Microbiology: Laboratory Theory and Application

786 Pages·2010·34.08 MB·English

by  Michael J. Leboffe

i-xii Front Matter 8/31/11 3:27 PM Page vi
i-xii Front Matter 8/31/11 3:27 PM Page i
M
ICROBIOLOGY
Laboratory Theory
& Application
THIRD EDITION
Michael J. Leboffe
San Diego City College
Burton E. Pierce
925 W. Kenyon Ave., Unit 12
Englewood, Colorado 80110
www.morton-pub.com
i-xii Front Matter 8/31/11 3:27 PM Page ii
Book Team
Publisher: Douglas N. Morton
Biology Editor: David Ferguson
Production Manager: Joanne Saliger
Production Assistant: Desiree Coscia
Patricia Billiot
Typography: Ash Street Typecrafters, Inc.
Copyediting: Carolyn Acheson
Illustrations: Imagineering Media Services, Inc.
Cover Design: Bookends Design
Copyright © 2010 by Morton Publishing Company
ISBN 10: 0-89582-830-8
ISBN 13: 978-0-89582-830-9
Library of Congress Control Number 2009944089
10 9 8 7 6 5 4 3
All rights reserved. No part of this publication may be reproduced,
stored in a retrieval system, or transmitted, in any form or by any
means, electronic, mechanical, photocopying, recording, or otherwise,
without the prior written permission of the copyright owners.
Printed in the United States of America
i-xii Front Matter 8/31/11 3:27 PM Page iii
Preface
T
his third edition in many ways is like another first edition. We have added 20 new
exercises, incorporated four more exercises from MLTA Brief Edition, and have
substantially rewritten several others. Every exercise has been screened and updated
for better clarity, comprehensiveness, and appropriate placement in the manual. We have
added three more of the boxed “A Word About . . .” features to offer expanded introduc-
tory material in Sections 4, 5, and 8. Finally, we replaced many older photographs, and
perhaps most important, employed a new artist to update the illustrations and enhance the
overall beauty of the book.
Following are major features of each section.
(cid:0) Introduction As in previous editions, the Introduction emphasizes safety. Refinements
include chemical safety awareness and examples of organisms to be handled at each BSL
level.
(cid:0) Section 1 Fundamental Skills Exercise 1-1 (The Glo-Germ™ Hand Wash Education
System) was brought over from the BriefEdition. This is a fun, eye-opening lab exercise,
raising consciousness about how easily the “unseen” can be overlooked. In Exercise 1-4
(Streak Plate Method of Isolation) we introduce alternative methods for streaking.
(cid:0) Section 2 Microbial Growth Exercises 2-5 (Evaluation of Media) and 2-12 (Steam
S terilization) have been added from the Brief Edition. Exercise 2-11 (The Effect of
Osmotic Pressure on Microbial Growth) has been rewritten to include Halobacterium.
Exercise 2-13 (The Lethal Effect of Ultraviolet Radiation on Microbial Growth) has
been renamed, and the procedure simplified.
(cid:0) Section 3 Microscopy and Staining Exercise 3-1 (Introduction to the Light Micro-
scope) has been rewritten to include an activity using the “Letter e” slide and the
“ Colored Thread” slide, both of which provide opportunities for novice microscopists
to learn more about how to operate the light microscope. Two new labs, Exercise 3-4
(Microscopic Examination of Pond Water) and Exercise 3-11 (Parasporal Crystal Stain),
round out the changes for this section.
(cid:0) Section 4 Selective Media This section has been updated for greater clarity and to
address reviewers’ concerns. The boxed item “A Word About Selective Media” has been
added as an adjunct to the Section introduction, and Bile Esculin Test (Exercise 4-3) has
been moved from Section 5 to Selective Media for the Isolation of Gram-Positive Cocci.
(cid:0) Section 5 Differential Tests This section has seen some reorganization and the addi-
tion of new material. The boxed item “A Word About Biochemical Tests and Acid-Base
R eactions,” has been added to supplement the introduction and help students better
understand the recurring theme of fermentation seen in differential media. Following
the introductory material, the section begins with an exercise demonstrating Reduction
Potential. It is designed to be an easy and fun introduction to the concept of energy
transformations in redox reactions, the understanding of which can be applied to many
exercises that follow. Novobiocin and Optochin susceptibility tests have been moved
from Gram-positive coccus identification and combined with Bacitracin (Exercise 5-24)
in the Antibacterial Susceptibility Testing subsection. Because of popular demand
to include both Kligler Iron Agar and Triple Sugar Iron Agar in the Combination
iii
i-xii Front Matter 8/31/11 3:27 PM Page iv
IV MICROBIOLOGY: LABORATORY THEORY AND APPLICATION
Differ ential Media subsection, we have written an exercise (5-21) that can be used with
either or both media.
