04 g
in 1 00 ml)

Equipment

37°C incubator with shaker platform
Water bath at 37°C
Water bath at 45 °C

Miscellaneous supplies
5 ml pipettes/pipettor
1 5 ml conical centrifuge tube
Tube for collection and storage of

phage filtrate
Sterile 0.45 Jim syringe tip filter
1 ml syringe without needle
1.5 ml microfuge tubes for preparing dilutions
1 .0 ml serological pipettes/pipettor or

micropipettor/tips (100-1,000 jll)
Laboratory marker

Procedure

Prior to today's lab, raw sewage was collected from a
local sewage treatment plant. Yesterday, 50 ml of lx
nutrient broth was inoculated with E. coli B for
overnight growth at 37 °C with shaking.

First Session: Amplification
of Bacterial Viruses

1 . Pipette 5 ml of 1 Ox nutrient broth into the flask
containing 40 ml of raw sewage.

2. Inoculate the sewage in the flask with 5 ml of an
overnight culture of E. coli B .

3. Inoculate a separate flask containing 45 ml of lx
nutrient broth with 5 ml of an overnight culture
of E. coli B (one per class).

4. Incubate both cultures at 37 °C, shaking for
24 hours.

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Bacteriophage from
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of Phage Titer

Companies, 2003

Isolation of Bacteriophage from Sewage and Determination of Phage Titer EXERCISE 37 281

Second Session: Bacteriophage
Isolation and Plating:

Prior to today's lab, 2 ml of lx nutrient broth was inoc-
ulated with E. coli B for overnight growth at 37 °C with
shaking. Earlier today, 100 ml of lx nutrient broth was
inoculated with a small volume of the overnight. This
was done to obtain a culture in log growth by class
time. Note: The instructor may choose to inoculate
today's culture with the day-old "overnight" stored in
the refrigerator.

1. Transfer 10 ml of the sewage-bacteria-
bacteriophage culture into a centrifuge tube, and
centrifuge the sample at 2,000 RPM for 5
minutes. Most of the remaining cells will be
pelleted. The supernatant contains bacteriophage.

2. Prepare a 10 ml storage tube for the collection of
bacteriophage supernatant as it is filtered. Then
pipette the supernatant into a 10 ml syringe barrel
fitted with a 0.45 micron filter. Gently slide the
plunger, allowing the flow-through to drip into

the storage tube. This step removes any
remaining bacteria from the phage sample. The
storage tube contains bacteriophage. It can be
stored at 4°C and is stable for several months.

3. Prepare a series of microfuge tubes for making
serial 1 0-fold dilutions of the bacteriophage
suspension (performing the same dilution
repeatedly in series is called serial dilution; see
figure 37.4). Label six tubes 1-6. Into each tube,
pipette 0.9 ml of sterile PBS.

4. Perform serial dilutions: Transfer 0.1 ml of
phage suspension (that has been mixed well) into
tube 1 , and mix. Using the same pipette, transfer
0. 1 ml of the sample from tube 1 into tube 2, and
mix. Repeat this process, transferring 0.1 ml from
tube 2 to tube 3, and so on, mixing each time, as
shown in figure 37.4. Store the remaining phage
suspension in the refrigerator.

5. Distribute 0.5 ml of log-phase E. coli into each of
six microfuge tubes, labeled 1-6.

0.1 ml

0.9 ml
(1:10)

Phage
suspension

0.1ml

+
0.9 ml
(1:10)

0.1 ml

+
0.9 ml
(1:10)

0.1 ml

+
0.9 ml

(1:10)

0.1ml

+
0.9 ml
(1:10)

0.1ml

+
0.9 ml
(1:10)

Final dilution

0.9 ml PBS

1:10

uo- 1 )

1:10 2

(io- 2 )

1:10 3

U<r 3 )

1:10 4

(i<r 4 )

1:10 5

U<r 5 )

Final dilution factor

0.9 ml PBS

1:10 6

(icr 6 )

Figure 37.4 Serial dilutions of bacteriophage suspension. First, pipette 0.9 ml of PBS (diluent) into each dilution tube
(numbered 1-6. Then transfer 0.1 ml of phage suspension in series, mixing each time.

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282 Section VIII Viruses

6. To each tube of bacteria, add 0.1 ml of the
corresponding phage dilution (0. 1 ml of dilution
6 to cell tube 6, and so forth). Note: If you work
from the most dilute to the least dilute, you can
use the same pipette. Cap the tubes, and mix
gently by inverting them.

7. Incubate at 37°C for 10 minutes to allow the
phage to adsorb (attach) to the bacteria. This is
your cell-phage mix.

8. In the meantime, label six warm, dry, nutrient
agar plates 1-6 (one for each infection). Write on
the bottom plate along the plate edge. Keep the
plates in the 37°C incubator until you are ready
to use them.

9. When you are ready to plate cell-phage mixes,
collect your warmed, labeled plates from the
incubator. Add the contents of cell-phage tube 1
to a vial containing 3 ml of top agarose (molten,
at 45 °C). Quickly cap the tube, and mix it by
gently inverting it three times. Quickly pour the
mixture onto warmed plate 1 (figure 37.5). You
can tip the plate slightly to spread the top
agarose. Push the plate aside, but do not pick it
up until the agarose solidifies.

10. Repeat step 9 for each of the remaining five
samples, 2-6.

1 1 . Allow the plates to cool without being disturbed
for approximately 10 minutes. When the top
agarose has solidified, incubate the plates,
inverted, at 37 °C for 24 hours.

Third Session: Examination of
Bacteriophage Plates, Phage Storage

1 . Record the number of plaques on each plate in
your laboratory report.

Using one of the least-crowded plates, pick an
isolated plaque for long-term storage: Pipette 1 ml
of PBS into a microfuge tube, and add 1 drop of
chloroform. Then, using either the large or small
end of a Pasteur pipette (depending on the size of
the plaque and the space around it), pierce the agar
surrounding the plaque, and pick out the agar
"plug" containing the plaque (figure 37.6). Place
the "plug," agar and all, into the 1 ml of PBS. The
phage will diffuse into the PBS over time, and the
chloroform will kill any remaining bacteria. Store
the plaque in the refrigerator (4-1 0°C).

Figure 37.5 Plating phage. Once you have gently mixed
the cell-phage-top agarose suspension by inverting it a few
times, quickly pour the mixture onto a warmed agar plate.

Figure 37.6 Picking a phage plaque for storage. Pierce the
agar surrounding the plaque, and pick out the agar "plug"
containing the plaque. Transfer the plug into a microfuge
tube containing 1 ml of PBS and a drop of chloroform.

Alexander-Strete-Niles:

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37. Isolation of

Lab Exercises in
Organismal and Molecular
Microbiology

Bacteriophage from
Sewage and Determination
of Phage Titer

Companies, 2003

Name

Lab Section

EXERCISE

Laboratory Report

Date

solation of Bacteriophage from Sewage and Determination
of Phage Titer

1. Count the plaques on each plate. Note: If the plate is very crowded, it may be easier to count if you
divide the plate in quarters or eighths and then multiply the count by 4 or 8, respectively. Then complete
the following table.

Plate
no.

Plaques
per plate

Dilution
factor

Volume of

phage plated

(ml)

Titer calculation

(number of plaques) (DF)

volume plated (ml)

Titer:
plaque-forming units

(PFU) per ml

2. Do the results in the far right-hand column agree? Should they agree? What is the average titer of the
amplified, filtered phage suspension?

3. Approximately how many bacteriophages are in the phage filtrate you collected?

4. A protocol calls for 10 9 phage particles as starting material. How much of your phage suspension would
you need to have 10 9 phages?

283

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37. Isolation of

Lab Exercises in Bacteriophage from Companies, 2003

Organismal and Molecular Sewage and Determination

Microbiology of Phage Titer

284 Section VIII Viruses

5. Why is bacteriophage titer expressed as PFU/ml and not bacteriophages/ml?

6. Take a look at one of the phage plates, and comment on the plaques you see with respect to their
appearance and dimensions. Do they all look alike? If two plaques differ in size or shape, what might
that indicate about the bacteriophages in the two plaques?

7. What is the purpose of the amplification step?

8. You used a 0.45 Jim filter to separate bacteriophages from any whole bacteria that remained after
centrifugation. Why was this a proper choice of filter pore size? How big is an E. coll cell? How
big is a typical bacteriophage? What else might be present in the bacteriophage filtrate?

9. You picked a single plaque from a phage plate for long-term storage. It is expected that all of the
phages in the storage tube are identical. Why?

10. Describe and diagram how a bacteriophage plaque arises on a bacterial lawn.

11. Bacteriophage X is a temperate phage. When X is plated with susceptible E. coll, the plaques
are visible but they are cloudy, not clear. Why are the plaques cloudy?

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38. The Virus Infection

Lab Exercises in
Organismal and Molecular
Microbiology

Cycle: The One-step
Growth Curve

The Virus Infection Cycle:
The One-step Growth Curve

The steps described for the bacteriophage T4 infection
cycle (see figure 37.2) are essentially the same for any
type of virus that elicits a productive infection, no mat-
ter what the virus-host combination is. The molecular
details of these steps (adsorption, entry, virus gene
expression, viral genome replication, virion assembly,
and release) are known for a number of bacterial, ani-
mal, and plant viruses.

In this exercise, you will observe the general fea-
tures of a productive viral infection by completing what
is known as a one-step growth curve experiment. One
step refers to the fact that a single round of virus infec-
tion is assessed, and growth refers to the increase in
numbers of virions that results from the round of infec-
tion. The experiment requires a synchronous culture —
in this case, a uniform group of cells that are infected
simultaneously with a uniform preparation of functional
virions. In a synchronous culture the events in the infec-
tion cycle are expected to occur in each cell at nearly
the same time. Therefore, the analysis of the whole cul-
ture over time reflects events occurring in a single cell.

The synchrony required for this kind of experiment
has been achieved only with bacteriophages and their
susceptible bacterial hosts in liquid culture. This is
mainly because bacteriophages are much more efficient
than plant or animal viruses at entering a cell once the
virus has made contact with the cell. While the effi-
ciency of infection for bacteriophages, expressed as a
ratio of virions to infections, is 1 : 1 to 2: 1 (for every one
or two virions present, one is successful), the efficiency
of plant and animal viruses ranges from 4: 1 to 10,000: 1 !

Recall that during adsorption and entry, the virion
attaches to a host cell, and its nucleic acid enters the
cell. In the next phase of infection, the first steps of
virus production occur: Virus genes are expressed, virus
proteins are synthesized, and the viral genome is repli-
cated. This phase is known as the eclipse period,
because new virions are not yet formed; if the cells are
taken from the culture and broken open chemically, no
infectious virions are found. The eclipse period also

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encompasses the bulk of the latent period, except that
the latent period lasts through virus assembly, until the
virions have formed and are released from the cell in the
final phase, called burst. The term burst refers to the
sudden increase in the number of free virions in the cul-
ture, not necessarily to the lysis that occurs in this — and
in many but not all — productive virus infections. A rep-
resentation of the one- step growth curve, based on the
number of free virions in the culture over time, is shown
in figure 38.1. The number of free virions at each time
point is estimated by performing a plaque assay on a
small sample of the liquid culture (figure 38.2).

Free

virions

(PFU/ml)

Adsorption
and entry

Latent period

Eclipse period

Burst

Virus
added

10 15 20 25 30 35 40 45 50

Minutes

Figure 38.1 The one-step growth curve. A schematic
representation of a virus infection cycle. The number of free
virions at each time point is estimated by performing a
plaque assay on a small sample of the culture. Keep in mind
that although eclipse and latent are terms that convey lack of
activity, there is much going on within the cell as virus
genes are expressed at high levels (proteins are synthesized)
and viral nucleic acid is replicated many times over.

285

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VIM. Viruses

38. The Virus Infection

Lab Exercises in
Organismal and Molecular
Microbiology

Cycle: The One-step
Growth Curve

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286

Section VIII Viruses

(a)

(b)

(c)

Figure 38.2 Result of plaque assays (phage plating) completed (a) 20 minutes, (b) 30 minutes, and (c) 40 minutes after
initial infection. Each plate contains 0.1 ml of phage diluted 1:10,000 (10~ 4 ).

Materials

Cultures

Overnight culture of E. coll B

Media

Nutrient broth (1 g Peptone, 0.5 g yeast

extract, 0.25 g NaCl, 0.8 g potassium

phosphate, dibasic in 100 ml distilled water)
Warmed nutrient agar plates (7, 100 x 15 mm

plates) (12-15 g agar/liter nutrient medium)
Tubes (7) containing 3 ml each of warm, top

agarose (7.5 g agarose/liter nutrient broth,

molten, cooled to 45 °C)

Reagents

T4 phage at 10 7 PFU per ml

or phage prepared in Exercise 37

Phosphate-buffered saline (PBS) (1.6 g sodium
chloride, 0.04 g potassium chloride, 0.22 g
sodium phosphate, dibasic, 0.04 g
potassium phosphate, monobasic in 1 00 ml)

Equipment

37°C incubator with shaker platform

(for overnight culture)
Water bath at 45 °C
Water bath at 37°C

Miscellaneous supplies

15 ml conical centrifuge tubes
1 .0 ml serological pipettes/pipettor
or micropipettor/tips (100-1,000 jll)
Laboratory marker
Linear graph paper

Prior to today's lab, 2 ml of nutrient broth was inocu-
lated with E. coll B for overnight growth at 37°C with
shaking. Earlier today, 100 ml of lx nutrient broth was
inoculated with a small volume of the overnight culture.
This was done to obtain a culture in log growth by class
time. Note: The instructor may choose to use the day-
old overnight culture stored in the refrigerator.

1 . Label seven warm, dry, nutrient agar plates with
your name and the date, and label each with a
time point: 20 minutes, 25 minutes, 30 minutes,
35 minutes, 40 minutes, 45 minutes, and 50
minutes. Keep the plates in the 37°C incubator
until you are ready to use them.

2. Pipette 1 ml of the E. coll B culture into a sterile
15 ml conical centrifuge tube, and add 0.1 ml

of bacteriophage (at about 10 7 PFU/ml:
suspension commercially prepared or saved
from Exercise 37). Mix well, and place the
cap on the tube loosely.

3. Place the cell-phage mixture into the 37°C bath,
and record the time. This is time zero. Make a
note of what time it will be 1 9 minutes from now.

4. Incubate the mixture in the bath for 6 minutes.
During this time, the bacteriophages adsorb to
their host cells. Prepare the tubes for the dilutions
you will do in the next step by labeling two 1 5 ml
tubes #1 and #2 and pipetting 9.9 ml of sterile
nutrient broth into each. Cap the tubes.

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38. The Virus Infection

Lab Exercises in
Organismal and Molecular
Microbiology

Cycle: The One-step
Growth Curve

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The Virus Infection Cycle: The One-step Growth Curve

Exercise 38 287

5 . After the 6-minute incubation, centrifuge the
cell-phage "adsorption" culture at 2,000 RPM for
5 minutes. Decant the supernatant into a waste
receptacle, and resuspend the pelleted cells in 1 ml
of fresh, sterile nutrient broth. Dilute the adsorption
culture (infected cells) 10 4 -fold by doing two 100-
fold serial dilutions: Pipette 0.1 ml of the cells into
dilution tube 1 , mix well, and transfer 0.1 ml of
cells from tube 1 to tube 2. Mix well.

6. Place the 10 4 -fold dilution culture into the 37°C
water bath. Check the time. When 19 minutes
have elapsed since "time zero," collect your
warmed, labeled nutrient agar plates, and go to
step 7.

7. Add 2 drops of the remaining E. coll B culture
(from step 2) to a tube containing 3 ml of top
agarose (molten, at 45 °C).

At exactly 20 minutes, transfer 0.1 ml of the 10 4
fold diluted culture to the tube of top agarose.
Quickly cap the tube, and mix it gently by
inverting it a few times; then immediately pour
the mixture onto the warmed plate labeled "20
minutes" (see figure 37.5). You can tip the plate
slightly to spread the inoculated top agarose.

Repeat step 8 at each of the subsequent time
points: 25, 30, 35, 40, 45, and 50 minutes.

Once all the plates have cooled and the top
agarose has solidified, incubate the plates,
inverted, at 37 °C overnight.

Count the plaques on each plate, and record
the data in your laboratory report.

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38. The Virus Infection

Lab Exercises in
Organismal and Molecular
Microbiology

Cycle: The One-step
Growth Curve

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Alexander-Strete-Niles:

VIM. Viruses

38. The Virus Infection

Lab Exercises in
Organismal and Molecular
Microbiology

Cycle: The One-step
Growth Curve

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EXERCISE

Laboratory Report

Name

Date

Lab Section

The Virus Infection Cycle: The One-step Growth Curve

1 . Count the plaques on each plate. Then complete the following table.

Plating
time
point

Number

of
plaques

Dilution
factor

Volume of

phage plated

(ml)

Titer calculation

(number of plaques) (DF)

volume plated (ml)

Titer:

(PFU) per ml

2. Plot the number of PFU/ml versus time on the graph paper provided. Be sure to title the graph,
label the axes, and include the units.

3. Using a different-colored ink, label the same graph with the phases of the virus infection cycle:
adsorption and entry period, eclipse period, latent period, and burst.

4. If you continued to assay this infection culture beyond the 50-minute mark, to 100 minutes or so,
what might the growth curve look like? Keep in mind that there are still plenty of cells remaining after
the first infection cycle. Diagram and label a growth curve representing 100 minutes of phage-cell
interaction, and briefly explain your answer. What would the growth curve look like if all the cells in
the culture were infected in the first round of infection?

289

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38. The Virus Infection

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Organismal and Molecular
Microbiology

Cycle: The One-step
Growth Curve

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290

Section VIII Viruses

5. Step 2 of the procedure called for 0.1 ml of a phage sample at 10' PFU per ml. Approximately how
many bacteriophages did you add to the E. coli B cells?

6. What was the titer of the bacteriophage you isolated in Exercise 37? How many milliliters of this
suspension would you need to obtain the number of phages you calculated in question 5?

7. Why is it necessary to have a synchronous culture in order to formulate a one-step growth curve?
Why is it impossible to generate a one-step growth curve using an animal virus?

8. In Exercises 37 and 38, bacteriophage suspensions were first diluted prior to plating with bacteria in top
agarose. Why are dilutions necessary?

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39. Infection of Plant

Lab Exercises in
Organismal and Molecular
Microbiology

Leaves with Tobacco
Mosaic Virus

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E X E R C I

Infection of Plant Leaves with Tobacco
Mosaic Virus

Background

The tobacco mosaic virus (TMV) is considered a pro-
totype plant virus. In fact, it is perhaps the most studied
and best understood of all the viruses. There are two
reasons for its fame: First, TMV has potential impact on
agriculture because of its wide range of hosts, includ-
ing tobacco, tomato, and potato plants as well as orna-
mentals such as impatiens, geraniums, coleus, and
African violets. Second, TMV is extremely stable — it
is readily isolated from infected plant tissue and eas-
ily stored and maintained for laboratory studies.

TMV, a member of a large group of related viruses
called tobamoviruses, is a rod-shaped, nonenveloped

virus with a single- stranded RNA genome (figure
39.1a). The stability of TMV arises from tightly packed
capsid proteins that make it resistant to conditions that
would destroy most other types of viruses. As you can
see in figure 39.1b, the tight association of TMV cap-
sid proteins results in a rigid structure.

Plant viruses require help to breach the plant cell
wall and gain access to the cytoplasm. This help comes
in the form of prior tissue damage, from insects (in fact,
some plant viruses are insect-transmitted) or from abra-
sions or wounds inflicted on the plant by weathering,
machinery, or tools. Once in the cytoplasm, the virus
replicates, and newly assembled TMV virions move
throughout the plant, infecting most of its cells. The

RNA

Capsid
protein

J I I L

J I I I I I I I L

J I I L

(a)

10 nm

20 nm

(b)

Figure 39.1 The structure of tobacco mosaic virus (TMV). (a) The capsid proteins are arranged in
a helical array, tightly associated with each other and with the RNA genome. The RNA is said to be
"positive sense" because it acts as a messenger RNA as soon as it enters the cell and associates with
ribosomes. (b) Electron micrograph of TMV (400,000x).