(cid:0) Section 6 Quantitative Techniques In an ongoing effort to help students understand
the dilutions and calculations necessary in quantitative techniques (and to simplify the
terminology), we have rewritten the introduction to this section and the theory portion
of Exercise 6-1 (Standard Plate Count). The terms “dilution factor” and “final dilution
factor” associated with dilutions and platings have been replaced with “dilution” and
“sample volume,” respectively. We hope this helps.
(cid:0) Section 7 Medical Microbiology In this section we have added one new lab demon-
strating clinical biofilms (Exercise 7-4). Exercise 7-3 (Antimicrobial Susceptibility Test-
ing) has been rewritten with new antibiotics and an optional exercise to demonstrate the
difference between bacteriostatic and bacteriocidal agents. Last, Exercise 7-9 (Identifi ca-
tion of Gram-Positive Rods) rounds out the series of three unknown identifications.
(cid:0) Section 8 Environmental Microbiology This is the section that has seen the most
growth. Of the 13 exercises, 9 are new. Exercise 8-1 (Winogradsky Column) should be
done near the beginning of the semester so it can be used as a source of microo rganisms
in other labs (Exercises 8-6 through 8-8). The next seven exercises provide activities
related to the Nitrogen Cycle (Exercises 8-2 through 8-5) and the Sulfur Cycle (Exercises
8-6 through 8-8). The other new lab is Exercise 8-10 (Soil Slide Culture), in which soil
microorganisms are grown and then can be viewed in their proper spatial orientation.
Also included in this section is a brief explanation of trophic group terminology (“A
Word About Trophic Groups”).
(cid:0) Section 9 Food Microbiology One exercise (that was problematic) has been removed
from this section. Otherwise, the content here is unchanged. You can expect a lot of
growth in the food microbiology section in subsequent editions of this manual.
(cid:0) Section 10 Microbial Genetics This section has been reorganized and three new
e xercises added to reflect the change in microbiology from traditional methods of iden -
tification to more sensitive techniques involving molecular biology. Exercises 10-2 and
10-4 address two important methods of molecular biology: performing a restriction
digest and polymerase chain reaction, respectively. Exercise 10-7 (Phage Typing of
E. coliStrains) introduces the student to using viral susceptibility as a tool in microbial
identification.
(cid:0) Section 11 Hematology and Serology Two of the seven exercises in this section are
new. Exercise 11-6 is a hemagglutination test used in the diagnosis of infectious m ono -
nucleosis. Exercise 11-7 is a quantitative ELISA that models the identification and
quantification of HIV antibodies.
(cid:0) Section 12 Eukaryotic Microbes This section continues to be a survey of the micro-
scopic eukaryotes encountered in a medical microbiology laboratory. One new exercise,
Exercise 12-2 (Fungal Slide Culture) provides an opportunity to cultivate fungi in a way
that their true structure can be observed microscopically.
(cid:0) Appendices Appendix G (Agarose Gel Electrophoresis) contains instructions for p re -
paring and (multiple techniques for) staining agarose gels used in the electrophoresis
portion of Exercises 10-2 and 10-5. Many additions have been made to Appendix H
(Medium, Reagent, and Stain Recipes) to accompany all the new exercises. The Glossary
also has been updated and expanded.
Our book is maturing, becoming more complete and polished. And as we use it, new
ideas for presentation or content will occur to us for future changes. That’s why new edi-
tions are numbered!
i-xii Front Matter 8/31/11 3:27 PM Page v
PREFACE V
Last, though we do get compensated for our work, we are primarily educators. We
take great satisfaction that our efforts may in some small way contribute to your successful
academic and professional careers.
All our best,
Mike and Burt
_______________________________
Acknowledgments
Thanks so much to all of you who had a part in making this project a success. We have
thanked all of you personally; this is our opportunity to let our readers know who you are
and why you are so appreciated. Listed below, in no particular order, are the people who
went out of their way to give time, advice, space, and patience to support us during this
project. We sincerely hope that, with the pages that follow, we have earned that support.
First of all, thanks to Debra Reed, Biology Laboratory Technician at San Diego City
College, for her longstanding support of our projects. Deb’s assistance spans more than a
decade and includes help with test media and cultures, hand modeling for photos, and
gentle directions when we occasionally forget where we are. We thank Muu Vu for her
assistance modeling for photos, and helping to make valuable contacts. Thanks very much
to Brett Ruston for the free lunch and the “loan” of several chemicals used in the Nitrogen
and Sulfur cycle exercises. Thanks to lab technician Laura Steininger for running interfer-
ence by listening to Burt’s sustained belly-aching about living in an RV for a month without
his dogs, Yancy, Megan, and Beau to keep him company. Thanks also to Ed Sebring of
the Chemistry Department who provided a pinch of this or that chemical when we were
desperate.