291

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39. Infection of Plant

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Organismal and Molecular
Microbiology

Leaves with Tobacco
Mosaic Virus

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292 Section VIII Viruses

(a)

(b)

Figure 39.2 Viral lesions on plant leaves, (a) Tobacco mosaic virus (TMV) on Nicotiana glutinosa. (b) TMV
infection of an orchid showing leaf color changes.

movement of virions to adjacent cells occurs through
plasmodesmata, while movement to distal leaves or
roots occurs via phloem. TMV infection stunts the
growth of its host plant, and causes light and dark mot-
tling (a mosaic pattern) on its leaves (figure 39.2).

In this exercise, you will extract TMV from dried
tobacco leaves and detect the virus by applying it to leaves
of a susceptible living plant such as coleus (figure 39.3).

Materials

Plants and reagents

Young tomato plant {Ly coper sic on esculentum)

or coleus plant {Coleus blumei)
1 g tobacco (from about 2 cigarettes)
Positive and negative control inocula:

TMV-infected and virus-free tobacco

homogenates

Miscellaneous supplies

Sharp knife, scissors, or razor blade

Mortar and pestle

Small piece of 600 grit sandpaper

Small paintbrush

Labeling tape

Laboratory marker

Small weighing dishes

10 ml graduated cylinder or 10 ml pipette

Cheesecloth

Small funnel

50 ml beaker

Figure 39.3 The effects of TMV on Coleus blumei.

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39. Infection of Plant

Lab Exercises in
Organismal and Molecular
Microbiology

Leaves with Tobacco
Mosaic Virus

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Infection of Plant Leaves with Tobacco Mosaic Virus EXERCISE 39 293

Procedure

Obtain two plants. Label both with the date and
the names of your group members. Also label one
plant "TMV inoculation"; it will be inoculated
with the tobacco extract you prepare. The second
plant will serve as either a negative control or a
positive control. Check with your instructor to
determine which control to use, and label the
second plant accordingly.

If cigarettes are to be the source of tobacco, slit
open two cigarettes using a sharp tool, and collect
the tobacco in a small weighing dish.

Using a second weighing dish, weigh out 1 g of
tobacco, and pour it into the mortar.

Add 10 ml of distilled water to the tobacco, and
let it stand for 10 to 15 minutes.

5. Grind the tobacco for a few minutes with the
mortar and pestle.

6. Separate the leaf extract (containing virions) from
the tobacco remnants by filtration: Place two
layers of cheesecloth into a small funnel
positioned over a 50 ml beaker. Pour the contents
of the mortar through the cheesecloth.

7. Make a 1:10 dilution of the extract: Pipette 0.9
ml of distilled water into a microfuge tube; then
pipette 0. 1 ml of extract from the beaker into tube
1 , and mix well.

8. Choose one leaf on each of the plants (TMV-
inoculated and control), and place a small piece
of label tape around the stem of each.

9. Using sandpaper, gently scrape off the surface of
a small area (about the size of a nickel) on each
of the chosen leaves.

10. With the paintbrush, apply either the undiluted
TMV suspension or the 1:10 dilution of tobacco
extract to the scraped area on the leaf of the plant
labeled "TMV-inoculated." Apply the prepared
control homogenate to the second leaf. Note: This
may be a positive control or a negative control.
Be sure to record which you are using.

1 1 . Place the plants in a greenhouse or other
appropriate space, keeping the control plants
away from the infected plants. The virus can be
transmitted from plant to plant if the leaves
touch. Observe the plant leaves every 2 or 3 days
for 14 days and record your observations in your
laboratory report.

Alexander-Strete-Niles:

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39. Infection of Plant

Lab Exercises in
Organismal and Molecular
Microbiology

Leaves with Tobacco
Mosaic Virus

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Alexander-Strete-Niles:

VIM. Viruses

39. Infection of Plant

Lab Exercises in
Organismal and Molecular
Microbiology

Leaves with Tobacco
Mosaic Virus

Companies, 2003

Name

Lab Section

EXERCISE

Laboratory Report

Date

Infection of Plant Leaves with Tobacco Mosaic Virus

1 . Record your observations at selected days after inoculation, commenting on the appearance of the
infected leaf and the control leaves. If your group prepared a negative control plant, take a look at a
positive control done by another group, and vice versa.

Days after
inoculation

TMV-inoculated leaf

Positive control leaf

Negative control leaf

2. What conclusions do you draw, based on the observations recorded in question 1?

3. What was the purpose of rubbing the leaf with sandpaper prior to the infection?

295

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39. Infection of Plant

Lab Exercises in
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Microbiology

Leaves with Tobacco
Mosaic Virus

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296

Section VIII Viruses

4. A plant leaf lesion assay is similar to a plaque assay. In order to titer a suspension of TMV, a researcher
spreads 0.1 ml of a 1:1,000 virus dilution evenly over the surface of a prepared leaf. After 3 weeks, she
counts a total of 22 lesions on the leaf. What is the titer (here, expressed in infectious units [IU] per
milliliter) of the original virus suspension?

5. The RNA genome of TMV is called "positive sense" because it acts as messenger RNA as soon as it
enters the cytosol and associates with the host's translation machinery. Compare the tobacco mosaic
virus and the banana streak badnavirus (a double- stranded DNA virus) with respect to gene expression.

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

IX. Hematology and
Serology

40. Identification and
Enumeration of White
Blood Cells

Identification and Enumeration

of White Blood Cells

In the preceding sections, you have learned how pro-
tozoans, fungi, multicellular parasites, bacteria, and
viruses reproduce within their hosts and sometimes
cause disease. We now shift our focus from the infec-
tious agent to the host — in particular, the human body —

and how it can remain healthy even as disease-causing
organisms gain access to it by way of air, food, and
water. Immunity, the state of protection from infectious
disease, is achieved by both nonspecific and specific
mechanisms. Nonspecific immunity is provided by the
skin and mucous membranes, which act as physical bar-
riers to infection, and by several types of white blood
cells (leukocytes), some of which are phagocytic,
capable of engulfing microorganisms. Specific immu-
nity, on the other hand, is conferred by lymphocytes,
including leukocytes that bind specifically to foreign
molecules (antigens) on the surfaces of invading organ-
isms and infected body cells.

There are five major leukocyte types, each classi-
fied as either a granulocyte or an agranulocyte. The
granulocytes (neutrophils, eosinophils, and basophils)
contain cytoplasmic granules that are packed with
degradative enzymes and mediators of inflammation.
The agranulocytes consist of two morphologically and
functionally distinct cell types (monocytes and lym-
phocytes) that have finer, less prominent granules. A
white blood cell can usually be identified by the shape
of its nucleus and by the presence or absence of gran-
ules. For example, a mature neutrophil has a distinc-
tive multilobed nucleus and fine granules in its
cytoplasm, while a lymphocyte has a rounded nucleus
that fills much of the cell interior, and no obvious gran-
ules. The morphology, function, and population size for
each leukocyte type is presented in table 40.1.

If you were to count all of the white blood cells in
a particular volume of blood, you would be determin-
ing the total white blood cell count, a value expressed
in number of cells per microliter (jil). If you were to
identify each cell type as you count it, you would be
generating a differential white blood cell count. A dif-
ferential count is a measure of each leukocyte type (both
mature and immature forms) and is likewise expressed
as cells/jil for each cell type. As you will see, however,
the most common cell in a sample of whole blood is the
red blood cell, or erythrocyte. Typical erythrocyte
counts range from 4,500,000 to 6,500,000 cells per (il,
while total white blood cell counts range from 4,500
to 11,000 cells per (il.

The finger-stick method of blood collection pro-
vides a small amount of mixed capillary, arteriole, and
venule blood. This type of blood collection is frequently
used when only a small amount of blood is needed. It is
also used on infants younger than 6 months of age, in
young children, and in adults who have poor veins or
whose veins cannot be used because of intravenous
infusions. As with any blood collection or invasive pro-
cedure, all materials that touch the subject must be ster-
ile (alcohol wipe, cotton ball, lancet, Band- Aid), and
blood-contaminated materials must be disposed of
properly. Review the universal precautions, in the lab-
oratory safety section of this manual (see p. xiii) to learn
the steps you must take when handling human source
samples.

Although automated methods are now available for
identifying and counting white blood cells, you will be
identifying and counting cells with the aid of staining
and light microscopy.

298

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IX. Hematology and
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40. Identification and
Enumeration of White
Blood Cells

Identification and Enumeration of White Blood Cells EXERCISE 40 299

Blood cell type

Functions

Characteristic features
(average diameter)

Number of cells/
mm 3 (julI) of

blood percent of
total WBC

Granulocytes

Neutrophil

Important phagocytic cells in blood
and tissues.

Multilobed nucleus,

small cytoplasmic

granules

(10-14 Jim)

3,000-7,000
cells/jil

35-71%

Eosinophil

Phagocytic cells that can migrate
from the blood into tissues. Granule
contents are particularly harmful to
parasitic worms.

Bilobed nucleus, large
cytoplasmic granules

(10-14 Jim)

100^00 cells/ jil

0-4%

Basophil

Nonphagocytic cells with granules
containing histamine and other
compounds that act against parasitic
worms. Basophils (in blood) and
mast cells, a related cell type in
tissues, also contribute to allergic
and inflammatory responses.

Pinched U- or S- shaped

nucleus, large

cytoplasmic granules

(10-12 Jim)

20-50 cells/ jil

0-2%

Agranulocytes

Monocyte

Moderately phagocytic in the blood,
these cells migrate into the tissues,
becoming large, highly phagocytic
cells called macrophages.

U- kidney- shaped

nucleus, no visible

granules

(15-20 Jim)

100-700 cells/ jil

1-10%

Lymphocyte

B and T lymphocytes are present in
the blood and in lymphoid tissues
(the spleen and lymph nodes, for
example). In response to contact with
specific antigens, T cells develop into
active killer cells, and B cells develop
into antibody- secreting plasma cells.

Large, rounded nucleus

with little visible

cytoplasm

(5-17 Jim)

1,500-3,000
cells/ jil

24-44%

Erythrocytes, or red blood cells, not shown here, are more numerous than white blood cell (4,500,000-6,500,000 cells/|il of blood.
Erythrocytes are small (7-7.5 |im diameter), non-nucleated, and are biconcave in shape.

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

IX. Hematology and
Serology

40. Identification and
Enumeration of White
Blood Cells

300 Section IX Hematology and Serology

Materials

Blood Collection

Miscellaneous supplies
Latex gloves
Sterile, disposable safety lancets

(Medi-Let®)
Sterile cotton balls
70% ethanol, or alcohol wipe
Two clean microscope slides (one slide for

sample, one spreader slide)
Orange biological disposal bag (one per lab)
Plastic sharps collector (one per lab)

Wright's Staining

Reagents

Wright-Giemsa stain and buffer
Distilled water
70% ethanol

Equipment

Light microscope

Miscellaneous supplies

Staining support and drip pan
Human blood film, smear
Latex gloves

Blood Collection

1 . Use the middle of the outer segment of the third
or fourth finger. To increase local blood flow
prior to the puncture, you can wrap the finger in a
warm, moist paper towel for 2-3 minutes.

CAUTION:

Human Blood Handling
Safety Note: Observe universal
precautions (see page xiii);
handle only your own sample.

Clean the site with an alcohol wipe, and allow it
to air-dry. Do not blow on the skin to dry the
alcohol. Blowing can contaminate the site. It is
important to completely air-dry the residual
alcohol because it may cause rapid hemolysis
when it contacts the blood.

3 . Follow the Medi-Let® procedure to obtain your
blood sample. Immediately dispose of the
cartridge in the plastic sharps collector.

4. Wipe away the first drop of blood with the sterile
cotton ball. The first drop of blood usually
contains excess tissue fluid.

5. Place 2 drops of blood near one end of a clean
slide. Take care not to touch your skin to the
slide. Place the short end of a second, spreader
slide into the blood drops, with a 30° to 40° angle
between the two slides, until the blood spreads
along the edge of the spreader slide (see figure
9.3). Just before the blood has spread completely
along the edge of the spreader slide, push the
spreader slide along the first slide to form a
smear. The smear should show a gradual
transition from thick to thin.

6. Label the slide with your name and the date, and
let the smear air-dry.

7. Dispose of all materials properly.

Wright's Staining

1 . Place slides on a staining support over a drip pan,
and apply enough drops of Wright-Giemsa stain
to cover the smear. Count the number of drops
you use. Incubate for 1 minute at room
temperature.

2. Add an equal number of drops of Wright's
phosphate buffer (pH 6.4). Gently blow on the
slide to mix the solutions, and incubate for 3-6
minutes at room temperature.

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

IX. Hematology and
Serology

40. Identification and
Enumeration of White
Blood Cells

Identification and Enumeration of White Blood Cells EXERCISE 40 301

3 . Rinse the slides with distilled water. Cleanse the
back of the slide with 70% ethanol.

4. Allow the smear to air-dry.

5. Examine the stained blood smear: Scan the
blood smear at 40x magnification. Erythrocytes
(red blood cells) are rose to salmon in color, are
biconcave, and have no nucleus. Most of the cells
you see are erythrocytes. The leukocytes (white
blood cells) are larger, nucleated cells, colored
shades of blue by the stain. The nuclei of white
cells will be blue to light purple; the cytoplasm
will vary from pale pink (neutrophils), to pale
gray (monocytes), to light blue (lymphocytes).
Eosinophils and basophils are discernible by their
granules (orange to rose for eosinophils; violet to
blue for basophils). Neutrophils also contain
granules that stain pink to purple.

6. Perform a differential white blood cell count:

Shift the magnification to lOOx. Identify and
record the white blood cell types you see, starting
from the sparse end and working toward the more
dense end of the smear. Use the cross-sectional
method of differential counting. Count from the

bottom right of the sparse end; count up, count
left, count down, count left, etc., as shown in
figure 40. 1 . When you see a white blood cell,
identify and record it in your laboratory report.
Design a data table that will allow you to easily
record your data. Stop when you have counted a
total 100 white blood cells.

Calculate the percentage of each leukocyte type
you counted. When counting 100 cells, the
percentage is easily discerned; a count of 65
neutrophils means that neutrophils make up 65%
of the total white cells in the sample.

Figure 40.1 The cross-section method of counting white
blood cells.

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

IX. Hematology and
Serology

40. Identification and
Enumeration of White
Blood Cells

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

IX. Hematology and
Serology

40. Identification and
Enumeration of White
Blood Cells

Name

Lab Section

EXERCISE

Laboratory Report

Date

Identification and Enumeration of White Blood Cells

1. Complete the following table based on your results.

White blood
cell type

Labeled diagram
of cell type

Number of
cells counted

Percent of
total cells

Typical percent
range for
cell type

2. Briefly state whether your results are within typical ranges for each cell type.

303

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Lab Exercises in
Organismal and Molecular
Microbiology

IX. Hematology and
Serology

40. Identification and
Enumeration of White
Blood Cells

304 Section IX Hematology and Serology

3. If you counted 100 total white cells over a portion of a smear that is equivalent to about 50 \i\ of blood,
would that white blood cell count be within normal range?

4. Identify each of the following cell types. Give one reason for your answer in each case.

Reasoning

5. A 22-year-old female comes into the emergency room complaining of severe abdominal pain in the right
lower quadrant. Her temperature is 39°C, and laboratory studies reveal a white blood cell count of
25 , 000/microli ter.

a. Is her total white blood cell count within normal range?

b. She is diagnosed with appendicitis. One of our defenses against an infection such as this is
phagocytosis. Thus, an important response to infection is the proliferation of phagocytic cells, in
particular a white blood cell type that can be found in both the blood and in tissues and that has a
multilobed nucleus. Given this, which of the five cell types might be most prominent in her blood?

6. White blood cells include those involved in nonspecific defenses such as phagocytosis and the release
of histamine as well as those that operate in specific defense. Name the type of white blood cell that
operates in specific defense. There are two subtypes in this category that function in particular ways
in specific defense. Name the two cell subtypes, and state the role of each.

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

IX. Hematology and
Serology

41. Antigen-Antibody
Precipitation Reactions
and Determination of
Antibody Titer

Antigen-Antibody Precipitation Reactions and
Determination of Antibody Titer

Specific immunity is conferred by T lymphocytes (T
cells) and B lymphocytes (B cells), two special classes
of leukocytes that bind to foreign antigens (similar to
enzyme- substrate binding) and become activated to help
clear the antigens from the body. T cell responses
include the development of active cytotoxic T cells that
destroy virus-infected cells and other abnormal body
cells. B cells respond to antigen by developing into
plasma cells that secrete high levels of proteins called
antibodies or immunoglobulins (figure 41.1a). Anti-
bodies circulate in the blood and tissue fluid, binding to
the precise antigens that induced their production in the
first place, and forming antibody-antigen complexes
(figure 41. lb). Once tethered in a large complex,
viruses, bacteria, and toxins are effectively blocked
from harming the body and are targeted for destruction
by a variety of mechanisms, such as phagocytosis.

Antibodies are present in the serum, the viscous
yellow fluid that remains after red and white blood cells
have been separated from the fluid portion of blood
(plasma) and a clot has formed. Because of its antibody
content, serum from an infected or immunized person
or animal is sometimes called antiserum. Antiserum

contains many antibody molecules specific for the
infecting or immunizing agent. In fact, the study of anti-
body-antigen reactions in vitro is known as serology.

In serological or immunological methods, antibod-
ies are used as tools for the detection and quantification
of specific molecules or microbes. The antibodies, col-
lected from the serum of an animal that has been immu-
nized with the selected antigen, are called polyclonal
because they are the products of many different B cells
(plasma cells) specific for different parts of the same
antigen. Monoclonal antibodies (Mabs), on the other
hand, cannot be collected directly from an animal. Mabs
are secreted from a cultured hybridoma — a fusion of
two cells, one a B cell from an immunized mouse and
the other a type of cancer cell called a myeloma. The
B cell partner provides the desired antibody specificity,
and the myeloma partner contributes immortality to the
hybridoma. The hybridoma becomes a continuous
source of monoclonal antibodies of singular specificity.
Figure 41.2 outlines the production of polyclonal anti-
bodies and monoclonal antibodies.

In addition to the appropriate polyclonal or mon-
oclonal antibody preparation, serological methods
require a way to detect antibody-antigen binding. Some
serological methods take advantage of the formation of

2 identical antigen-
binding sites, each
formed by heavy-light
chain combination

r Disulfide bridges

link the chains together.

2 identical
light chains

(a)

Protein
antigen

Antibody

(b)

Figure 41.1 Antibodies are secreted by a type of differentiated B cell called a plasma cell, (a) Diagram of an antibody
molecule, (b) Each antibody has two binding sites for antigen. As a result, multiple antibody molecules can link multiple
antigen molecules to form a large antibody- antigen complex.

305

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

IX. Hematology and
Serology

41. Antigen-Antibody
Precipitation Reactions
and Determination of
Antibody Titer

306 Section IX Hematology and Serology

1. Immunization
Antigen of interest (X)

2. Collect BLOOD.

3. Centrifuge and clot blood.

Serum contains antibodies specific for antigen
}■ X as well as other antibodies and serum proteins

Cells and clotting factors

4. Purify antibodies from serum.

5 . Purify X-specific antibodies from other antibodies

The X-specific antibodies are polyclonal because they come
from many different plasma cells (B cells).

2. Collect SPLEEN CELLS. (The spleen cells include B cells
specific for antigen X.)

\_/

3 . Fuse spleen cells with mutant myeloma cells

B cell partner Myeloma partner

provides antibodies provides immortality,
specific for X.

4. Select for hybrid cells (hybridomas).

5. Screen for hybridomas secreting X-specific antibodies

6. Grow a clone of hybridoma

cells secreting X-specific antibodies.

The X-specific antibodies are monoclonal because they come
from a single clone of hybridoma cells.

Figure 41.2 The production of antibodies for serological methods, (a) Diagram of polyclonal antibody preparation,
(b) Diagram of monoclonal antibody preparation.

visible antibody- antigen complexes (agglutination and
precipitation tests; see Exercises 42 and 43). Others
make use of antibodies that have been covalently linked
to a detectable label (Enzyme- linked immunosorbent
assay and Western blotting; see Exercises 44 and 45).
In this exercise, you will use a precipitation test to
determine the titer of antibodies in serum from an
immunized animal. An antibody titer is a measure of the

relative strength of an antiserum, and is expressed as the
reciprocal of the greatest dilution of antiserum still
capable of mediating a detectable effect (here, precip-
itation). For example, if antiserum A activity is
detectable up to a dilution of 1:200 and antiserum B
activity is detectable up to a dilution of 1 :400, then the
strength, or titer, of antiserum B (400) is greater than
that of antiserum A (200).

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

IX. Hematology and
Serology

41. Antigen-Antibody
Precipitation Reactions
and Determination of
Antibody Titer

Antigen-Antibody Precipitation Reactions and Determination of Antibody Titer EXERCISE 41 307

The precipitation test is among the simplest and
quickest of the serological methods and is based on the
propensity of antibodies to form complexes with their
corresponding antigens (see figure 4 1. lb). When anti-
bodies attach to antigen molecules in solution, the mol-
ecules become part of an insoluble antibody- antigen
complex, and a visible precipitate forms. Thus, the pres-
ence of a precipitate is a positive result; the fact that the
antibody molecules have bound specifically to the anti-
gen molecules is obvious. (Similarly, when the anti-
gen is located on a cell surface, the cells become
clumped together, or agglutinated, by antibodies). Here,
you will use a series of precipitation tests to determine
the titer of a polyclonal antiserum raised in rabbits
against the protein albumin from cows (known as BSA,
for bovine serum albumin).

Procedure

Reagents

Antigen: bovine serum albumin (BSA)
(25 jig/ml)

Antiserum: rabbit anti-BSA

Phosphate-buffered saline (PBS) (sodium
chloride 1.6 g, potassium chloride 0.04 g,
sodium phosphate-dibasic 0.22 g, potassium
phosphate-monobasic 0.04 g in 100 ml)

Saline (0.9% solution of NaCl)

Miscellaneous supplies

10 serological (precipitin) tubes (6 x 50 mm)

Precipitin tube rack

1 .0 ml serological pipette/pipettor

Pasteur pipette/bulb

1.5 ml microfuge tubes

Laboratory marker

1. Prepare tubes for antiserum dilutions: Label
nine microfuge tubes with a lab marker,
numbering them 1-9. Using a 1.0 ml serological
pipette, transfer 0.9 ml of 0.9% saline into tube 1
Then pipette 0.5 ml of 0.9% saline into each of
the remaining tubes, 2-9.

2. Perform antiserum dilutions (figure 41.3):
Pipette 0.1 ml of rabbit anti-BSA into tube 1.
Mix by pipetting gently up and down about five
times, trying to avoid bubbles. With the same
pipette, transfer 0.5 ml from tube 1 into tube 2.
Again, mix the antiserum and saline in tube 2 by
pipetting gently up and down about five times,
trying to avoid bubbles. With the same pipette,
transfer 0.5 ml from tube 2 into tube 3, and so
forth, repeating the mixing and transfer of

0.5 ml- volumes in series to tube 9. (Tube 9
contains 1 .0 ml of diluted antiserum.)

3. Label 10 serological tubes, 1-10. Using a
Pasteur pipette, transfer 8 drops of 0.9% saline
into serological tube 10. Using the same Pasteur
pipette, transfer 8 drops of antiserum from
dilution tube 9 to serological tube 9. Do the
same for samples 8 through 1 , in that order
(you can use the same Pasteur pipette because
you are handling samples from the most dilute
to the least dilute).

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

IX. Hematology and
Serology

41. Antigen-Antibody
Precipitation Reactions
and Determination of
Antibody Titer

308 Section IX Hematology and Serology

4. The antigen sample (BSA) is provided in 0.9%
saline at a concentration of 25 jig/ml. Using a
Pasteur pipette, gently layer about 1 ml of the
BSA preparation over the antiserum in each of
the 1 precipitin tubes . It is important not to
mix the two layers together. You are looking for
local precipitate forming at the interface, where
antigens meet antibodies (see figure 41. 3b).

5. Watch for a reaction in the tubes after you are
finished adding the antigen to all of them. Check
for the formation of precipitate at the interface
every 3 minutes for 15 minutes.

6. Record your results in the laboratory report,
indicating the extent of precipitate formation, if
any (- [no ppt], +, ++, +++).

(1:10)

0.1 ml
in 1 ml

(1:2)
0.5 ml
in 1 ml

Antiserum

0.9 ml
saline

0.5 ml
saline

1:10

1:20

1:40

1:80

1:160

1:320

1:640

1:1,280

1:2,560

Final dilutions

(a)

(b)

Figure 41.3 Titering antibodies, (a) In this procedure for making dilutions, one 1:10 dilution is
followed by a series of two-fold dilutions. (A series of the same dilution is called serial dilutions.)
(1) Pipette the appropriate volume of saline (diluent) into each tube. (2) Pipette undiluted serum into
tube 1, and proceed with serial dilutions, (b) A set of nine precipitation tests corresponding to dilu-
tions 1-9. The titer of this antiserum is 160.

Alexander-Strete-Niles:

IX. Hematology and

41. Antigen-Antibody

Lab Exercises in

Serology

Precipitation Reactions

Organismal and Molecular

and Determination of

Microbiology

Antibody Titer

EXERCISE

Laboratory Report

Name

Date

Lab Section

Antigen-Antibody Precipitation Reactions
and Determination of Antibody Titer

1. Complete the following table: Record the extent of precipitate formation, if any (- [no ppt], +,
for each antiserum dilution.

<-+)

Antiserum: Antigen:

Dilution

Results

2. What is the titer of the antiserum?

3. What is the purpose of reaction tube 10?

4. Multiple molecules of BSA have been covalently linked to tiny beads the size of sand particles. Samples
of the BSA-beads are each mixed with antiserum specific for BSA, prepared at dilutions of 1:1,000 to
1:10,000. You observe the beads clumping (agglutinating) at dilutions 1:100 and 1:200, but not at greater
dilutions. Is this antiserum stronger or weaker than the one you used? Briefly explain your answer.

5. Diagram the antibody- antigen complex in question 4.

309

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

IX. Hematology and
Serology

41. Antigen-Antibody
Precipitation Reactions
and Determination of
Antibody Titer

310 SECTION IX Hematology and Serology

6. The following is a diagram of the immunization of a mouse to generate polyclonal antibodies specific for
human insulin. Complete the diagram by labeling it with the terms listed below. Also, define each term.

Human insulin

2 to 3 weeks later

Activity
detectable
to a dilution
of 1:2,000

Clotted blood

antigen

antibody

antiserum

serum (contrast with antiserum)

polyclonal antibodies

titer

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

IX. Hematology and
Serology

42. Agglutination
Reactions: ABO Blood
Typing

Agglutination Reactions: ABO Blood Typing

As noted in Exercise 41, the highly specific and selec-
tive binding of antibodies to antigens has led to the
development of a number of antibody-based diagnos-
tic and research methods. In these methods (aggluti-
nation, precipitation, ELISA, Western blot, and others),
antibodies are used as tools for the detection and quan-
tification of drugs, hormones, and other molecules, as
well as for the identification and characterization of
viruses, cells, and tissues. Alternatively, such molecules,
viruses, and cells (antigens) can be used to detect the
presence of particular antibodies in a test sample. In the
HIV screening test, for example, a person's serum is
tested for the presence of antibodies specific for HIV,
and not for the virus itself. In this case, the antigen is
the tool, or the "known," and the antibody specificity
is the "unknown."

When antibodies bind to cells, such as bacteria,
yeast, or red blood cells, the cells clump together, or
agglutinate. A visible agglutination reaction indicates
that antibodies are binding specifically to cells, link-
ing them together to form a large complex. So, just as
antibodies bind to soluble molecules to form an insol-
uble precipitate, they bind to cell-bound molecules to
form a clump of cells. Agglutination reactions are rou-
tinely used to type blood, to identify microorganisms,
and to test serum samples for the presence of antibod-
ies reactive against a particular microbe.

In blood typing, antibodies are used to detect red
blood cell surface antigens such as those of the ABO

system. These antigens consist of a core glycolipid
called substance H. If substance H is modified by the
addition of another sugar, N-acetylgalactosamine, it is
an A antigen. If substance H instead has an attached
galactose, it is a B antigen. If substance H stands alone,
unmodified, it is neither A nor B, and is known as O
(figure 42.1). A antigens are found on type A and type
AB red blood cells, B antigens are found on type B
and type AB red blood cells, and neither is found on
type O red blood cells. Each of these blood types may
be Rh + or Rrr, depending on the presence or absence of
another antigen, a protein called the Rh factor.

It may seem odd that normal molecules such as
these are called antigens. In fact, all macromolecules
are potential antigens, especially if they are transferred
into a nonidentical person or to another animal through
transfusion or transplantation. And, in autoimmune dis-
orders, normal self-molecules such as these may be
treated as foreign antigens. So, macromolecules can
be called antigens because they have the potential to
generate antibodies.

In this exercise, you will determine the blood type
of either an aseptic blood sample or your own blood
sample using the agglutination reaction. As shown in
figure 42.2, agglutination, or hemagglutination (the
term for red blood cell clumping), occurs when a blood
sample is mixed with antibodies specific for its type. For
example, if a sample of blood agglutinates when treated
with antibodies to B but not when treated with anti-
bodies to A or to the Rh factor, the sample is type B~.

311

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

IX. Hematology and
Serology

42. Agglutination
Reactions: ABO Blood
Typing

312 SECTION IX Hematology and Serology

Short chain of sugars

Membrane lipid
(outer leaflet of RBC membrane)

Substance H: "O" antigen

A antigen

B antigen

Key:

\_/ Glucose

<(_/ N -acetylgalactosamine

\_/ Galactose

\__) N-acetylglucosamine

\_/ Fucose

Figure 42.1 Illustration of the H (O), A, and B antigens. The antigens, which belong to a class of macromolecules called
glycolipids, consist of a short chain of sugars covalently attached to membrane lipids.

BLOOD TEST CARD

Af4T%A

SEFUJIVi BLOCG

KAME

TYPE

ANTI-B

SERUM BLOOD

Mil

ANTMJ

SERUM BLOOO

DATE

GMOLHIA BIOLOGICAL SUPflY COfflrW

(a)

BLOOD TEST CARD

AMI B

ffi R! if.1 BLOOO

ANT1-0

SERUM BLCQD

DATE

CAROUiu noioacm suppw compAnv

(b)

Figure 42.2 ABO-Rh blood typing with the antibody agglutination test, (a) Left: Type A~ blood reacts with antibodies to
A (notice the clumped, particulate appearance of the samples) but not with antibodies to B or Rh (notice the smooth appear-
ance). Right: Type B~ blood reacts only with antibodies to B. (b) A drawing of red blood cells agglutinated by antibodies.

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

IX. Hematology and
Serology

42. Agglutination
Reactions: ABO Blood
Typing

Agglutination Reactions: ABO Blood Typing EXERCISE 42 313

Blood Collection

Miscellaneous supplies
Latex gloves
Sterile, disposable safety lancets

(Medi-Let®)
Sterile cotton balls
70% ethanol, or alcohol wipe
Two clean microscope slides (one slide for

sample, one spreader slide)
Orange biological disposal bag (one per lab)
Plastic sharps collector (one per lab)

Aseptic Blood and ABO-Rh
Blood Typing

Reagents

Aseptic blood samples, anti-A, B, and Rh
antisera and materials provided in a Blood
Cell/Antisera BioKit
Miscellaneous supplies
Latex gloves

Procedure

Blood Collection

CAUTIO IM

#* *

(

Un i versa I Preca utions
xiii); handle your own

sample.

Have a test card ready. Label it with your name
and the date. If you are using an aseptic sample
of blood, go to step 5. To prepare for the blood
collection, consider using the middle of the outer
segment of the third or fourth finger. To increase
local blood flow prior to the puncture, you can
wrap the finger in a warm, moist paper towel for
2-3 minutes.

2. Clean the site with an alcohol wipe, and allow it
to air-dry. Do not blow on the skin to dry the
alcohol. Blowing can contaminate the site. It is
important to completely air-dry the residual
alcohol because it may cause rapid hemolysis
when it contacts the blood.

3. Follow the Medi-Let® procedure to obtain your
blood sample. Immediately dispose of the
cartridge in the plastic sharps collector.

4. Wipe away the first drop of blood with the sterile
cotton ball. The first drop of blood usually
contains excess tissue fluid. Allow the blood to
drop onto each of the three areas on the test card
marked "blood." Go to step 6.

ABO-Rh Blood Typing

5. If you are using a provided, aseptic blood sample,
place 1 drop of the blood onto each of the three
areas on the test card marked "blood."

6. Place 1 drop each of the anti-A serum, anti-B
serum, and anti-D (Rh) serum onto the designated
areas of the test card.

7. Mix each of the blood drop-anti serum sets, using
a fresh mixing stick for each sample. Dispose
of each mixing stick in the sharps container as
soon as you are done with it.

8. Gently rock the test card for 1 minute, without
allowing the samples to flow out of their
designated test areas.

9. Examine the samples for agglutination. The
anti-D reaction may take longer than the others. It
may be easier to see agglutination if you tilt the
card slightly.

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

IX. Hematology and
Serology

42. Agglutination
Reactions: ABO Blood
Typing

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

IX. Hematology and
Serology

42. Agglutination
Reactions: ABO Blood
Typing

EXERCISE

Laboratory Report

Name

Date

Lab Section

Agglutination Reactions: ABO Blood Typing

1. Record your agglutination results in columns 1-4 of the following table

Sample I.D.

Anti-A reaction

(+ or -)

Anti-B reaction

(+ or -)

Anti- (Rh) reaction

(+ or -)

Phenotype
(blood type)

Genotype(s)

2. A person's ABO phenotype (A, B, AB, or O) arises from the expression of two alleles (alternative genes),
one inherited from each parent. For example, the genotype of someone with type A blood may be either
AO or AA.

If we know the genotype of each parent, we can use a Punnett square to predict the possible genotypes of
their offspring. Complete the following Punnett square, remembering that each parent contributes a single
allele to each child. What is the genotype of their child with type A blood? What are the chances that they

will have a child with type AB blood?

Maternal
ABO genotype

Paternal
ABO genotype

3. In the far-right-hand column of the table in question 1, indicate the genotype or possible genotypes for
each phenotype you determined.

4. A child has type O blood, her mother is type A, and her father is type AB. Which parent could be a
biological parent? Which parent cannot be a biological parent?

315

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

IX. Hematology and
Serology

42. Agglutination
Reactions: ABO Blood
Typing

316 SECTION IX Hematology and Serology

5. If a person is transfused with mismatched blood, an immediate transfusion reaction (an immune response
against foreign blood group antigens) can occur. This happens because antibodies specific for foreign
blood group antigens already exist in the recipient's blood. A person with type A blood, for example, has
antibodies to the B antigen, even if he has never been exposed to type B blood. These antibodies arise in
response to bacteria (normal flora) that have antigens very similar to the A and B antigens. Thus, this
person with type A blood does not make antibodies to A- like bacterial antigens — the immune system
considers these self — but does make antibodies to B-like bacterial antigens. Therefore, if a person with
type A blood receives a transfusion of type B blood, the preexisting anti-B antibodies will induce an
immediate and devastating transfusion reaction.

For each of the following blood types, indicate whether the blood would also contain antibodies to A,
antibodies to B, antibodies to both A and B, or no antibodies to A or B.

Type A

Type B

Type AB

Type O

6. A person with type O blood is considered a universal donor, while a person with type AB blood is a
universal recipient. When type O blood is donated to a person with type A, AB, or B, packed cells are
used rather than whole blood. Why?

7. Consider a sample of type AB + blood. Each red blood cell in the sample has many copies of the
A and B antigens on its surface but few copies of the Rh factor. Explain why this results in an
anti-Rh agglutination reaction that is slower to form and less pronounced than either the anti-A
or anti-B reaction.

8. A person's serum is mixed with Salmonella typhimurium cells on a slide. After a few minutes,
particulates or clumps can be seen on the slide. What do you conclude about this result?

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

IX. Hematology and
Serology

43. Immunodiffusion:
Antigen-Antibody
Precipitatioin Reactions in
Gels

mmunodiffusion: Antigen-Antibody
Precipitation Reactions in Gels

As you observed in Exercise 41, when soluble antigen
molecules become linked together by multiple anti-
bodies, an insoluble precipitate forms. This precipitate,
visible to the naked eye, can reveal the identity of an
antigen (if the antibody specificity is known) or the
specificity of the antibody (if the antigen is known).
A precipitate also indicates that antibody and antigen
molecules are present at optimal proportions for the for-
mation of a large complex, or lattice. In this equiva-
lence zone, there are about two to three antibody
molecules for every one antigen molecule, leaving no
free antigens or antibodies (figure 43.1).

In immunodiffusion tests, antibodies and/or solu-
ble antigens are loaded into separate wells of a gel and
are allowed to diffuse, each reagent moving radially into
the gel. An immobile precipitate, visible as a band (pre-
cipitin line) in the gel, develops if specific antibody-
antigen binding takes place, and if antibody and antigen
components are present at optimal proportions. Dou-
ble immunodiffusion, also known as Ouchterlony, is the
most widely used gel precipitation technique in the
research laboratory, while radial immunodiffusion and
Immunoelectrophoresis are principally used in clinical
labs to test serum protein levels.

In double immunodiffusion, antigen and antibody
preparations are loaded into separate wells of an agarose
gel as shown in figure 43.2. In this example, the anti-
bodies (specific for human serum proteins) are located
in the center well, and the antigens (serum proteins) are
located in the outer wells. Each substance diffuses from
its well, and in time, white lines of insoluble precipi-
tate appear at positions where antibodies have bound
to their specific antigens at optimal proportions (the

Amount of
precipitate
formed

#* *

** + * < 5

Antibody excess

Antigen excess

Low

Antigen concentration

High

(a)

Antigen concentration
Low ► High

(b)

Figure 43.1 The precipitin curve, (a) The curve repre-
sents a series of antigen- antibody precipitin reactions
showing that if either antibody or antigen is in excess, a
complex does not form. The region of the curve in which
complexes form is called the equivalence zone, (b) There is
no visible precipitation in samples 1 and 2 because the
antibody is in excess, and there is no precipitation in sam-
ples 8 and 9 because the antigen is in excess. Samples 3 to
7 represent the equivalence zone.

317

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

IX. Hematology and
Serology

43. Immunodiffusion:
Antigen-Antibody
Precipitatioin Reactions in
Gels

318 SECTION IX Hematology and Serology

Human
albumin

(a)

Horse
albumin

(b)

Bovine
albumin

Figure 43.2 Double immunodiffusion assay, (a) Anti-
bodies specific for human serum proteins were loaded into
the center well, and antigens (human, bovine, and horse
albumin) were loaded into the outer wells. After a few
hours of diffusion, antigen- antibody complexes formed.
The complexes do not diffuse, and they appear as white
lines in the gel. The results indicate that the antibodies spe-
cific for human serum proteins bind with human albumin
but not with horse or bovine albumin, (b) Diagram of the
results in (a).

equivalence zone). In radial immunodiffusion, the anti-
bodies are evenly distributed within a preformed 1 %
agarose gel. Therefore, in this test, only the antigen sam-
ple, loaded into a well, diffuses. As antigen molecules
move through the gel, they bind to and carry antibod-
ies, until the ratio of antigens to antibodies is optimal for
complex formation. At this point, a ring of precipita-
tion forms, and its diameter is proportional to the con-
centration of antigen loaded into the well; at higher
concentrations, the diameter of the ring is greater
because antigen molecules must migrate farther before
they gather up enough antibodies to form a complex.
Figure 43.3 shows the results of a radial immunodiffu-
sion test for the human serum protein IgA (the antigen
in this case).