Additionally, thank you to Dr. Carla Sweet for helping with some new photographs as
a hand model, Gary Wisehart for assisting with the Winogradsky column, Alicia Leboffe
for editorial work, Nathan Leboffe for help in photography, and Dr. Steven J. Byers for
piloting Mike around San Diego County so he could get aerial photographs.
Thanks to Dr. Donna DiPaolo, Dr. Anita Hettena, Dr. Roya Lahijani, Erin Rempala,
Dr. David Singer, and Gary Wisehart of the San Diego City College Biology Department
for patience and understanding about losing territory in the Biology Resource Center due
to our set-up and for putting up with some of the foul odors produced by the cultures.
Thanks to Sonia Bertschi co-owner and manager of the Jacumba Hotsprings Spa and
Lodge in Jacumba, California for a tour of her lovely resort and donation of natural min-
eral hot springs water.
Thanks to Jerry Davis, San Diego City College Vice President of Administrative
Services and Joyce Thurman, San Diego City College Business Services Administrative
Secretary, for expediting use of college facilities. Thanks also to Dr. Steve Barlow, Associate
Director of the San Diego State University Electron Microscope Facility for his patience
and humor as one of us learned how to use the transmission electron microscope.
A very special thank you goes to Dr. Radu Popa, Portland State University Microbiology
Professor for taking time on numerous occasions to give expert advice on many issues, but
most specifically, sulfur biogeochemical transformations. Also thank you to Jane Boone,
Portland State University Biology Laboratory Coordinator, for giving Burt a workspace of
his own and occasional needed advice. Both Dr. Popa and Ms. Boone, who were working
on their own advanced microbiology laboratory manual during this time, generously pro-
vided a collegial and sharing environment.
Thanks to Imagineering Media Services Inc. from Toronto, Ontario, Canada for the
new artwork included in this edition. Thanks also to Bob Schram of Bookends Design
for the cover design. And, as always, thanks so much to Joanne Saliger at Ash Street
i-xii Front Matter 8/31/11 3:27 PM Page vi
VI MICROBIOLOGY: LABORATORY THEORY AND APPLICATION
Typecrafters, Inc., who capably (and cool-handedly) managed to produce a beautiful book
out of moderately well organized scraps. Thanks to Carolyn Acheson, who copyedited
(and removed most of the split infinitives from) the manuscript. We also are indebted to
reviewers and students who provided valuable suggestions for improving our book. Special
recognition and thanks go to Dr. Amy Warenda Czura of Suffolk County Community
College and Dr. Lisa Lyford of University of the Cumberlands for their thorough input.
A special thanks to Dr. Jack G. Chirikjian, Chairman of Edvotek, Inc. for agreeing to
the use of his company’s kits in Exercises 10-2, 10-4, 11-7, and Appendix G.
We, of course, remain grateful to the Morton Publishing team for their support and
patience. Specifically, thanks to Doug Morton, President, Chrissy Morton DeMier, Business
Manager, David Ferguson, Acquisitions Editor, Carter Fenton, Sales and Marketing Man-
ager, and Desiree Coscia, Publisher’s Assistant.
And as always, thanks to our wives Karen Leboffe and Michele Pierce for their contin-
ued support and understanding. If writing a book is difficult, being married to an author is
worse.