The third type of immunodiffusion is called Immu-
noelectrophoresis. Antigens are first loaded into wells
of an agarose gel and are separated by charge in an elec-

(a)

log 10 [IgA] A
(mg/ml)

12 3 4 5 6
Precipitation ring diameter (mm)

(b)

Figure 43.3 Radial immunodiffusion, (a) A photograph
of a radial immunodiffusion gel. The agarose gel contains
antibodies to immunoglobulin (Ig) A, one of the five
classes of antibodies. Wells 7, 8, and 14 contain control
IgA samples at 0.54 mg/ml, 5.4 mg/ml, and 1.3 mg/ml,
respectively. Well 6, 10, and 14 contain no sample, (b) A
graph of the log of the IgA concentration (mg/ml) versus
the diameter of the precipitation ring using the standard
samples in wells 7, 8, and 14 in (a).

trophoresis chamber. Antibodies are then used to detect
the separated antigens; after being loaded into a trough
that runs the length of the gel, they diffuse toward and
complex with the antigens, and form visible lines of
precipitate. Figure 43.4 shows the results of an Immu-
noelectrophoresis analysis of human serum.

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

IX. Hematology and
Serology

43. Immunodiffusion:
Antigen-Antibody
Precipitatioin Reactions in
Gels

Immunodiffusion: Antigen-Antibody Precipitation Reactions in Gels EXERCISE 43 319

Figure 43.4 A photograph of an Immunoelectrophoresis
assay to detect serum proteins. Whole serum was loaded
into wells A and B, and albumin was loaded into well C.
The proteins migrate according to their net charges. For
example, proteins that have a net negative charge migrate
to the right, toward the positively charged anode. After
electrophoresis, troughs 1 and 2 were both loaded with
antibody specific for whole- serum proteins. The curved
precipitin lines reveal the relative position of the major
types of serum proteins.

Double Immunodiffusion (Ouchterlony)

Note: As an alternative to the following reagents and
procedure, the Ouchterlony procedure can be accom-
plished using a kit (Edvotek #270).
Reagents

Agarose: 40 ml 1% (w/v) molten agarose in
0.05 M Tris-Cl, pH 8.6 (per pair)

Antibodies (table 43.1)

Serum antibody set (Carolina Biological

Supply: #RG-20-2102)
Goat anti-bovine albumin
Goat anti-horse albumin
Goat anti- swine albumin

Antigens

Serum antigen set — bovine serum, horse
serum, swine serum
Equipment

Microwave oven
Water bath at 55°C

Miscellaneous supplies

60 mm diameter petri dishes
Covered box for gel storage
Label tape
Laboratory marker
10 ml pipette/pipettor
Glass dropper (well cutter)
Micropipettor/tips (1-10 Jill)

Radial immunodiffusion

Reagents

Human IgG, IgA, and IgM "NL" Bindarid™

radial immunodiffusion kit
Human serum

Miscellaneous supplies

Micropipettor/tips (1-10 jil)

Alexander-Strete-Niles:
Lab Exercises in
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IX. Hematology and
Serology

43. Immunodiffusion:
Antigen-Antibody
Precipitatioin Reactions in
Gels

320 SECTION IX Hematology and Serology

Table 43.1 Sample Loading Order for Double Immunodiffusion Assays

Center well*

Outer wells**

Pattern A

Pattern B

Pattern C

Goat anti-bovine albumin

Goat anti-horse albumin

Goat anti-swine albumin
and goat anti-bovine albumin

1. Bovine serum

2. Horse serum

3. Swine serum

4. Swine serum

The contents of the outer wells are the same for all three assays.
The center well antibodies are different for each assay.

Immunoelectrophoresis

Reagents

High-resolution electrophoresis buffer, pH 8.8
1% agarose in high-resolution buffer, pH 8.8
Antigens: bovine serum

bovine albumin, 10 mg/ml
Antibodies: anti-bovine albumin

anti-bovine serum

Equipment

Horizontal gel electrophoresis box
Power supply

Miscellaneous supplies

1 60 mm diameter petri dish
Tape

2 glass slides

Glass or plastic dropper (well cutter)
Micropipettor/tips (10-100 jll)
Grade no. 1 Whatman paper or
3MM paper

Double Immunodiffusion (Ouchterlony)

1. Prepare 40 ml of 1% agarose: Add 0.4 g of

agarose to 40 ml of 0.05 M Tris-Cl, pH 8.6, in a
1 25 ml flask. Microwave the mixture for about 30
seconds, checking to make sure it does not boil
over. Using a hot glove, gently swirl the flask,
and return it to the microwave. Heat for 15
seconds, repeating this until no flecks of agarose
are visible in the flask. Let the molten agarose
cool until the flask is comfortable to handle, but
still warm.

Obtain three 60 mm diameter petri dishes.
Writing with a lab marker on the plate bottom,
label the three plates A, B, and C, respectively.
Write your initials on all three plates. Pipette 5 ml
of slightly cooled molten agarose into each dish.
Allow the agarose to solidify, about 20 minutes.

Using the large end of a plastic or glass dropper
(a diameter of about 0.5 cm), cut wells into each
gel as shown in the following template. (See also
figure 43.2.)

Label the outer wells 1, 2, 3, and 4 by writing on
the plate bottom.

Changing micropipette tips between different
reagents, pipette 20 jal of the appropriate antigen
and 20 jll of antibody to the designated wells
according to the loading order in table 43.1.

Line the bottom of the storage box with a moist
paper towel, and place the dishes into the storage
container. Make sure the dishes are level.
Incubate the gels for 24 to 48 hours at room
temperature to allow diffusion and banding. The
gels can be stored in the refrigerator for several
weeks if the box is kept moist.

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

IX. Hematology and
Serology

43. Immunodiffusion:
Antigen-Antibody
Precipitatioin Reactions in
Gels

Immunodiffusion: Antigen-Antibody Precipitation Reactions in Gels EXERCISE 43 321

Radial Immunodiffusion

1 . Using a micropipettor, obtain 5 \xl of human
serum, and pipette it into a designated well of the
RID assay gel.

Human Blood Handling
Note: Observe universal
precautions, (see page xiii)

2. Reserve three wells for standard concentrations: a
high standard, a low standard, and a serum
control (provided in the RID kit).

3. Place the gel into the moist box, and incubate
for 24 hours at room temperature to allow
diffusion and banding. Again, the gels can be
stored in the refrigerator for several weeks if
the box is kept moist.

4. Read the results of your assay: Measure
the diameter of the circle of precipitate (in
centimeters) for the sample you loaded and
for the three standard samples. Record these
results in your laboratory report.

Immunoelectrophoresis

1 . Prepare 40 ml of 1% agarose: Add 0.4 g of

agarose to 40 ml of high-resolution buffer, pH
8.8, in a 125 ml flask. Microwave the mixture for
about 30 seconds, checking to make sure it does
not boil over. Using a hot glove, gently swirl the
flask, and return it to the microwave. Heat for 15
seconds, repeating this until no flecks of agarose
are visible in the flask. Let the molten agarose
cool until the flask is comfortable to handle, but
still warm.

2. While the agarose cools, prepare a horizontal gel
electrophoresis box by putting the dams securely in
place. Also prepare a trough-forming apparatus:
Obtain a 60 mm petri dish with lid, and tape a slide
to each side as shown in figure 43.5.

3. Once the agarose has cooled so that the flask is
comfortable to hold, pour the agarose into the
unit until it completely covers the platform. Place
the trough-forming apparatus at the center of the
platform (see figure 43.5). Allow the agarose to
solidify, about 10 minutes.

Figure 43.5 In preparation for immunoelectrophoresis, the trough-forming
apparatus is placed at the center of the molten agarose.

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

IX. Hematology and
Serology

43. Immunodiffusion:
Antigen-Antibody
Precipitatioin Reactions in
Gels

322 SECTION IX Hematology and Serology

4. When the gel is solid, gently remove the dams
and the trough- forming apparatus. Using
the large end of a plastic or glass dropper
(a diameter of about 0.5 cm), cut wells into each
gel as shown in the following template.
(See figure 43.4.)

5. Pour high-resolution buffer, pH 8.8, into the
electrophoresis box on either side of the gel,
being careful not to pour onto the gel itself, 1 or
2 inches deep. Cut two pieces of Whatman

chromatography paper wicks, and place them into
the apparatus as shown in figure 43.6. Be sure
that the paper is in contact with the gel and the
buffer at both ends of the gel.

6. Load the antigens: Changing micropipette tips
between samples, load 20 (il of bovine serum into
wells A and C, and 20 jil of bovine albumin into
well B. Do not load the troughs (figure 43.6Z?).

7. Electrophorese samples at 70 volts for 1.5 hours
(figure 43.6c).

8. Load the antibodies: After electrophoresis is
complete, load 50 jil of anti-bovine albumin into
trough 1 and 50 (il of anti-bovine serum into
trough 2. Again, change tips between samples.

9. Leave the gel in the electrophoresis apparatus,
and wrap a moist paper towel and plastic wrap
around it to create a moist container. Incubate for
24 to 48 hours at room temperature to allow
diffusion and banding.

(a)

" *■■>"--

{JC Sum

««P

•Aa-M*

(C)

(b)

Figure 43.6 Immunoelectrophoresis, (a) The wicks must be in contact with the buffer and the gel. (b) Load antigens into
the wells. Do not load the antibodies into the troughs until electophoresis is complete, (c) Electrophorese the samples at 70
volts for 1.5 hours.

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

IX. Hematology and
Serology

43. Immunodiffusion:
Antigen-Antibody
Precipitatioin Reactions in
Gels

Name

Lab Section

EXERCISE

Laboratory Report

Date

mmunodiffusion: Antigen-Antibody Precipitation Reactions in Gels

Double Immunodiffusion (Ouchterlony)

1 . A precipitin line represents a specific antibody-antigen reaction occurring between the antibodies
diffusing from the center well with antigen diffusing from one of the outer wells. The precipitin line
should be perpendicular to an imaginary straight line drawn from an outer well to the center well.
The predicted results for pattern A are shown here. Predict the results for patterns B and C.

Pattern A

Pattern B

Pattern C

2. The double immunodiffusion assay also can be done to test the relatedness of antigens loaded into

adjacent wells. If the two antigen samples are identical, a smooth corner forms where the two lines meet
(identity). If the two antigen samples are not identical but related, then a spur forms at the corner (partial
identity). Finally, if the two adjacent antigen samples are not related at all, two spurs form at the corner
(nonidentity ) .

Antibodies specific for all human serum proteins were loaded in the center well.

Cytochrome c

Hen egg
lysozyme

IgG

f j— Albumin

Partial identity

Albumin

Nonidentity

IgG, IgA, IgM

No reaction is expected between the center well and the antigen wells containing hen egg lysozyme
or cytochrome c.

3. Return to question 1, and consider whether spurs should be included in your predicted results.
Add spurs to the diagram if they are expected.

323

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

IX. Hematology and
Serology

43. Immunodiffusion:
Antigen-Antibody
Precipitatioin Reactions in
Gels

324 SECTION IX Hematology and Serology

4. Diagram your double immunodiffusion results. Do they agree with your predictions?

Pattern A

Radial Immunodiffusion

Pattern B

Pattern C

1 . Record the three standard immunoglobulin (Ig) concentrations and the diameter of each resulting
precipitin ring. Also record the diameter of the precipitin circle for the sample you loaded.

Ig standard
concentration

Diameter of
precipitate circle

Sample precipitin ring diameter:

2. Graph the standard results: On semilog paper, plot the three known, standard Ig concentrations (on the
log scale) versus the diameter of the corresponding precipitin rings (on the linear scale). If you did not
run standards, analyze the gel shown in figure 433a.

Comment on your results. The normal mean concentration of each antibody in serum is presented in
table 43.2. The radial immunodiffusion test is often done to determine the concentrations of IgG, IgA,
and IgM.

Table 40.2

The Five Classes or Isotypes of
Antibodies (Immunoglobulins)

Antibody isotype

Mean concentration
(mg/ml)

IgG

13.5

IgA

3.5

IgM

1.5

IgD

0.03

IgE

0.0005

Source: The Difco Manual. Eleventh Edition. Difco Laboratories.

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

IX. Hematology and
Serology

43. Immunodiffusion:
Antigen-Antibody
Precipitatioin Reactions in
Gels

Immunodiffusion: Antigen-Antibody Precipitation Reactions in Gels EXERCISE 43 325

Immunoelectrophoresis

1 . Diagram the results of the IEP assay.

2. The following diagrams represent the results of two IEP assays done on the serum of a young child who
has experienced frequent infections since infancy. What is your diagnosis?

Child's serum

Rabbit anti-human serum

Control serum

Child's serum

Control serum

Rabbit anti-human IgG

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

IX. Hematology and
Serology

44. Enzyme-linked
Immunosorbent Assay
(ELISA)

Enzyme-linked Immunosorbent Assay (ELISA)

In this laboratory experiment, we will simulate the trans-
mission of a hypothetical infectious disease among mem-
bers of the class. The infectious agent or antigen we are
using is a harmless protein. However, for our purposes,
consider it contagious and dangerous ! The transmission
of this hypothetical disease sets the stage for an ELISA
(enzyme-linked immunosorbent assay), an antibody-
based test that is commonly used as a research and diag-
nostic tool, and is the basis of the screening test for HIV.
The ELISA takes advantage of the strong and specific
attachment that occurs between an antibody and an anti-
gen (thus the term immunosorbent). It is enzyme -linked
because an enzyme is covalently attached to the tail por-
tion of the antibody. The enzyme linked to the antibody is
one that catalyzes the conversion of a colorless substrate
into a colored product.

In an ELISA, the test sample, here simulated body
fluid, is loaded into one well of a 96- well microliter plate.
Next, the enzyme-linked antibody specific for the infec-
tious agent is added to each well. After washing to remove
nonspecifically bound antibodies, the chromogenic (color-
generating) substrate is added. The development of color
in a well indicates a positive result; if the sample remains
colorless, it is negative (figure 44.1). In addition, the inten-
sity of color is an indication of the amount of reaction
product in that well, which in turn correlates with the
amount of enzyme-linked antibody — and so, the concen-
tration of antigen — in the well.

Like the precipitin reactions (ID, IEP, and RID) and
agglutination reactions, the ELISA takes advantage of
a specific interaction between an antibody and an anti-
gen, but unlike these, detection by ELISA doesn't
require the formation of a large antibody-antigen com-
plex. Therefore, the ELISA is much more sensitive than

• •

precipitin- type tests.

*Adapted from "Simulating the Spread of HIV" at The Biology
Project, an interactive online resource for learning biology, developed
at the University of Arizona: www.biology.arizona.edu

To carry out the experiment,
given a solution representing your own body fluid. You
will exchange some of your body fluid with three other
randomly chosen members of the class. Then you will
perform an ELISA to test for the simulated disease
agent in your "exposed" body fluid. Given the class
results, it will be possible for you to trace the pathways
of transmission and identify the original carrier or car-
riers of the disease.

Once-clear substrate
converted to a colored product
in the presence of enzyme, E

Enzyme-linked antibody
specific for the antigen

Antigen

(a)

(b)

Figure 44.1 The enzyme-linked immunosorbent assay,
(a) A diagram of the components of a typical ELISA
depicting what would be a positive result, (b) A photo-
graph of an ELISA plate after development. Row A 1-6
contains a negative control, and row Bl-6 contains a posi-
tive control. The test samples are in rows D (1-6), E (1-6),
G (1-6), and H (1-6). No other wells on the plate contain
test samples. The samples in rows D, E, and H are positive.

327

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

IX. Hematology and
Serology

44. Enzyme-linked
Immunosorbent Assay
(ELISA)

328 Section IX Hematology and Serology

Materials

Exchange of "Body Fluids"

Reagents (see table 44.1)

Two microfuge tubes that contain the solution

representing body fluid (per person)
1 .0 ml serological pipette/pipettor or Pasteur

pipette/bulb

Analysis of Samples by ELISA

Reagents (per pair)

Positive control (contains infectious agent)
Negative control (contains no infectious agent)
Nonsharing fluid (partner A sample)
Sharing fluid (partner A sample)
Nonsharing fluid (partner B sample)
Sharing fluid (partner B sample)
Washing buffer

Enzyme-linked antibody reagent
Substrate (color-change reagent)

Miscellaneous supplies (per pair)
96-well ELISA microtiter plate
Micropipettor/tips (100 jll)
10 ml pipette/pipettor

Pasteur pipettes/bulb

One piece of bench-coat absorbent paper

Procedure

Exchange of "Body Fluids"

1 . Label the two body fluid tubes with your name,
and place one of them (labeled "nonsharing") in
the rack at the front of the room. Use the second
body fluid tube (labeled "sharing") for the
following steps.

Proceed with steps 2 through 4 using your
"sharing" tube. At the end of each exchange, you
should have about the same volume of fluid you
started with.

2. Using a transfer pipette, exchange about one-half
of your sharing fluid with another person in the
room. In table 44.2, record the name of the
person you first made contact with.

NOTE: Make sure you share with people in
different parts of the room to prevent a local
epidemic — spread "it" around.

Table AAA Table of Reagent Recipes for Simi

ilated Infectious Disease Transmission

Simulated

Identity

Recipe

substance

Body fluid

Sodium carbonate buffer

0.16 g sodium carbonate
0.27 g sodium bicarbonate
in 100 ml distilled water

Viral antigen

Biotinylated albumin

1 ul of biotinylated albumin at 6 mg/ml

(Sigma- Aldrich #A 8549)

in 10 ml of sodium carbonate buffer

Wash buffer

PBS/0.1% Tween-20

32 g sodium chloride

0.8 g potassium chloride

4.48 g sodium phosphate, dibasic

0.8 g potassium phosphate, monobasic

2 ml Tween-20

Distilled water to 2 liters

Enzyme-linked

Streptavidin peroxidase

5 ul of streptavidin peroxidase (0.5 mg/ml

antiviral antibody

(Sigma- Aldrich #5512)

50% glycerol) in 50 ml of wash buffer

Substrate

TMB (tetramethylbenzidine)

Phosphate citrate solution: Combine 25.7 ml

in phosphate citrate solution

0.2 M dibasic sodium phosphate and 24.3 ml
0. 1 M citric acid solution with 50 ml
distilled water.

TMB tablets

Dissolve 3 mg TMB in 30 ml of phosphate

(Sigma- Aldrich #T 3405)

citrate solution. Add 5 ul 30% hydrogen
peroxide. (Use same day; keep cold and dark.)

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

IX. Hematology and
Serology

44. Enzyme-linked
Immunosorbent Assay
(ELISA)

Enzyme-linked Immunosorbent Assay (ELISA) EXERCISE 44 329

Table 44.2 ELISA Results and Potential Transmission Events

Name

Sharing
ELISA
results

Exchange
1

Exchange

Exchange
3

Analysis (excluded or
not excluded as
original carrier)

Non-
sharing
ELISA
results

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

IX. Hematology and
Serology

44. Enzyme-linked
Immunosorbent Assay
(ELISA)

330 Section IX Hematology and Serology

3. At the instructor's signal, find a different person
to exchange one-half of your sharing fluid with.
Record the name of your second contact.

4. At the instructor's signal, find a third person to
exchange about one-half of your fluid with.
Record the name of your third contact.

Analysis of Samples by ELISA

7 8

10 11 12

Join with a partner to proceed with the ELISA.
The ELISA is designed to establish whether or
not the infectious agent is present in your body
fluid samples.

Write your names or initials on the plate edge.
If you are using a Pasteur pipette to add
samples and reagents to the ELISA wells,
pipette 2 drops of each sample or reagent.
Always change pipettes between different
samples and reagents. If you are using a
micropipettor to add samples and reagents,
set it at 100 jil. Always change tips between
different samples and reagents. Load controls
and "body fluid" samples as shown in the
following list and in figure 44.2. Pipette
carefully and accurately.

To wells:
A1-A6 (row A)
Bl-B6(rowB)
D1-D6 (row D)
E1-E6 (row E)
G1-G6 (row G)
H1-H6 (row H)

Add 2 drops or 100 fll of:
negative control
positive control
nonsharing fluid (partner A)
sharing fluid (partner A)
nonsharing fluid (partner B)
sharing fluid (partner B)

3. Incubate the samples at room temperature for
10 minutes, undisturbed.

4. Discard the liquid contents into the sink, and then
place the plate facedown on absorbent paper with
some force to remove any remaining liquid from
the wells.

5 . Using a 1 ml pipette, fill each of the wells that
you used with wash buffer. Discard the wash
solution into the sink.

6. Repeat step 5 twice (for a total of three washes).

7. Place the plate facedown on absorbent paper with
some force to remove any remaining liquid from
the wells.

8. Add 2 drops (or 100 jil) of enzyme- linked
antibody to each of the wells (all the ones you
used).

9. Incubate the samples at room temperature for
10 minutes, undisturbed.

C^\ Negative
control

Positive
control

Q Body fluid
samples

Figure 44.2 Diagram of a 96- well microtiter plate
format and numbering system; colored areas represent
loaded samples.

10. Wash the plate thoroughly by repeating steps 4-7

11. Add 2 drops (or 100 jil) of substrate to each of
the wells you used Except those in rows D and
G. Rows D and G contain nonsharing fluids and
will be assayed later.

12. After about 5 minutes of development, examine
the qualitative results with respect to sample
color changes.

Analyze Data; Determine
Original Carrier(s)

1. Record your results in table 44.2 and on the
board, providing your name, the results of the
ELISA of your own "sharing sample" (+ or - ),
and the three people with whom you exchanged
fluid, in order.

2. Join with two other pairs of students. As a
group, work through the path of transmission to
determine who the original carrier(s) might be.

3. Working in your original pairs, go back to your
plate and add 2 drops (or 100 jil) of substrate to
the wells in rows D and G (Dl-6 and Gl-6).
Examine the sample results as in step 12.
Remember that these are the original

nonsharing" fluid samples.

4. Record the results of the nonsharing fluid
samples in table 44.2 and on the board.
Determine if your group's conclusions were
correct regarding the original carrier(s).

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

IX. Hematology and
Serology

44. Enzyme-linked
Immunosorbent Assay
(ELISA)

Name

Lab Section

EXERCISE

Laboratory Report

Date

Enzyme-linked Immunosorbent Assay (ELISA)

1 . What happened in each test well? In the wells depicted below, diagram a positive ELISA and
a negative ELISA. Include:

• infectious agent (antigen)

• enzyme-linked antibody where appropriate

• the substrate and whether it is clear or colored

Positive ELISA result

Negative ELISA result

Microtiter well

2. Describe the path of transmission that occurred in the class. In addition, formulate a flowchart that
depicts the path.

3. It has been said that, "When you share fluid with someone, you are also sharing fluid with everyone they
have previously shared fluid with." Addressing your results, discuss whether you agree or disagree with
this statement.

331

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

IX. Hematology and
Serology

44. Enzyme-linked
Immunosorbent Assay
(ELISA)

332 SECTION IX Hematology and Serology

4. In the ELISA, what would happen if you eliminated the washing step prior to adding substrate?

5. The ELISA screening test for HIV actually detects antibodies to HIV, not HIV itself, in a serum sample

a. Why does the presence of antibodies specific for HIV in serum indicate that a person is infected
with the virus?

b. In the HIV antibody ELISA, HIV antigens are first loaded into wells of a microtiter plate. The serum
sample to be tested is then loaded into the well. If the serum contains antibodies specific for HIV, they
will remain in the well, bound to HIV, even after the wash step. However, the antibodies are not
enzyme- linked. A second-step antibody, specific for human antibody tails (IgG), is therefore added to
the well next. This second antibody is enzyme-linked. An ELISA that requires two antibody steps is
referred to as an indirect ELISA. Diagram a positive HIV antibody ELISA, including HIV, serum
antibody, enzyme-linked second antibody, and substrate.

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

IX. Hematology and
Serology

45. Bacterial Protein
Fingerprinting and Western
Blotting

Bacterial Protein Fingerprinting
and Western Blotting

Just as any organism has a distinctive genome, it like-
wise has a proteome, a particular set of proteins char-
acteristic of its species. The proteins can be extracted
from cells and separated by gel electrophoresis to gen-
erate a pattern of bands, or a protein fingerprint, that
is unique to that species. Although a typical bacterial
cell contains about 2,000 different proteins, the finger-
print procedure outlined here reveals only those proteins
present at high concentrations in the cells. Therefore,
it will be possible for you to discern the fingerprint of
each bacterial strain tested and compare it with others.
It should be noted that different bacterial species have
a number of proteins in common. However, these shared
proteins will likely exhibit strain- specific differences in
concentration, size (molecular weight), charge, shape,
and reactivity to antibodies.

As you saw in Exercise 43, proteins separate by
charge when exposed to an electric field. In order to
separate proteins electrophoretically by size, they are
first mixed with SDS (sodium dodecyl sulfate), a neg-
atively charged detergent. SDS binds to all proteins in
the mixture and denatures them so that each molecule
assumes a random coil configuration — and becomes
negatively charged. Thus, each protein will migrate
toward the anode during electrophoresis, and its rate
of migration will depend on its size. Larger random coil
chains take longer to slither through the gel matrix
(there is more drag), while smaller random coil chains
migrate more rapidly through the gel matrix. Therefore,
the mobility of each protein in an SDS-polyacrylamide
gel (PAGE) is inversely proportional to the log of its
molecular weight. The proteins in the gel are then visu-
alized by staining, and the particular banding pattern, or
fingerprint, of each bacterial strain can be discerned
(figure 45.1).

While SDS -PAGE provides information about a
protein's molecular weight — and here, a fingerprint —
the identification of a specific protein within the pro-
teome can be accomplished by following SDS-PAGE
with Western blotting. In this method, proteins sepa-
rated by SDS-PAGE are transferred from the gel onto the

Molecular Weight
(Daltons)

206,000 (blue)

124,000 (magenta)
83,000 (green) —

42,000 (violet) -
32,200 (orange)

18,800 (red)
7,000 (blue)

Figure 45.1 A stained gel containing bacterial proteins.
A protein size marker (BioRad #161-0324) was loaded into
lane 1. Proteins from E. coli B (lanes 2 and 6), S. marces-
cens (lane 3), M. luteus (lane 4), and B. subtilis (lane 5)
were run on a 12% polyacrylamide gel, and the gel was
stained with Coomassie blue. E. coli and S. marcescens are
Gram-negative rods, M. luteus is a Gram-positive coccus,
and B. subtilis is a Gram-positive rod.

surface of a membrane such as nitrocellulose or nylon.
The membrane is then flooded with a solution contain-
ing labeled antibodies specific for a particular protein or
proteins. In this exercise, the antibody label is a cova-
lently linked enzyme, horseradish peroxidase, that con-
verts a colorless substrate into a colored product. So, the
Western is similar to an ELISA, except that the antigen
is bound to a membrane instead of a plastic well, and
a positive result appears as a colored band on the mem-
brane instead of a colored liquid in a well. In this exer-
cise, you will subject a duplicate, unstained fingerprint
gel to Western blotting, and detect E. coli proteins with
E. co//-specific horseradish peroxidase-linked antibod-
ies (figure 45.2). At the completion of the Western blot
procedure, the only bands you will see on the mem-
brane will represent E. coli proteins. Keep in mind
that some of these proteins (bands) may appear in
non-£. coli samples, since different strains of bacteria
have some proteins in common.

333

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

IX. Hematology and
Serology

45. Bacterial Protein
Fingerprinting and Western
Blotting

334 Section IX Hematology and Serology

Molecular Weight
(Daltons)

206,000 (blue)
124,000 (magenta)

32,200 (orange)
18,800 (red)

7,000 (blue)

Figure 45.2 Photograph of a Western blot. Proteins
were subjected to SDS-PAGE as described in figure 45.1,
and transferred to a membrane for the detection of E. coli
proteins with specific antibodies. The samples are a pro-
tein size marker (lane 1) and proteins from E. coli B (lane
2), S. marcescens (lanes 3 and 6), M. luteus (lane 4), and
B. subtilis (lane 5). Note that antibodies have bound to
proteins in the E. coli and S. marcescens samples (both
Gram-negative), but not to proteins of M. luteus or
B. subtilis (Gram-positive).

First Session: Preparation
of Crude Protein Extracts

The following cultures and reagents are available in the
Identification of Bacterial Protein Profiles Kit.

Cultures

Bacterial strains grown as lawns on LB agar

plates
E. coli B, Serratia marcescens, Micrococcus

luteus, Bacillus subtilis

Reagents

Tris-EDTA-glucose (TEG) solution (25 rnM

Tris-Cl, pH 8.0, 50 mM glucose, 10 mM

EDTA)
TEG containing lysozyme 5 mg/ml, prepared

the day of lab and stored cold
Sample loading buffer (table 45.1)

Equipment

Microcentrifuge

37°C water bath or heat block

Boiling water bath with microfuge tube rack

Miscellaneous supplies (for all parts of this
exercise)
Latex gloves

Laboratory marker

Micropipettor/tips (10-100 |il, 100-1,000 jil)

1 ml and 1 ml pipettes/pipettor

100 ml beaker

1 cc syringe, 1 8 g needle

1.5 ml microfuge tubes

Large weigh dishes for gel staining and

Western blot incubations
Bench-coat absorbent paper
Receptacle to collect used antibody-blotto

Second Session: SDS-Polyacrylamide
Gel Preparation and Electrophoresis

Unpolymerized acrylamide is a
neurotoxin. Always wear gloves
and a lab coat when handling it.
Since unpolymerized acrylamide
may be present at the edges of
polymerized gels, always handle gels
with gloves.

Reagents

Precast 12% poly acrylamide gels

Or, to cast gels: reagents for poly acrylamide

gel formation
Or, prepare gel solutions per table 45.1

Equipment

Mini Protean II Cell
Power supply

Gel preparation kit (included in the BioRad
Mini Protean II system)

Gel Staining/Western Blot Assembly
and Transfer

Reagents

Western transfer buffer (table 45 . 1 )
Blocking buffer (blotto) (table 45.1)

Equipment
Power supply
White light box

Mini Transblot Electrophoresis Transfer Cell
Optitran BA-S supported nitrocellulose

membranes
3MM chromatography paper

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

IX. Hematology and
Serology

45. Bacterial Protein
Fingerprinting and Western
Blotting

Bacterial Protein Fingerprinting and Western Blotting

Exercise 45 335

Table 45.1 Reagents for SDS-Polyacrylamide Gel Electrophoresis, Staining, and Western Blotting

SDS-Polyacrylamide Gel Electrophoresis

4x electrophoresis "running" buffer

Tris-base

90.85 g

10% SDS

20 ml

dH 2

to 500 ml

Adjust pH to 8.8 with HC1; store at 4°C.

Sample loading buffer

4x running buffer

1.1ml

Glycerol

1.75 ml

dH 2

2.4 ml

2-mercaptoethanol

0.5 ml

0. 1 % bromphenol blue

0.25 ml

10% SDS

4ml

30% acrylamide: bis aery lamide 29:1

Acrylamide

60 g

Bis-acrylamide

1.6 g

CAUTION: Wear gloves and goggles whenever

dH 2

to 200 ml

handling acrylamide solutions; wear

a mask when working with dry powder.

Filter through .45 Jim filter; store at 4°C.

10% sodium dodecyl sulfate (SDS)

SDS 50g

in 500 ml dH 2

Acrylamide gel solution

30% acrylamide:bisacrylamide 29:1

20 ml

(50 ml for 6, 12% gels)

4x running buffer

12.5 ml

dH 2

16 ml

10% ammonium per sulfate

1.5 ml

Gently swirl to mix; when the pouring apparatuses

have been prepared, add TEMED, swirl (

gently, and

immediately pour the gels.

TEMED

25 pi

lx running buffer

4x running buffer

250 ml

10% SDS

10 ml

Use this concentration for electrophoresis

dH 2

to 1 liter

Gel staining (Do not stain a gel if you plan to

proceed with Western blotting.)

Coomassie stain

Coomassie brilliant blue (250)

1.25 g

Glacial acetic acid

50 ml

Isopropanol

125 ml

dH 2

325 ml

Destain

Methanol

100 ml

Glacial acetic acid

140 ml

Destain can be regenerated by running it through

dH 2

to 2 liters

activated carbon in a filter funnel.

Gel storage solution

dH 2

425 ml

Glacial acetic acid

50 ml

Glycerol

25 ml
(Continued)

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

IX. Hematology and
Serology

45. Bacterial Protein
Fingerprinting and Western
Blotting

336 Section IX Hematology and Serology

Table 45.1 Reagents for SDS-Polyacrylam

ide Gel Electrophoresis,

Staining,

and Western Blotting

(continued)

Western transfer and blot development

Western transfer buffer

Methanol optima
Tris base
Glycine
dH 2

200 ml
3.03 g
14.4 g
to 1 liter, CHILL

Tris-buffered saline (TBS)

Tris-Cl
NaCl

dH 2

15.7 g

to 7.5
to 1 liter

Blocking buffer (5% blotto)

Carnation instant nonfat dried milk 25 g in 500 ml TBS;

store cold.

Third Session: Membrane Treatment
and Development

Reagents

Blocking buffer (blotto) (table 45.1)

TBS (table 45.1)

Antibody: rabbit anti-is. coli antigens,

HRP-linked
Substrate: TMB 3,3,5,5,-tetramethylbenzidine

First Session: Preparation
of Bacterial Protein Extracts

1 . Obtain a bacterial plate culture. The plate should
have a confluent or nearly confluent lawn of
bacteria (figure 45.3). Obtain a 10 ml test tube
and a microfuge tube. Label each tube the same
way the plate is labeled. Weigh the empty
microfuge tube.

2. Pipette 4 ml Tris-EDTA-glucose onto the plate.
Using a sterile inoculating loop or rubber
policeman, gently scrape the entire plate to
release the cells.

3 . Transfer the fluid containing the released cells to
the 1 ml tube, tilting the plate as needed to
collect as much of the liquid and cells as
possible. Once the liquid is in the 10 ml test
tube, gently pipette up and down to disperse any
cell clumps. You can also vortex the capped tube
to suspend the cells.

4. Once there are no remaining clumps of cells,
transfer 1 ml of the suspension into the

microfuge tube. Cap the tube, and centrifuge it
for 1 minute at 14,000 RPM.

5. Decant the supernatant into a waste receptacle,
and drain the remaining liquid onto a tissue.
Weigh the tube once again to determine the
weight of the cell pellet. Dispose of the tissue in
a biohazard bag.

6. Resuspend the pellet with TEG so that the final
concentration is 100 mg cells/ml. (For example, if
you have 50 mg cells, suspend the cells in 0.5 ml
TEG.) Mix the cells well by pipetting or
vortexing, until there are no clumps.

7. Add one-tenth volume of TEG containing
lysozyme. For example, if you have a 0.5 ml cell
suspension, add 50 jil TEG-lysozyme. Mix the
sample by pipetting up and down.

8. Incubate the sample at 37°C for 30 minutes.

9. Transfer 250 jil of your sample into a fresh,
labeled microfuge tube, and add 750 (il of sample
loading buffer to it. If you have 250 jlxI of sample
or less, keep the sample in the original tube and
add three times the volume of sample loading
buffer to it. For example, if you have 130 jil, add
390 (il of sample loading buffer to the tube.

10. Cap the microfuge tube, and poke a small hole in
the top using a needle. This will prevent the cap
from popping open during the boiling step.
Alternatively, use a screw-cap microfuge tube.

1 1 . Place the capped tube into a boiling water bath
for 10 minutes.

12. Allow the sample to cool, and then centrifuge it
for 5 minutes at 14,000 RPM. Transfer most of

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

IX. Hematology and
Serology

45. Bacterial Protein
Fingerprinting and Western
Blotting

Bacterial Protein Fingerprinting and Western Blotting

Exercise 45 337

(a)

(b)

(c)

(d)

Figure 45.3 Confluent or nearly confluent growth of various bacterial strains on LB agar plates. The cultures were grown
overnight at 30 °C. (a) Escherichia coli. (b) Serratia marcescens. (c) Micrococcus luteus (d) Bacillus subtilis.

the liquid into a fresh, labeled tube, leaving a
small volume in the original tube. There may or
may not be a visible pellet. Discard the tube
containing the small volume along with any pellet

13. Store the protein samples in the freezer, or
proceed to the next step.

Second Session: SDS-Polyacrylamide
Gel Preparation

1. If you are using precast gels, proceed to step 3.
If you are using Reagents for Polyacrylamide
Gel Formation (Edvotek #251), work on
absorbent paper, wear gloves and a lab coat,
and follow the instructions included in the kit;
then proceed to step 3. To prepare the gel from
scratch, go to step 2.

2. Working on absorbent paper and wearing gloves
and a lab coat, pipette the appropriate volumes
of 30% acrylamide:bis-acrylamide, 4x running
buffer, distilled water, and 10% ammonium
persulfate into a 100 ml beaker (see table 45.1).
Do not add TEMED, until you are ready to pour
the gel. When the gel-pouring apparatuses have
been prepared, add TEMED and swirl gently.
Using a 10 ml syringe fitted with a needle,
aspirate about 8 ml of the gel solution. Place the
bevel of the needle against the longer of the two
plates, and push the plunger gently, allowing the
solution to flow down between the plates. Place
the comb, and allow the gel to polymerize (about
5 minutes). The BioRad gel apparatus and
pouring procedure are shown in figure 45.4.

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

IX. Hematology and
Serology

45. Bacterial Protein
Fingerprinting and Western
Blotting

338 Section IX Hematology and Serology

Electrophoresis
chamber

Plate clamp
assembly

Casting stand

Glass plate
sandwich

Combs

(a) MiniProtean II electrophoresis cell components. The glass plate sandwiches consist of one long plate, one short plate,
and two spacers.

(b) Assemble the glass plate sandwich, and insert it into
the clamp assembly. Place it onto the stage of the casting
stand. Align the spacers and two plates so they are flush
with the stage platform. Be sure that the spacers are
straight and positioned at the outer edges of the plates.
Tighten the two upper knobs slightly to hold the plates and
spacers in place.

Figure 45.4 Assembly of the gel apparatus and
preparation of the gel for SDS-polyacrylamide gel
electrophoresis.

(d) Place the clamp assembly/plate sandwich onto the
gasket of the casting stand, holding the assembly at an
angle with its bottom end against the wall of the casting
stand. Secure the assembly in place by pressing down on
the white plastic clamp assembly (not the plates) and
bringing the assembly upright beneath the plastic over-
hang. The assembly should snap into position.

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

IX. Hematology and
Serology

45. Bacterial Protein
Fingerprinting and Western
Blotting

Bacterial Protein Fingerprinting and Western Blotting

Exercise 45 339

(e) Position the comb at an angle between the glass plates.
Pour the prepared gel solution with a syringe fitted with a
needle.

(f ) Position the comb so that there is still space between
teeth above the level of the short plate.

(g) Once the acrylamide has polymerized, gently remove the
comb. Clean the long plate, above the level of the short plate,
with a tissue. Dispose of the tissue in acrylamide waste.

(h) Attach the clamp assembly/plate sandwich/gel to the
cooling core. Slide the two wedges at the top of the clamp
assembly into the two small slots in the cooling core, and
snap the bottom of the clamp assembly into place.

(i) Snap a second clamp assembly/plate sandwich/gel onto
the cooling core, and place the entire assembly into the
electrophoresis chamber.

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

IX. Hematology and
Serology

45. Bacterial Protein
Fingerprinting and Western
Blotting

340 SECTION IX Hematology and Serology

3. If the protein samples you are working with were
previously frozen, boil them for 10 minutes prior
to loading the gel.

4. In consultation with the instructor, determine
the appropriate loading for your particular gel.
Record the loading order on a piece of notebook
paper. The samples you load should include at
least one "unknown" protein sample.

5. Load 20 \i\ of each sample into the designated
lanes. Note: Change micropipette tips between
samples. If possible, run each gel in duplicate,
one for staining and protein fingerprinting, and
one for Western blotting. Do not stain a gel if it is
to be analyzed by Western blotting.