i-xii Front Matter 8/31/11 3:27 PM Page vii
Contents
Introduction Safety and Laboratory Guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
A Word About Experimental Design. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Data Presentation: Tables and Graphs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Data Presentation: Be Creative, But Complete!. . . . . . . . . . . . . . . . . . . . . . . . . . 8
SECTION 1 Fundamental Skills for the Microbiology Laboratory . . . . . . . . . . . . . . . . . . . . . . . 11
EXERCISE 1-1 Glo-Germ™ Hand Wash Education System. . . . . . . . . . . . . . . . . . 12
Basic Growth Media. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
EXERCISE 1-2 Nutrient Broth and Nutrient Agar Preparation . . . . . . . . . . . . . . . 14
Aseptic Transfers and Inoculation Methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
EXERCISE 1-3 Common Aseptic Transfers and Inoculation Methods . . . . . . . . 18
EXERCISE 1-4 Streak Plate Methods of Isolation . . . . . . . . . . . . . . . . . . . . . . . . . 25
EXERCISE 1-5 Spread Plate Method of Isolation. . . . . . . . . . . . . . . . . . . . . . . . . . 29
2 Microbial Growth 33
SECTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Diversity and Ubiquity of Microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
EXERCISE 2-1 Ubiquity of Microorganims. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
EXERCISE 2-2 Colony Morphology. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
EXERCISE 2-3 Growth Patterns on Slants. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
EXERCISE 2-4 Growth Patterns in Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
Environmental Factors Affecting Microbial Growth. . . . . . . . . . . . . . . . . . . . . . . . . . . 46
EXERCISE 2-5 Evaluation of Media. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
Aerotolerance. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
EXERCISE 2-6 Agar Deep Stabs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
EXERCISE 2-7 Fluid Thioglycollate Medium. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
EXERCISE 2-8 Anaerobic Jar. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
EXERCISE 2-9 The Effect of Temperature on Microbial Growth . . . . . . . . . . . . . 54
EXERCISE 2-10 The Effect of pH on Microbial Growth . . . . . . . . . . . . . . . . . . . . . . 56
EXERCISE 2-11 The Effect of Osmotic Pressure on Microbial Growth. . . . . . . . . 58
Control of Pathogens: Physical and Chemical Methods. . . . . . . . . . . . . . . . . . . . . . . . 60
EXERCISE 2-12 Steam Sterilization. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
2-13 The Lethal Effect of Ultraviolet Radiation
EXERCISE
on Microbial Growth. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
EXERCISE 2-14 Chemical Germicides: Disinfectants and Antiseptics. . . . . . . . . 66
vii
i-xii Front Matter 8/31/11 3:27 PM Page viii
VIII MICROBIOLOGY: LABORATORY THEORY AND APPLICATION
3 Microscopy and Staining 69
SECTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Microscopy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
EXERCISE 3-1 Introduction to the Light Microscope. . . . . . . . . . . . . . . . . . . . . . . 70
EXERCISE 3-2 Calibration of the Ocular Micrometer. . . . . . . . . . . . . . . . . . . . . . . 76
EXERCISE 3-3 Microscopic Examination of Eukaryotic Microbes. . . . . . . . . . . 79
EXERCISE 3-4 Microscopic Examination of Pond Water. . . . . . . . . . . . . . . . . . . 84
Bacterial Structure and Simple Stains. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
EXERCISE 3-5 Simple Stains. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
EXERCISE 3-6 Negative Stains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
Differential and Structural Stains. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
EXERCISE 3-7 Gram Stain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
EXERCISE 3-8 Acid-Fast Stains. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
EXERCISE 3-9 Capsule Stain. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115
EXERCISE 3-10 Endospore Stain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117
EXERCISE 3-11 Parasporal Crystal Stain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
EXERCISE 3-12 Wet Mount and Hanging Drop Preparations. . . . . . . . . . . . . . . . 122
EXERCISE 3-13 Flagella Stain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124
EXERCISE 3-14 Morphological Unknown. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126
4 Selective Media 129
SECTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
A Word About Selective Media. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130
Selective Media for Isolation of Gram-Positive Cocci . . . . . . . . . . . . . . . . . . . . . . . . 131
EXERCISE4-1 Phenylethyl Alcohol Agar. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131
EXERCISE4-2 Columbia CNA With 5% Sheep Blood Agar. . . . . . . . . . . . . . . . . 133
EXERCISE4-3 Bile Esculin Test. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135
EXERCISE4-4 Mannitol Salts Agar. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137
Selective Media for Isolation of Gram-Negative Rods. . . . . . . . . . . . . . . . . . . . . . . . 139
EXERCISE4-5 MacConkey Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139
EXERCISE4-6 Eosin Methylene Blue Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142
EXERCISE4-7 Hektoen Enteric Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
EXERCISE4-8 Xylose Lysine Desoxycholate Agar. . . . . . . . . . . . . . . . . . . . . . . . 147
5 Differential Tests 149
SECTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
A Word About Biochemical Tests and Acid-Base Reactions . . . . . . . . . . . . . . . . . . . 150
Introduction to Energy Metabolism Tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151
EXERCISE5-1 Reduction Potential. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152
EXERCISE5-2 Oxidation–Fermentation Test. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155
Fermentation Tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158
EXERCISE5-3 Phenol Red Broth. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158
EXERCISE5-4 Methyl Red and Voges-Proskauer Tests . . . . . . . . . . . . . . . . . . . 161
Tests Identifying Microbial Ability to Respire . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165
EXERCISE5-5 Catalase Test. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165
EXERCISE5-6 Oxidase Test. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168
EXERCISE5-7 Nitrate Reduction Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171