6. Run the gel at 70 volts for 1.5 hours.

For a Gel to be Coomassie-stained:
Staining and Destaining the Gel

1. When SDS-PAGE is complete, place the gel into
a large weigh dish containing Coomassie blue
stain. Be sure that your gloves are wet with
running buffer when you pick up the gel. Lift
the gel by its two bottom corners.

2. Make sure that the gel is submerged in the stain,
and cover the dish with plastic wrap. Place the
dish on a rocker platform at a low setting for 1
hour. The gel can also be left overnight but will
require more extensive destaining.

3 . Place the gel into destain in a fresh weigh dish,
cover the dish with plastic wrap, and return the
gel to the rocker platform. After 5 minutes, pour
the destain off into the proper receptacle and add
fresh destain. Return the dish to the rocker
platform for 1 hour, or until the destain solution
is as blue as the gel itself. Repeat this until the
blue protein bands begin to appear. Destaining
can proceed overnight in a covered weigh dish.

4. When destaining is complete, soak the gel in
storage solution (see table 45.1).

5 . Place the gel on a light box, and examine the
banding patterns. Record your observations in
your laboratory report.

For a Gel to be Used for
Western Blotting and Detection
of E. coli Proteins

1 . While the gels are running, prepare material for
the transfer of proteins from the gel to the
nitrocellulose membrane (Western transfer).
Wearing clean gloves, cut one piece of the
membrane to the dimensions of the gel (8 cm

x 6 cm) and two pieces of Whatman paper
(9 cm x 7 cm). Place these into a large weigh
dish containing Western transfer buffer.

2. When SDS-PAGE is complete, place the gel into
another large weigh dish containing Western
transfer buffer.

3. Assemble the transfer apparatus. The assembly of
the Western apparatus is shown in figure 45.5.

4. Place the assembled cassette into the transfer
tank, and fill the tank with Western transfer
buffer. Allow the transfer of proteins to proceed
at 100 volts for 1 hour (or 30 volts overnight).

5. Disassemble the apparatus. The gel may be
discarded, or it can be stained to confirm the
transfer of proteins (protein bands should be
absent or significantly weaker than those in the
gel you stained).

6. Place the membrane, protein side up, into a large
weigh dish, and pipette 20 ml of blotto over it. Be
sure the membrane surface is covered with blotto.
Blotto acts as a blocking agent because milk
proteins bind to the membrane wherever proteins
(here, bacterial proteins) are not already bound.
Blocking is important to prevent the antibody
from binding nonspecifically to the membrane in
the next step. Incubate it at room temperature for
30 minutes with constant, gentle agitation on a
rocking platform. The membrane can also be left
in the blocking agent, covered with plastic wrap,
and stored in the refrigerator for up to a week.

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

IX. Hematology and
Serology

45. Bacterial Protein
Fingerprinting and Western
Blotting

Bacterial Protein Fingerprinting and Western Blotting

Exercise 45 341

(a)

(b)

(c)

(d)

(e)

(f)

Anode (+)

Direction of
electrophoresis

Clear cassette panel

Fiber pad

Blotting paper
Nitrocellulose membrane
Gel

Blotting paper
Fiber pad

Gray cassette panel

Cathode (-)

(g)

Figure 45.5 Preparation of the SDS-polyacrylamide gel for Western transfer, (a) The gel is soaked briefly in Western
transfer buffer placed onto a piece of wet 3MM paper atop a fiber pad and gray cassette panel. On top of this are layered
(b) nitrocellulose, (c) a second piece of wet 3MM paper, and (d) a second fiber pad. (e) The sandwich is then closed
between cassette panels, and (f) placed into the electrophoresis chamber, (g) Diagram of assembly components.

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

IX. Hematology and
Serology

45. Bacterial Protein
Fingerprinting and Western
Blotting

342 Section IX Hematology and Serology

Third Session: Membrane Treatment
and Development

1 . Holding the membrane with a gloved hand, pour
the blotto into the sink, and pipette about 5 ml of
blotto containing antibody (rabbit anti-£. coli
antigens, HRP-linked, diluted as suggested by
manufacturer) onto the membrane, completely
covering it. Incubate at room temperature for 30
minutes with constant, gentle agitation on a
rocker platform.

2. Holding the membrane with a gloved hand, pour
the antibody-blotto into a receptacle (the antibody
can be re-used). Wash the membrane three times
with blotto: Pour about 50 ml blotto onto the
membrane, and place it on the rocker platform for
10 minutes. Pour the blotto wash into the sink.
Repeat this twice for a total of three washes.

3. Be sure the blotto has been fairly well drained
from the dish. Pipette about 50 ml of TBS
(without milk) onto the membrane, and incubate
at room temperature on a rocker platform for 10
minutes. Pour the TBS off, and repeat the wash
once with fresh TB S .

4. Pour off TBS, and pipette 5 ml of TMB substrate
onto the membrane. The time necessary for color
development will vary, usually from 3 to 1
minutes. Terminate the reaction by rinsing the
blot with distilled water.

5. Allow the membrane to air-dry. Examine the
banding patterns, and record your observations in
your laboratory report.

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

IX. Hematology and
Serology

45. Bacterial Protein
Fingerprinting and Western
Blotting

EXERCISE

Laboratory Report

Name

Date

Lab Section

Bacterial Protein Fingerprinting and Western Blotting

Preparation of Crude Protein Extracts, Electrophoresis, and Gel Staining: Protein Fingerprinting

1 . Compare the protein banding pattern in each lane of the stained gel. Provide a brief discussion
of your results.

2. Given that the mobility of a protein in an SDS-polyacrylamide gel (PAGE) is inversely proportional to
the log of its molecular weight, it is possible to determine the approximate size of an unknown protein
or band using a standard graph. Measure the distance migrated by each band in the standard lane, and
complete the following table.

Standard protein
molecular weight (KD)

Distance migrated (cm)

3. Use these values to produce a graph on semilog paper. Graph the molecular weight of each standard
protein (log scale) versus the distance migrated (linear scale).

343

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

IX. Hematology and
Serology

45. Bacterial Protein
Fingerprinting and Western
Blotting

344 Section IX Hematology and Serology

4. Use the standard graph to determine the size of one of the proteins in the E. coli sample. Choose one of
the strongest- staining bands. Once you have determined the approximate molecular weight of the E. coli
protein, do some research on bacterial proteins to see if you can suggest the protein's identity.

Western Transfer and Membrane Treatment and Development: E. coli Protein Detection
by Western Blotting

1 . Which lane or lanes do you expect to be positive in the Western blot? Does this agree with your results?

2. Do any of the proteins in the other lanes appear on the Western blot? Briefly discuss this result.

3. What would have happened if you had eliminated the blocking step? What would your developed
membrane have looked like?

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

IX. Hematology and
Serology

46. The Neutralization of
Viruses by Antibodies

The Neutralization of Viruses by Antibodies

In Exercises 41-45, you learned about a number of
serological methods, so called because each, in some
way, takes advantage of antibody- antigen binding, usu-
ally employing antibodies to detect antigens. Let us now
return to antibodies in their natural locale, circulating in
the blood and tissue fluid, having been secreted from
plasma cells (differentiated B cells) in response to for-
eign antigens. While it is obvious that these antibod-
ies attach specifically to foreign substances, such as
viruses, bacteria and bacterial toxins in the blood and
tissue spaces, their effects upon these substances are not
as apparent. How do antibodies help clear microbes,
toxins, and foreign debris from the body?

As noted previously, antibodies tend to form com-
plexes with their corresponding antigens, agglutinating
cells and viruses, or precipitating free-floating molecules.
These antibody- antigen complexes are at the core of three
important infection-defeating mechanisms: neutraliza-
tion, opsonization, and complement activation.

Neutralization is the simplest of the three mecha-
nisms because it occurs simply as a consequence of anti-

bodies binding to antigens. For exampl
bound to a bacterial toxin effectively block the toxin from
contacting its target tissue and generating symptoms of
the infection (figure 46.1a). Similarly, antibodies block,
or neutralize, a virus by attaching to the molecules that
the virus must use to attach to its host cell.

In opsonization, the tail portions of antibodies in
the antibody-antigen complex become attached to
receptors on macrophages, greatly enhancing the effi-
ciency of phagoctyosis (figure 46.1b). In fact, a
macrophage takes in and destroys a substance about
4,000 times faster when it is coated with antibodies.
Complement activation can lead to the death of bac-
terial cells (mainly Gram-negative cells) and the
destruction of enveloped viruses (figure 46.1c). Com-
plement is a set of about 20 serum proteins that act in
a cascade of steps. The cascade begins with the binding
of the first component, CI, to two adjacent antibodies
(class IgM or IgG) that are in turn attached to antigen
on the surface of a bacterium; the cascade ends with the
formation of a large pore in the bacterial membrane.
With this loss of membrane integrity, fluid rushes into
the bacterial cell, which then bursts, or lyses.

345

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

IX. Hematology and
Serology

46. The Neutralization of
Viruses by Antibodies

346 SECTION IX Hematology and Serology

(a) Neutralization Antibodies block viruses from
infecting cells and block the effects of
bacterial toxins.

(b) Opsonization Antigen-antibody complexes are
effectively marked for efficient phagocytosis
by macrophages.

Antigen-antibody
complexes

Toxin

Water

(c) Complement activation Antibodies
bound to cell surface antigens activate
the complement system, leading to
lysis of the cell.

Cell swells.

Cell lyses.

Figure 46.1 Antibody-mediated mechanisms of antigen disposal. The binding of antibody to foreign cells and molecules
results in agglutination, the formation of large complexes. Antibodies mark these substances for defeat by (a) neutralization,
(b) opsonization, and (c) complement activation.

In this exercise, you will observe the impact of
antivirus antibodies on the capacity of viruses to infect
cells. The virus in this case is bacteriophage T4, and the
susceptible host cell is E. coli B (figure 46.2). Keep in
mind that, although bacteria have ways of defeating
virus infections (namely, restriction endonucleases),

they do not exhibit specific immunity, and they do not
make antibodies. For our purposes, however, consider
the bacteriophage a pathogenic human virus, and the
antibodies a result of a specific immune response to
the virus.

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

IX. Hematology and
Serology

46. The Neutralization of
Viruses by Antibodies

The Neutralization of Viruses by Antibodies EXERCISE 46 347

Figure 46.2 The effects of antibodies on virus infectivity.
On the left is a photograph of bacteriophage T4-E. coli
plating in the absence of antibody treatment. On the right is
a photograph of bacteriophage T4-Zs. coli plating after the
phage suspension was incubated with antibodies to T4 for
8 minutes. Viral neutralization is apparent in the diminished
number of plaques on the plate on the right.

Materials ^^^^^^B^l

Reagents

Disease Prevention Kit

Equipment

37°C incubator with shaker platform
Microwave oven or Bunsen burner
Water bath at 48°C

Miscellaneous supplies
Laboratory marker
100 ml beaker
Test tube rack

Procedure

First Session

Prepare Overnight Culture of E. coli B, and Label
Dilution Tubes, Soft Agar Tubes, and Agar Plates

1 . Inoculate 5 ml culture broth with E. coli B .
Incubate the culture overnight at 37°C with
shaking.

2. Label the 17 dilution broth tubes as shown:

P-l, P-2, P-3, P-4, P-5, P-6, A-l, AP-2, 2AP-3,
2AP-4, 2AP-5, 4AP-3, 4AP-4, 4AP-5, 8AP-3,
8AP-4, 8AP-5

• P stands for Phage (T4 bacteriophage), and
A stands for Antiserum (antibody to
bacteriophage T4).

• The negative number to the right indicates the
dilution of the sample: -1 means 10" 1 , or a 1:10
dilution; -2 means 10~ 2 , or a 1:100 dilution, and
so forth.

• The number to the left of the letters A/P refers to
the time (in minutes) that the antiserum and
phage will be allowed to interact prior to plating.

3. Place the AP-2 tube into the 37°C incubator, and
leave the others at room temperature.

4. Label the caps of the six tubes of soft agar: 1, 2,
3, 4, 5, X.

5. Label six agar plates: P-5, P-6, 2AP-5, 4AP-5,
8AP-5, 8AP-5X.

Second Session:

Dilute Stocks of Phage and Antiserum

1 . Measure 1.1 ml of the T4 phage stock, and
transfer 0.1 ml into the tube labeled AP-2 located
in the 37°C incubator and 1 ml into the tube
labeled P-l. Cap the tubes, and mix well. Return
the AP-2 tube to the incubator.

2. Pour the entire contents of the anti-T4 antiserum
container into the tube labeled A- 1 . (The
antiserum is now diluted 1:10 [10 -1 ]. Cap the
tube, and mix well.

Prepare Soft Agars, and Prepare and Plate
Phage Dilutions (P-l to P-6)

3 . Place the six tubes of soft agar into a 1 00 ml
beaker containing about 2 inches of water. Boil
the water using a microwave oven or a Bunsen
burner to melt the soft agar. Once the soft agar is
molten, let it cool until you can touch it, but it is
still quite warm (about 50°C). Place the tubes
into the 48 °C water bath. Once the tubes have
been at 48 °C for about 10 minutes, loosen each
cap, but keep the contents covered.

4. Transfer 0.1 ml of the overnight culture of
bacteria into each of the soft agar tubes except
tube X. You may use the same pipette to inoculate
tubes 1, 2, 3, 4, and 5.

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

IX. Hematology and
Serology

46. The Neutralization of
Viruses by Antibodies

348 SECTION IX Hematology and Serology

5. Complete the serial dilutions of phage (P-2 to
P-6), using a fresh pipette for each of the
following transfers: Transfer 1 ml of the P-l
mixture (from step 1) into tube P-2 , cap the tube,
and mix by shaking; transfer 1 ml from P-2 into
P-3, cap it, and mix by shaking; transfer 1 ml
from P-3 into P-4, cap it, and mix by shaking;
transfer 1 ml from P-4 into P-5, cap it, and mix
by shaking; transfer 1 ml from P-5 into P-6, cap
it, and mix by shaking.

6. If the molten soft agar tubes have been inoculated
with bacteria (step 4), transfer 1 ml of phage from
dilution tube P-5 into soft agar tube 1 . Quickly
cap the soft agar tube, and gently invert it a few
times to mix. Immediately pour the contents of
the tube onto the agar in the plate labeled P-5
(see figure 37.5). Cover the plate, and tilt it
slightly to spread the soft agar evenly. Allow the
soft agarose to solidify (about 10 minutes).

7. Repeat step 6, transferring 1 ml of phage from
dilution tube P-6 into soft agar tube 2, and plating
the phage as before.

Prepare Antiserum-Phage Reaction; Dilute
and Plate the Reaction at 2, 4, and 8 Minutes

8. Transfer 1 ml of antiserum from tube A-l (from
step 2) into tube AP-2 (from step 1) taken from
the 37°C incubator. Cap the tube, and mix by
shaking. Record the exact time, or set a timer for

8 minutes. Three teams (A, B, and C) should be
ready to plate phage (team A at 2 minutes: steps

9 and 10; team B at 4 minutes: steps 11 and 12;
and team C at 8 minutes: steps 13 and 14).

Team A

9. At exactly 2 minutes, quickly prepare serial
dilutions (AP-2 to 2AP-5), using a fresh pipette
for each of the following transfers: Transfer 1 ml
of the AP-2 mixture into tube 2AP-3, cap the
tube, and mix by shaking; transfer 1 ml from
2AP-3 into 2AP-4, cap it, and mix by shaking;
transfer 1 ml from 2AP-4 into 2AP-5, cap it, and
mix by shaking.

10. Transfer 1 ml of sample from the dilution tube
2AP-5 into the inoculated molten soft agar tube
3. As in step 6, quickly cap the soft agar tube,
and gently invert it a few times to mix it. Then
immediately pour the contents of the tube onto
the agar plate labeled 2AP-5 as described in
step 6.

TeamB

1 1 . At exactly 4 minutes, quickly prepare serial
dilutions (AP-2 to 4AP-5), using a fresh pipette
for each of the following transfers: Transfer 1 ml
of the AP-2 mixture into tube 4AP-3, cap the
tube, and mix by shaking; transfer 1 ml from tube
4AP-3 into tube 4AP-4, cap it, and mix by
shaking; transfer 1 ml from tube 4AP-4 into tube
4AP-5, cap it, and mix by shaking.

12. Transfer 1 ml of sample from the dilution tube
4AP-5 into the inoculated molten soft agar tube
4. As in step 6, quickly cap the soft agar tube,
and gently invert it a few times to mix it. Then
immediately pour the contents of the tube onto
the agar plate labeled 4AP-5 as described in
step 6.

TeamC

13. At exactly 8 minutes, quickly prepare serial
dilutions (AP-2 to 8AP-5), using a fresh pipette
for each of the following transfers: Transfer 1 ml
of the AP-2 mixture into tube 8AP-3, cap the tube
and mix by shaking; transfer 1 ml from tube
8AP-3 into tube 8AP-4, cap it, and mix by
shaking; transfer 1 ml from tube 8AP-4 into tube
8AP-5, cap it, and mix by shaking.

14. Transfer 1 ml of sample from the dilution tube
8AP-5 into the inoculated molten soft agar tube
X and another 1 ml of the 8AP-5 dilution into
molten soft agar 5. You can use the same pipette
for both transfers. As in step 6, quickly cap both
soft agar tubes, and gently invert them a few
times to mix them. Then immediately pour the
contents of each tube onto the corresponding
plates, labeled 8AP-5 and 8AP-5X, as described
in step 6.

15. Once the soft agar on each plate has solidified,
place them, inverted, into the 37°C incubator for
24 hours.

Third Session

Analysis of Antibody Neutralization

Examine the plates, and record the results in your
laboratory report.

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

IX. Hematology and
Serology

46. The Neutralization of
Viruses by Antibodies

Name

Lab Section

EXERCISE

Laboratory Report

Date

The Neutralization of Viruses by Antibodies

1 . Describe the appearance of each of the bacteriophage plating control plates, P-5 and P-6

2. Plate P-6 should contain identifiable plaques. Count the plaques, and complete the following table

Plate
designation

Number
of plaques

Dilution
factor

Volume of phage
suspension
plated (ml)

Phage titer
(PFU/ml)

3. Given the number of plaques on the P-6 plate, approximately how many plaques (or originally,
plaque-forming units) must there be on plate P-5, even though you may not be able to count them?

4. Considering the deduced number of plaques (infection events) on plate P-5, determine the extent of
phage neutralization, if any, by the antiserum at the designated times. Complete the following table.

Plate
designation

Antiserum- phage

incubation time

(minutes)

Number of

plaques per

plate

PFU/ml

remaining

infective

Percent successful
phage infection

Percent phage
inactivation

5. Given the data presented in the table in #4, discuss the impact that anti-T4 antibodies have on the ability
of T4 phage to infect E. coll cells.

349

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

IX. Hematology and
Serology

46. The Neutralization of
Viruses by Antibodies

350 SECTION IX Hematology and Serology

6. Describe the appearance of the control plate labeled 8AP-5X. What is the purpose of this control?

7. Diagram a T4 phage, and depict its attachment to and entry into its host cell. Where do you expect the
anti-T4 antibodies to be binding? Draw and briefly explain your answer.

8. A teenager comes into the emergency room complaining of headache and spasms of the jaw muscles. A
few weeks before, he stepped on a dirty nail, cutting his foot. Because he has never been immunized
against tetanus, his physician suspects that he has a Clostridium tetani infection. She orders a blood
culture and an injection of tetanus antitoxin, antibodies specific for the tetanus toxin. What is the purpose
of tetanus antitoxin in this case?

Alexander-Strete-Niles:
Lab Exercises in
Organismal and Molecular
Microbiology

IX. Hematology and
Serology

46. The Neutralization of
Viruses by Antibodies

The Neutralization of Viruses by Antibodies EXERCISE 46 351

9. Three 0.01 ml samples are taken from a liquid culture of E. coll. One sample, labeled A, is spread
directly onto an agar plate. Another sample, labeled B, is first treated with antibodies specific for
E. coli surface molecules, and then spread on a separate plate. The third sample, labeled C, is treated
with the same antibodies and complement proteins, and is spread on a third plate. The plates are placed
at 37 °C overnight.

The next day, there are about 30 colonies on plate A. Predict the results for plates B and C.
Explain your answer.

Plate A

Plate B

Plate C

10. Antibodies help dispose of foreign antigens by targeting them for destruction by opsonization or
complement activation. Briefly describe each of these processes.

11. In this exercise, you used polyclonal antibodies specific for bacteriophage T4. Draw a flowchart showing
how these antibodies might have been generated.

Alexander-Strete-Niles:

Back Matter

Credits

Lab Exercises in
Organismal and Molecular
Microbiology

Companies, 2003

Credits

Photographs

Figure 1.5

Courtesy Tom King, Scott & White
Hospital and Clinic, Temple, TX.

Figure 1.6

Courtesy Tom King, Scott & White
Hospital and Clinic, Temple, TX.

Figure 1.7

Courtesy Gerald V. Stokes and
Robyn Rufner, George Washington
University Medical School, Washing
ton, D.C.

Figure 1.8

Courtesy Gerald V. Stokes and
Robyn Rufner, George Washington
University Medical School, Washing
ton, D.C.

Figure 33.1

Huntington Potter and David
Dressier/Life Magazine 1980, Cour-
tesy Time Inc.

Figure 33.4

Lifetime Technologies.

Figure 35.1

Courtesy Charles C. Brinton and
Judith Carnahan.

Figure 36.1a

SPL/Photo Researchers.

Figure 36.1b

Science VU-NIH, R. Feldman/Visu-
als Unlimited.

Figure 36.2a

K.G. Murti/Visuals Unlimited.

Figure 37.1b

Thomas Broker/Phototake.

Figure 37.2b

Lee D. Simon/Photo Researchers,
Inc.

Figure 39.1b

Dennis Kunel/Phototake.

Figure 39.2

Runk/Schoenberger/Grant Heilman
Photography, Inc.

Figure 39.2b

Charles Marden Fitch/Talisman Cove
Productions.

Line Art

Figure 13.1

Alexander, Stretey 'Microbiology:
A Photographic Atlas for the
Laboratory with permission Pearson
Education.

Figure 17.5

Wistreich, Microbiology Laboratory
Fundamentals & Applications, with
permission Pearson Publishing.

Figure 17.6

Wistreich, Microbiology Laboratory
Fundamentals & Applications, with
permission Pearson Publishing.

Figure 25.2

Johnson, Case, Laboratory Experi-
ments in Microbiology with permis-
sion Pearson Education.

353

Alexander-Strete-Niles:

Back Matter

Index

Lab Exercises in
Organismal and Molecular
Microbiology

Companies, 2003

Index

ABO blood typing, 311-13
Acid-fast bacteria, 85
Acid-fast stain

morphology of bacteria, 65

staining procedures, 85, 86
Acidic stains, 66
Adenovirus, 268
Adonitol, and Enterotube II, 175
Adsorption, of virions, 285
Agar deep cultures

media preparation, 119

obligate aerobes and facultative
anaerobes, 135

stab technique for, 125
Agarose gel electrophoresis

plasmid isolation and restriction
mapping, 248-50

polymerase chain reaction, 27 1 ,
272-73

pulsed-field gel electrophoresis,
270

Southern blotting, 225, 226, 228,
231
Agar plates

culture characteristics of colonies,
135

inoculation, 113, 126

media preparation, 119, 122
Agar slants

culture characteristics of colonies,
135

inoculation, 124

media preparation, 119
Agglutination, of antibodies, 307,

311-13
Agranulocytes, 298, 299
AIDS, 268

Alcali genes denitrificans, 95, 102
Alcaligenes faecalis, 144, 148
Alcohol, as antiseptic, 205
Algae

classification of, 39

distinguishing traits, 40

size of, 1 8
Alkyldimethylbenzyl, 211
Allergies, and mast cells, 299

Alpha-hemolysis test, 164

Amebas, 39

Ames, Bruce, 239

Ames test, for mutagenesis of bacteria,

239^-1
Ammonium chloride, 211
Amoeba proteus, 18
Amoeba spp., and pseudo podia, 39
Amphitrichous flagella, 96
Amphotericin B, 112
Ampicillin

assessing effectiveness of, 185,

189
bacterial resistance, 255, 256,
261, 262
Amplification, of bacterial viruses, 280
Anthrax, 89
Antibiosis, 111, 114
Antibiotic resistance. See also
Antibiotics
bacterial conjugation, 261-62
bacterial transformation, 253-57
Escherichia coli, 247, 261, 262
Antibiotics. See also Antibiosis;

Antibiotic resistance; Penicillin
bacterium as producers of, 111-12
broad- spectrum, 185
Kirby-Bauer method for

assessment of effectiveness,
185-89
Antibodies

agglutination reaction, 311
antigen precipitation reactions and

titer, 305-8
neutralization of viruses by,
345^8
Antigens

antigen- antibody precipitation and

antibody titer, 305-8
antigen- antibody precipitation and

immunodiffusion, 317-22
immunity and immune response,
298
Antimicrobial chemical agents, 211
Antiseptics, 205-8. See also Aseptic

procedures; Disinfectants
Antiserum, 305, 306, 307
Antiserum-phage reaction, 348
Apicomplexa, 39

Apoptosis, 239

Aquatic ecosystems, and food chains,

55. See also Algae

Arabinose, and Enterotube II, 176

Arachnids, as disease vectors, 61

Arthropods, as disease vectors, 61

Ascariasis, 47

Ascaris lumbricoides, 47, 50

Ascospore, 33

Aseptic blood samples, 313

Aseptic procedures

media inoculation, 119, 120
removing specimens from broth
culture, 67

Aspergillus, 18

Autoclave, and sterilization of media,

119, 120,200

Autoimmune disorders, 311

Autotrophic bacteria, 25

Bacillus amyloliquefaciens, 246
Bacillus anthracis, 89
Bacillus cereus

cell size, shape, and arrangement,

102
gram stain, 78
negative stain, 66
spore stain, 90
Bacillus spp.

antibiotics and antibiosis, 111-12,

114
endospores, 89
size of, 1 8
Bacillus subtilis, 333, 334, 337
Bacitracin

assessing effectiveness of, 185,

189
Bacillus as source of, 112
beta-hemolytic streptococci, 164
Back mutations, 239
Bacteria. See also Bacterial cultivation;
Bacterial identification;
Bacterial infections; Cultures;
Genetics, bacterial; Staining
techniques; specific species

Alexander-Strete-Niles:

Back Matter

Index

Lab Exercises in
Organismal and Molecular
Microbiology

Companies, 2003

356 Index

Bacteria — Cont.
capsule, 65, 95
cell shapes and arrangements,

25-26
classification of, 25
endoscopes, 89
flagella arrangements, 96
laboratory environment, 1 10-11,

113-14
morphology, 65, 66
protein fingerprinting and Western

blotting, 333^2
size of, 1 8
Bacterial cultivation

aseptic technique and laboratory

environment, 110-14
culture characterization, 133-35
growth media preparation and
inoculation, 119-27
Bacterial identification

biochemical tests, 142^48
clinical unknowns, 193-95
enteric bacteria, 169-76
noncritical unknowns, 151-52
staining characterization, 101-3
Staphylococcus spp. from skin,

156-60
Streptococcus spp. from throat,

163-66
urinary tract and isolates,
179-81
Bacterial infections, controlling risk

and spread of. See also
Disease, human; Pathogens
countertops and disinfectants,

211-12
MPN method for evaluation of

drinking water, 215-18
skin disinfection, 205-8
sterilization with high
temperature, 200-202
Bacteriology, and drinking water,

215-18
Bacteriophages

bacterial transformation, 253
Escherichia coli, 277, 278, 346,

347
isolation of from sewage,
277-82
Basic stains, 66, 71
Basidiospore, 33
Basophils, 299
Benthic marine animals, 55
Benzalkonium chloride, 205
Beta-hemolysis test, 163-64
Bile esculin agar (BEA), 179
Biochemical tests, for identification of

bacteria, 142^8
Biology Project, The (website), 327
Biotinylated albumin, 328

Blocking buffer, and SDS-PAGE, 336

Blood. See also Body fluids;

Hematology; Serology
ABO typing, 311-13
collection of, 298, 300, 313
formed elements, 4
Wright's staining, 300-301

Blood agar plate, 165

B lymphocytes (B cells), 299, 305

Body fluids, 327, 328, 330.
See also Blood

Boiling water, and sterilization, 200

Botulism, 89

Broad-spectrum antibiotics, 185

Broth culture, aseptic procedure for
removal of specimens, 67

Broth inoculation, 123

Broth tubes, 119

Brown algae, 40, 41

Buds, of yeast cells, 33

Burst, and viral infection, 285

Cancer, 239, 268

Capillary blotting, and Southern

analysis, 233, 234
Capsid, of virus

structure of viruses, 268, 269

tobacco mosaic virus, 291
Capsule, of bacteria, 65, 95
Capsule-forming rod, 95
Capsule stain, 65, 95, 96-97
Carbofuchsin, and acid-fast stain, 85, 86
Carcinogens, and Ames test, 239
Catalase test, 142, 143
Cell arrangements, 25, 26, 102
Cell membrane, and morphology of

bacteria, 65
Cell morphology, of bacteria, 65, 66, 71
Cell shapes, 25-26, 102
Cell wall

capsule of bacteria, 95

gram-negative and gram-positive
bacteria, 77

morphology of bacteria, 65
Central endospore, 90
Central spores, 89
Centric diatoms, 18, 41
Ceratium, 41

Cercariae, and Schistosoma mansoni, 48
Cetylpuridinium chloride, 205
Chemical agents, in disinfectants, 211
Chlamydia, 8

Chloramphenicol, 112, 185, 189
Chloroform, and DNA isolation,

229-30
Chlorophyta, 40, 41

Cholera, 215

Chromogenic substrate, 327
Chromosomes, 245. See also DNA;

Genetics, bacterial
Chrysophyta, 40, 41
Cilia and ciliates, 39
Ciliophora, 39

Citrate, and Enterotube II, 176
Classification, of microorganisms. See
also Kingdoms; Phylums

bacteria and cyanobacteria, 25

flatworms and roundworms, 47

systems of, 17
Cloning, and plasmids, 246
Clonorchiasis, 47
Clonorchis sinensis

life cycle, 48

as pathogen, 47

size of, 1 8
Clostridium botulinum, 89
Clostridium perfringens, 89
Clostridium spp., and endoscopes, 89
Clostridium tetani, 89
Club (cell shape), 25
Coagulase, 156, 160
Coccus (cell shape), 25
Coleus blumei, 292

Coliforms, and water tests, 215-16, 278
Collection, of urine, 1 80
Competent cells, 254, 256-57
Complement activation, 345, 346
Completed test, and MPN method,

216,218
Complex media, 119
Confirmed test, and MPN method,

216,218
Congo red, 66
Conjugation, of bacterial DNA, 253,

261-62
Coomassie stain, 335, 340
Cords, cell arrangements, 25, 26
Coryne bacterium pseudodiphtheriticum,

102
Counterstain

acid-fast stain, 86

gram stain, 78, 79

spore stain, 90, 91
Countertops, cleaning with

disinfectants, 211-12
Cross-section method, of counting

white blood cells, 301
Crude protein extracts, and bacterial

protein fingerprinting, 334
Cryptosporidium, 85
Crystal violet

capsule stain, 97

gram stain, 77, 79

negative stain, 66

simple stain, 7 1
Culex, 61

Alexander-Strete-Niles:

Back Matter

Index

Lab Exercises in
Organismal and Molecular
Microbiology

Companies, 2003

Index

357

Cultures. See Bacterial cultivation;
Growth media, for cultures

Cup fungi, 33

Cyanobacteria

autotrophic organisms, 25

cell shapes and arrangements, 26

size of, 1 8

Cyclops, 18. See also Zooplankton

Cytoplasm, and morphology of
bacteria, 65

Daphnia, 18. See also Zooplankton
Decolorization

acid-fast stain, 85, 86

gram stain, 79

spore stain, 90
Denaturation, of DNA, 226, 27 1
Denitrification test, 142, 143-44
Dermacentor, 61
Destain, and gel staining for Western

blotting, 335, 340
Diarrhea, and Escherichia coli, 200
Diatoms, 18, 40, 41
Differential white blood cell count,

298, 301
Differentiation, of enteric bacteria,

173
Dinoflagellates

distinguishing traits, 40, 41

size of, 1 8
Dipicolinic acid, 89
Diplobacilli (cell shape), 25, 26
Diplococci (cell shape), 25, 26
Diphtheroids, 156, 163
Dipylidium caninum, 18
Disease, human. See also Bacterial

infections; Medical
microbiology; Pathogens;
specific diseases

antibiotic resistance, 253

autoimmune disorders, 311

bacterial pneumonia, 95

drinking water and coliforms, 215

endoscopes of bacteria, 89

immunity, 298, 305

multicellular parasites, 47

mycobacterium, 85

vectors, 61

viral, 268
Disinfectants, 205, 211-12. See also

Antiseptics
Disinfection, of skin, 205-8. See also

Sterilization
Dissecting microscope, 113
DNA. See also Genetics, bacterial

morphology of bacteria, 65

plasmid isolation and restriction

mapping, 245-50
polymerase chain reaction and

synthesis, 271
Southern Analysis, 224-35
viruses and fingerprinting,
268-73
Double immunodiffusion, 317-18,

319, 320
Drinking water, bacteriological
examination of, 215-18
Dry heat, and sterilization, 200
Dulcitol, and Enterotube II, 176
Dysentery, 169

Ebola fever, 268

Eclipse period, of viral infection, 285
E. coli. See Escherichia coli
Electrophoresis. See Agarose gel

electrophoresis;
Immunoelectrophoresis ;
Pulsed-field electrophoresis;
SDS-PAGE
ELISA. See Enzyme-linked

immunosorbent assay
Endospore, of bacteria, 65, 89
Enteric bacteria, identification of,

169-76
Enterobacter aerogenes
capsule stain, 95
cell size, shape, and arrangement,

102
gram stain, 78
indole production, 145
methyl red test, 147
as pathogen of urinary tract, 179
Enterobacteriaceae, 169
Enterobiasis, 47
Enterobius vermicularis, 47, 50
Enterococcus faecalis
catalase test, 143
cell size, shape, and arrangement,

102
gram stain, 78

as pathogen of urinary tract, 179
Enterotube II, rapid test system,

169-71, 174-76
Entry, of viral infection, 285
Envelope, viral, 269
Environmental contaminants, in

laboratory cultures, 111-12
Environmental microbiology

isolation of bacteriophages from

sewage, 277-82
MPN method for examination
of drinking water, 215-18

Enzyme-linked immunosorbent assay

(ELISA), 327-30
Eosin methylene blue (EMB) agar,

216,218
Eosinophils, 299
Episome, 245
Equivalence zone, 317
Erythrocytes, 298, 299
Erythromycin, 185, 189
Escherichia coli

antibiotic resistance, 247, 261, 262

bacteriophages, 277, 278, 346, 347

chromosomes, 245

coliforms, 215, 216, 278

conjugation, 261

genetic map of K12 strain, 224

gram stain, 78

lactose utilization test, 146

methyl red test, 147

mutagenesis, 239

oxidation-fermentation test, 148

pathogenic strains, 179, 200

phage plaques, 279

plasmids, 246

protein fingerprinting, 333,
334, 337

restriction endonucleases, 246

size of, 1 8

spore stain, 90

transformation of competent cells,
254, 255, 257

Western blotting, 340
Ethyl alcohol, and gram stain, 78
Ethylhydrocupreine, 1 64
Eucaryotes and eucaryotic cells, 6,

33-34, 39^2. See also Algae;
Fungi; Protozoans
Euglena, 41
Euglenoids, 40, 41
Euglenophyta, 40, 41
Examination, of growth media, 121,

125-27
Exotoxins, 156
Extract dilution, and plant viruses, 293

Fecal contamination, coliforms as

indicators of, 215, 278

Fertility factor (F factor), 261

Filter paper method, for evaluation of

antiseptics, 205, 206, 207

Fingerprinting

bacterial protein, 333^-2
viral DNA, 268-73

Finger- stick method, of blood
collection, 298

Five-kingdom classification system, 17

Alexander-Strete-Niles:

Back Matter

Index

Lab Exercises in
Organismal and Molecular
Microbiology

Companies, 2003

358 Index

Flagella. See also Flagella stain

characteristics of protozoans, 39

morphology of bacteria, 65, 95-96
Flagella stain

applications of, 95-96

morphology of bacteria, 65

procedure, 97
Flagellates, 39
Flatworms, 47
Flea, as disease vector, 61
Fleming, Alexander, 111, 1 85
Flukes, 47, 48

Food chain, of aquatic ecosystems, 55
Foot-and-mouth disease, 268
Fractional sterilization, 200
Free spore, 89, 90
Fungi

growth forms, 33-34

hyphae, 9

laboratory environment, 1 10-11,
113-14

pressure-tape preparation, 34

size of, 1 8

Gas gangrene, 89

Gas production, and Enterotube II, 175

Gel staining, and Western blotting,

334, 335
Genetic maps, of Escherichia coli, 224,

247. See also Genome
Genetics, bacterial. See also DNA; RNA
Ames test and mutagenesis, 239^-1
antibiotic resistance and
conjugation, 261-62
antibiotic resistance and

transformation, 253-57
DNA isolation and Southern

Analysis, 224-35
plasmid isolation and restriction
mapping, 245-50
Genome. See also Genetic maps
influenza virus, 269
K12 strain of Escherichia coli, 224
tobacco mosaic virus, 291
Gentamicin, 185, 189
Germination, of endoscopes, 89
Glucose test, and Enterotube II, 175
Gram-negative and gram-positive
bacteria
bacterial identification, 103
gram stain, 77, 78, 79
Gram-positive pleomorphic rods, 156
Gram stain

applications of, 77
bacterial unknowns, 103
cell wall of bacteria, 65
MPN method, 216

procedure, 79

skin isolates, 156

smear preparation, 79

throat isolates, 163

urinary tract isolates, 179
Granulocytes, 298, 299
Green algae, 40, 41
Griffith, Frederick, 253
Group A streptococci, 1 64
Growth curve, and virus infection

cycle, 285-87
Growth forms, of fungi, 33-34
Growth media, for cultures

composition of biochemical
test, 144

examination, 121, 125-27, 134

incubation, 121, 123-25, 127, 133

inoculation, 119, 120, 133

preparation, 119

Haemophilus influenzae, 246
Hand sanitizers and washing, 205-8
Health. See Disease, human; Medical

microbiology; Pathogens;

Serology
Heat

fixation of smear prior to stain, 7 1
sterilization by high temperature,

200-202
Hemagglutination, 311
Hematology, identification and

enumeration of white blood

cells, 298-301. See also Blood
Hemolysins, 156
Heterotrophic bacteria, 25
Hexachlorophene, 205
His+ phenotype, 239
Histidine, 239
Holoplankton, 55
Hot-air oven, 200
Hybridization, and Southern blotting,

226, 234-35, 269
Hybridoma, 305
Hydrogen peroxide, 205
Hydrogen sulfide production

biochemical tests for bacterial

identification, 142, 144, 145
Enterotube II reactions, 175
Hyphae, 9, 33

Immunodiffusion, and antigen- antibody
precipitation reactions, 317-22
Immunoelectrophoresis, and serum
protein levels, 317, 318, 319,
320, 321-22
Immunoglobulins, 305
Incineration, and dry heat, 200
Incubation, of growth media, 121,

123-25, 127
India ink, 66

Indicators, of fecal contamination, 215
Indole production, 142, 144-^5, 175
Infection cycle, of viruses, 285-87
Influenza, 268, 269
Inhalation anthrax, 89
Inoculation, of growth media, 119,

120, 123-25
Insects, as disease vectors, 61
Intestinal tract, identification of enteric

bacteria, 169-76
Invertebrates, microscopic
characteristics of, 55
size of, 1 8
Iodine, tincture of, 205
Isolation

of bacteria from urinary tract,

179-81
of DNA for Southern blotting,

224-35
of plasmids for restriction

mapping, 245-50
of Staphylocci from skin, 156-60
of Streptococci from throat,
163-66

K12, strain of Escherichia coli, 224
Kingdoms. See also Classification,
of microorganisms
Monera, 25
Protista, 39, 40
Kirby-Bauer method, for assessing
effectiveness of antibiotics,
185-89
Klebsiella pneumoniae, 95
Koch, Robert, 268

Identification. See Bacterial

identification
Immunity, to infectious disease,

298, 305

Laboratory environment, bacteria and
fungi in, 110-11, 113-14. &?e
also Bacterial infections; Safety

Lactose, and Enterotube II, 175

Lactose broth, 144

Lactose fermenters and nonfermenters,

173, 175

Alexander-Strete-Niles:

Back Matter

Index

Lab Exercises in
Organismal and Molecular
Microbiology

Companies, 2003

Index

359

Lactose utilization, 143, 145^46

LacZ gene, 225

Lambda infections, 277

Larvae, of benthic marine animals, 55

Latent period, of viral infection, 285

Latex agglutination test, 160

Leprosy, 85

Leukocytes, 298

Lice, as disease vectors, 61

Lichens, 33

Life cycle

of endospore-forming bacteria, 89

of flukes, 48

of roundworms, 50

of tapeworm, 49
Light microscope (LM). See also
Microscope

magnification, 3

method used to carry, 10

positions of objectives, 11

steps in use, 4

types of microscopes, 2
Lipopolysaccharide, 77
Lophotrichous flagella, 96
Lymphocytes

formed elements of blood, 4

immune system, 298, 299
Lymphoid tissues, 299
Lysine decarboxylation, and

Enterotube II, 175
Lysis, 277
Lysogen, 277

MacConkey agar, 169
Macrophages, 299

Magnification, and light microscope, 3
Malachite green, and spore stain, 89,

90,91
Mannitol salt agar (MSA), 156, 157
Marker genes, and plasmids, 246
Mast cells, 299
Mastigophora, 39

Media. See Growth media, for cultures
Medical microbiology. See also

Disease, human; Hematology;
Pathogens; Serology
antibiotic effectiveness, 1 85-89
clinical bacterial unknowns, 1 93-95
enteric bacteria, 169-76
Staphylococcus spp. from skin,

156-60
Streptococcus spp. from throat,

163-66
urinary tract bacteria, 179-81
Membrane baking, and Southern

blotting, 234
Mercurochrome, 205

Meroplankton, 55
Merthiolate, 205
Methylene blue

acid-fast stain, 85, 86

negative stain, 66

simple stain, 7 1
Methyl red test, 143, 146
Micrometer, 18, 19
Micrococcus luteus, 334, 337
Micrococcus spp., and normal flora

of skin, 156
Microorganisms

classification systems, 17, 25, 47

disease vectors, 61

eucaryotes, 33-34, 39^-2

flatworms and roundworms,
47-50

microscopic comparisons, 17-20

procaryotes, 25-26

zooplankton, 55
Microscope. See also Dissecting

microscope; Light microscope

comparisons of microorganisms,
17-19

micrometer, 18, 19

structure, function, and use,
2-11
MiniProtean II electrophoresis, 338
Molds, 18,33
Monera (Kingdom), 25
Monoclonal antibodies, 305, 306
Monocytes, 299
Monotrichous flagella, 96
Morphological stains, 66
Morphology

algae, 40

bacteria, 65, 66, 71

white blood cells, 299
Mosquito, as disease vector, 61
Motile bacteria, 96
Motility test agar, 133, 135
MPN (most probable number) method,
for examination of drinking
water, 215-18
MR-VP medium, 144, 169
Mueller-Hinton agar, 186, 188
Multicellular parasites. See also
Flatworms; Roundworms

classification of, 47

human diseases, 47

size of, 1 8
Multidrug-resistant (MDR) strains, of

bacteria, 253
Mushrooms, 33

Mutagenesis, and Ames test, 239-41
Mycobacteria, and acid-fast stain, 85
Mycobacterium leprae, 85
Mycobacterium phlei, 85, 102
Mycobacterium tuberculosis, 85
Mycolic acid, 85
Myeloma, 305

Narrow-spectrum antibiotics, 1 85
Nauplius larvae, 1 8
Negative stain

bacterial unknowns, 103

definition of, 66

morphology of bacteria, 65

procedure, 68
Neisseria gonorrhoeae, 261
Neisseria sicca, 78, 102
Neisseria spp., and normal flora

of throat, 163
Nematoda (Phylum), 47
Neutralization, of viruses by

antibodies, 345-48
Neutrophils, 4, 299
Nicotiana glutinosa, 292
Nigrosin, 66
Nitrate broth, 144
Nitrocefin, 157

Nitrocellulose membrane, 233
Nocardia asteroides, 85
Nocardia rubral, 246
Nocardiosis, 85
Non-acid-fast bacteria, 85
Non-capsule-forming bacteria, 95
Nonmotile bacteria, 96
Nonpotable water, 215
Non-spore-forming rod, 90
Normal flora

of human skin, 156

of human throat, 163

of intestinal tract, 1 69

of urinary tract, 179
Nutrient agar, 110-11, 112-14
Nystatin, 112

Objective lenses, 3, 11
Ocular micrometer, 18, 19
One- step growth curve, and virus

infection cycle, 285-87
Opsonization, 345, 346
Optochin, 164
Oral suckers, of flukes, 47
Original carriers, and ELISA, 330
Origin of replication (ORI), 245
Ornithine decarboxylation, and

Enterotube II, 175
Orthophenylphenol, 211
Oscillatoria, 18

Ouchterlony procedure, 319, 320
Oxidase test, 143, 146, 147
Oxidation-fermentation (O-F) glucose

test, and identification of

bacteria, 143, 144, 146, 148

Alexander-Strete-Niles:

Back Matter

Index

Lab Exercises in
Organismal and Molecular
Microbiology

Companies, 2003

360

Index

Paramecium caudatum, 18
Paramecium spp., and cilia, 39
Pasteur, Louis, 268
Pasteurization, 200
Pathogens. See also Bacterial

infections; Disease, human

of human skin, 156

of human throat, 1 63

of intestinal tract, 1 69

strains of Escherichia coli, 200

of urinary tract, 179
Pediculus, 61
Penicillin. See also Antibiotics

assessing effectiveness of, 185, 189

discovery of, 111, 1 85

resistance in Staphylocci,
156-57, 160

spectrum of activity, 185
Penicillinase, 156-57
Penicillium, 111
Pennate diatoms, 18, 41
Peplomers, 268, 269
Peptidoglycan layer, 77
Peptidoglycan synthesis, 256
Peritrichous flagella, 96
pH, of nutrient agar, 111
Phaeophyta, 40, 41
Phage plaques, 279
Phage plating, 282, 286
Phage titer, 277-82
Phagocytic cells, 298, 299
Phenol, and DNA isolation, 229-30
Phenylalanine deaminase, and

Enterotube II, 176
Photo synthetic pigments, of algae, 40
Phylums. See also Classification,
of microorganisms

Arthropoda, 61

Platyhelminthes and
Nematoda, 47
Phytoplankton, 55
Pine oil, 211
Pink-eye infection, 268
Plankton, 55
Plants. See also Lichens

fungal hyphae, 9

viruses, 291-93
Plaque, viral, 277, 279
Plaque assay, 278
Plaque-forming units (PFU), 278
Plasma cells, 299, 305
Plasmids, isolation of, 245-50
Plasmodium, and ring stage, 39
Plating, of bacteriophages, 282, 286
Platyhelminthes (Phylum), 47
Pneumococcal pneumonia, 1 63
Pneumonia, 95, 163
Poliomyelitis, 253, 268

Polyacrylamide gel electrophoresis.

See SDS-PAGE
Polyclonal antigens, 305, 306
Polymerase chain reaction (PCR),

269, 271
Polymyxin B

assessment of effectiveness,
185, 189

Bacillus as source of, 112
Polysiphonia, 41
Pond water, and wet mount

preparation, 42
Posthybridization, and Southern

blotting, 229
Potable water, 215
Potato, and fungal hyphae, 9
Precautions. See Safety
Precipitation, of DNA, 230
Precipitation test, and serological

methods, 307, 308
Precipitin curve, 317
Prehybridization, and Southern

blotting, 234-35
Pressure cooker, 200
Pressure-tape preparation, 34
Presumptive test, and MPN method,

215-16,218
Primer annealing, and DNA

synthesis, 271
Probe labeling, and DNA, 234
Procaryotic microorganisms, 25
Pro-mutagens, 239
Protein

extracts, 336-37

fingerprinting of bacterial, 333-42

immunoelectrophoresis and serum
levels of, 317, 318, 319, 320,
321-22
Proteome, 333

Proteus spp., and enteric bacteria, 169
Proteus vulgaris

gram stain, 78

hydrogen sulfide production, 145

lactose utilization, 146

as pathogen of urinary tract, 179

peritrichous flagella, 96

triple sugar iron agar, 170
Protista (Kingdom), 39, 40
Protozoans

classification, 39

representative types, 39

size of, 1 8

wet mount preparation, 42
Providencia stuartii, 246
Pseudomonas aeruginosa

acid-fast stain, 85

denitrification test, 144

gram stain, 78

monotrichous flagella, 96

oxidase test, 147

oxidation-fermentation test, 148

as pathogen of urinary tract, 179
simple stain, 7 1
Pseudomonas marginalis, 96
Pseudopodia, 39
Pulsed-field gel electrophoresis

(PFGE), and viral DNA
fingerprinting, 269, 270
Pyrrophyta, 40, 41

Rabbit plasma, 160
Rabies, 268

Radial immunodiffusion, and antigen-
antibody precipitation
reactions, 317, 318, 319, 321
Rapid agglutination test, 160
Rapid test system, and Enterotube II,

169-70
Red algae, 40, 41
Red blood cells, 4, 39
Rep gene, 247
Resident flora, 156
Resistence factor (R factor), 261
Restriction digestion

bacterial DNA isolation, 227,
228-30

Southern blotting, 226

viral DNA fingerprinting, 272
Restriction endonuclease, 225, 246
Restriction enzymes, 246, 270
Restriction fragments

bacterial DNA isolation, 225,
226

plasmid isolation, 246
Restriction mapping, and plasmid

isolation, 245-50
Restriction pattern, 225
Rh factor, and blood typing, 311, 312
Rhizopus, 18
Rhodophyta, 40, 41
Ribosomes, and morphology

of bacteria, 65
RNA. See also Genetics, bacterial

influenza virus, 269

tobacco mosaic virus, 291
Rod (cell shape), 25
Rop gene, 247
Roundworms, 47, 50
R strain, of Streptococcus
pneumoniae, 254

Sabouraud dextrose agar, 110-11,

112-14
Saccharomyces, 18

Alexander-Strete-Niles:

Back Matter

Index

Lab Exercises in
Organismal and Molecular
Microbiology

Companies, 2003

Index

361

Safety

acrylamide solutions, 334, 335
agarose gel electrophoresis, 23 1
bacteriological examination of

drinking water, 217, 218
blood collection and handling,

298,300,313,321
ethanol and Bunsen Burner,

188, 207
inoculation and heating of broth

tubes, 201
Southern blotting, 233
urine collection and inoculation, 1 80
Safranin

gram stain, 78, 79
negative stain, 66
simple stain, 71
spore stain, 90, 91
Salmonella enterica

Ames test and mutagens, 239
fecal contamination of water, 215
as pathogen, 215
triple sugar iron agar and
differentiation of, 170
Salmonella spp. See also Salmonellosis
identification of, 169-76
as pathogens, 169, 200, 215
Salmonella typhi, 215
Salmonella typhimurium. See

Salmonella enterica
Salmonellosis, 169, 200, 215
Sarcodina, 39
Sargassum, 41
Scanning electron microscope (SEM),

2,5
Schaeffer-Fulton endospore stain

method, 90, 91
Schistosoma mansoni, 47, 48
Schistosomiasis, 47
Scolex, of tapeworm, 49
SCSI, strain of Escherichia coli, 261, 262
SDS. See Sodium dodecyl sulfate
SDS-PAGE (polyacrylamide gel

electrophoresis), 333, 335-36,
337-40
Semmelweis, Ignaz, 205
Serial dilutions, 281, 308
Serology. See also Blood

agglutination reactions and blood

typing, 311-13
antigen-antibody precipitation
reactions and antibody titer,
305-8
antigen-antibody precipitation
reactions and immunodiffusion,
317-22
bacteria protein fingerprinting and

Western blotting, 333-42
definition of, 305
enzyme-linked immunosorbent
assay (ELISA), 327-30

neutralization of viruses by
antibodies, 345-48
Serratia marcescens, 333, 334, 337
Serum, 305
Sewage, isolation of bacteriophages

from, 277-82
Sex pilus, 253, 261
Shigella flexneri, 170
Shigella sonnei, 215
Shigella spp., identification of, 169-76

See also Shigellosis
Shigellosis, 169, 215
SIM medium, 144, 169
Simmons citrate agar, 195
Simple stain

bacterial unknowns, 103

cell morphology of bacteria,
65,71

smear preparation, 71, 72, 73
Size

of bacteria cells, 65, 102

of selected microorganisms, 1 8

of white blood cells, 299
Skin, human

disinfection, 205-8

identification of isolates from,
156-60

normal flora, 156

pathogens, 156
Slide preparation, and wet mounts
for live specimens in pond
water, 42
Smallpox, 253, 268
Smear preparation

acid-fast stain, 86

gram stain, 79

simple stain, 71, 72, 73

spore stain, 91
Sodium carbonate buffer, 328
Sodium dodecyl sulfate (SDS), 333
Sodium hypochlorite, 211
Soft agars, 347-48
Sorbitol, and Enterotube II, 176
Southern Analysis, and DNA isolation,

224-35, 269
Specific immunity, 298, 305
Spirillum (cell shape), 25
Spirillum spp., 18
Spirillum volutans, 96
Spirochete (cell shape), 25
Spirogyra, 18, 41
Spleen cells, 306
Spore-forming rod, 90
Spore stain

applications of, 89

endospore of bacteria, 65, 89

procedure, 89-91
Sporogenesis, 89
Sporozoans, 39
S strain, of Streptococcus
pneumoniae, 254

Stab technique, for agar deep

cultures, 125
Staining techniques

acid-fast stain, 85-86

bacterial unknowns, 101-3

capsule and flagella stains, 95-97

gel staining and Western blotting,
334, 335

gram stain, 77-79

negative stain, 66-68

simple stain, 71-73

spore stain, 89-91

Wright's staining, 300-301
Staphylococci (cell arrangement),

25,26
Staphylococcus aureus

discovery of penicillin, 111

normal flora of throat, 163

pathogens of skin, 156
Staphylococcus epidermidis

catalase test, 143

cell size, shape, and arrangement,
102

characteristics of, 156

gram stain, 78

negative stain, 66

normal flora of skin, 156

simple stain, 71
Staphylococcus saprophyticus, 179
Staphylococcus spp.

isolation and identification of from
skin, 156-60

size of, 1 8
Sterilization. See also Aseptic
procedures

of growth media, 119, 120, 121

high temperature, 200-202
Storage, of bacteriophage plaques, 282
Streak-plate method, of media

examination, 127
Streptavidin peroxidase, 328
Strep throat, 163
Streptobacilli (cell arrangement),

25,26
Streptococci (cell arrangement), 25, 26
Streptococcus pneumoniae

capsule, 95

identification of, 164, 166

normal flora of throat, 163

transformation and virulence of,
253, 254
Streptococcus pyogenes, 163, 164, 166
Streptococcus spp., isolation and

identification of from throat,
163-66
Streptomycin

assessment of effectiveness,
185, 189

Streptomyces as source of, 112
Streptomyces spp. 111-12, 114
Subterminal spores, 89

Alexander-Strete-Niles:

Back Matter

Index

Lab Exercises in
Organismal and Molecular
Microbiology

Companies, 2003

362 Index

Swab inoculation, of agar plate, 113
Synchronous culture, 285

Taenia saginata, 49

Taenia solium, 49

Taenia spp., and life cycle, 49

Tapeworms, 18, 47, 49

Terminal sports, of Clostridium

tetani, 89
Tetanus, 89
Tetracycline

antibiotic resistance in Escherichia

coli, 255, 262
assessing effectiveness of, 185, 189
Streptomyces spp. as source of, 112
Tetrads (cell arrangement), 25, 26
Tetramethylbenzidine (TMB), 328
Throat, isolation and identification of

Streptococci from, 163-66
Throat swab, 164
Ticks, 18,61

Titer, of antibodies in serum, 305-8
T lymphocytes (T cells), 299, 305
TMB . See Tetramethylbenzidine
Tobacco mosaic virus (TMV), 291-93
Tobamo viruses, 291
Total white blood cell count, 298
Toxins, neutralization of viral

by antibodies, 345
Transduction, of bacterial DNA, 253
Transformation, bacterial and antibiotic

resistance, 253-57
Transmission electron microscope

(TEM), 2, 5
Treponema pallidum, 18
Trichinella spiralis, 47, 50
Trichinosis, 47

Trichomonas, and flagella, 39
Triclosan, 205

Triple sugar iron (TSI) agar, 169, 170
Tris-buffered saline (TBS), 336
Tryptic soy agar, 119, 180

Tryptic soy broth, 119
Tuberculosis, 85, 253
Typhoid fever, 215

Universal precautions, for blood

collection, 298
University of Arizona, 327
University of Wisconsin, Madison, 224
Unpolymerized acrylamide, 334
Urea, and Enterotube II, 176
Urea broth, 194, 195
Urinary tract, identification of bacteria

from, 179-81
Urinary tract infection (UTI), 179

Vancomycin, 185, 189
Vectors, and disease-causing

microorganisms, 61
Ventral suckers, of flukes, 47
Vibrio (cell shape), 25
Vibrio cholerae, 215
Virions, 268, 285
Viruses

antigens, 328
bacteriophage isolation from

sewage and phage titer, 277-82
DNA fingerprinting, 268-73
infected cells, 7
infection cycle and one- step

growth curve, 285-87
neutralization of by antibodies,

345-48
plant leaves and tobacco mosaic
virus, 291-93
Voges-Proskauer test, and Enterotube

II, 176
Volvox, 18,41
V-shapes (cell arrangement), 25, 26

Water

MPN method for examination

of drinking, 215-18
slide preparation for live

specimens in pond, 42
Western blotting, 333-42
Wet heat, and sterilization, 200
Wet mount preparation, of live

specimens in pond water, 42
White blood cells, 6, 298-301
Whittaker, R. H., 17
Whittaker classification system,

17,39
Woese, C, 17

Woese classification system, 17
Wright's staining, 300-301

Xenopsylla, 61

Yeasts

bud, 33
cell shape, 4
size of, 1 8

Ziehl-Neelsen acid-fast staining
procedure, 86

Zones of inhibition

antibiotics, 111, 186, 189
evaluation of antiseptics, 205

Zooplankton, 55

Zygospore, 33

Alexander-Strete-Niles:

Back Matter

Index

Lab Exercises in
Organismal and Molecular
Microbiology

Companies, 2003

Class Notes

Alexander-Strete-Niles:

Back Matter

Index

Lab Exercises in
Organismal and Molecular
Microbiology

Companies, 2003

Alexander-Strete-Niles:

Back Matter

Index

Lab Exercises in
Organismal and Molecular
Microbiology

Companies, 2003

Class Notes

Alexander-Strete-Niles:

Back Matter

Index

Lab Exercises in
Organismal and Molecular
Microbiology

Companies, 2003

Alexander-Strete-Niles:

Back Matter

Index

Lab Exercises in
Organismal and Molecular
Microbiology

Companies, 2003

Class Notes

Alexander-Strete-Niles:

Back Matter

Index

Lab Exercises in
Organismal and Molecular
Microbiology

Companies, 2003

Alexander-Strete-Niles:

Back Matter

Index

Lab Exercises in
Organismal and Molecular
Microbiology

Companies, 2003

Class Notes

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