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Each exercise includes a Lab and Lecture: Putting it all together activity available through MasteringMicrobiology.® These activities are designed to help students see how lab and lecture are integrat-ed. Students will use their new information and incor-porate their new knowledge into lab.

With a strong emphasis on laboratory safety, this laboratory manual encourages students not only to learn but also to practice safety techniques so that safety becomes part of their professional behavior. We have included a safety contract that students can hand in to their instructors to indicate that they understand safety requirements. (For specific safety suggestions, see the section titled Specific Hazards in the Labora-tory on page 4 and the sections of the Introduction that follow it.) We also alert students with yellow safety boxes at key points in the exercises. Biosafety levels (BSLs) are noted. Every effort has been made to use biosafety level 1 (BSL-1) organisms. BSL-2 organisms are required in a few exercises to demonstrate specific principles and processes.

These safety boxes are marked with either a bio-hazard icon , a general safety icon , or a biosafe-ty level 2 icon BSL-2 indicating appropriate safety techniques.

NEW TO THIS EDITIONFor the Twelfth Edition, the overall goal was to make this manual even more navigable and visually effective for students and instructors. The following changes have helped us fulfill that goal.

The American Society for Microbiology en-dorses inclusion of laboratory experience as an integral part of all microbiology courses and identifies topics and techniques consid-ered essential to teach in every microbiol-ogy laboratory. When an exercise directly addresses one of the guidelines, readers will see the ASM icon, along with a summary of the relevant statement.

Now in its Twelfth Edition, Laboratory Experiments in Microbiology is valued by instructors and students for its comprehensive coverage of every area of micro-biology, its user-friendly laboratory reports, and its clear, straightforward organization. Containing 57 thoroughly class-tested exercises, this manual pro-vides basic microbiology techniques with applications for undergraduate students in diverse areas, including the biological sciences, the allied health sciences, agri-culture, environmental science, nutrition, pharmacy, and various preprofessional programs. It is designed to supplement any nonmajors microbiology textbook. Coauthored by Christine L. Case, this manual is an ideal companion to Microbiology: An Introduction, Thirteenth Edition, by Gerard J. Tortora, Berdell R. Funke, and Christine L. Case.

OUR APPROACHExercises have been designed to include the American Society for Microbiology laboratory core curriculum, which is considered essential to teach in every introduc-tory microbiology laboratory, regardless of its emphasis.

This laboratory manual has two primary goals—teaching microbiology techniques and showing students the importance of microbes in our daily lives and their central roles in nature. Most of the exercises are inves-tigative by design and require the students to evalu-ate their experimental results and draw conclusions. We hope in this way to promote analytical reasoning and provide students with a variety of opportunities to reinforce the technical skills they have learned. We also highlight practical uses of microbiology by includ-ing material with direct applications to procedures per-formed in clinical and commercial laboratories.

Each part begins with a case study to give students a real-world example of the applications of the material they are learning. The solution to the case study requires content from the laboratory exercises in that part.

Each exercise reflects the American Society for Microbiology Guidelines for Biosafety in Teaching Laboratories.

Preface

ASM

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Laboratory exercises that address the human or environmental microbiome are marked with an icon.

Laboratory exercises that provide an investigative experiment are marked with an icon. Students use their new microbiological information to explore the microbial world.

ORGANIZATION

ASM: Thinking skills

Each exercise is designed to promote Laboratory Thinking skills:

• Predict expected results• Follow an experimental protocol• Collect and organize data• Draw appropriate conclusions based on the results• Work effectively in teams or groups so that the task,

results, and analysis are shared• Effectively manage time and tasks allowing con-

current and/or overlapping tasks to be done simul-taneously, by individuals and within a group

This manual is divided into 14 parts. The introduc-tion to each part explains the unifying theme for that part. Each Part begins with a Case Study that relates the exercises with clinical applications. Exercises in the first four parts provide sequential development of fundamental techniques. The remaining exercises are as independent as possible to allow instructors to select the most desirable sequence for their course. The exercises are organized as follows.

OBJECTIVES This introductory section defines the specific skills or concepts to be mastered in the exercise. The objectives can easily be used as the basis for assessment.BACKGROUND This narrative section provides definitions and explanations for each exercise. Stu-dents should refer to their text for more detailed explanations of the concepts introduced in the lab-oratory exercises.MATERIALS This comprehensive list includes sup-plies, media, and equipment needed for the exercise.

MICROBIOME

M

INVESTIGATIVE EXERCISE

NVESTIGATIVE

CULTURES This list identifies the living organisms required for the exercise.TECHNIQUES REQUIRED This section provides a list of techniques from earlier exercises in the manual needed to complete the current exercise.PROCEDURE At the core of each exercise, this sec-tion provides step-by-step instructions, stated as simply as possible and frequently supplemented with diagrams. Questions are occasionally asked in the Procedure section to remind the student of the rationale for a step.LABORATORY REPORT Designed to help students learn to collect and present data in a systematic fashion, the laboratory report concludes each ex-ercise. Students are first asked to write the Pur-pose of the exercise so they can relate their labo-ratory work to their learning. Students are asked to formulate their Hypothesis or write their Ex-pected Results using information provided in the Background and their own experience. Tables are provided to record Results. The questions in Con-clusions are designed to lead the student from a collection of data or observations to a conclusion. In most instances, the results for each student team will be unique; they can be compared with the information given in the Background and other references but will not be identical to that informa-tion. The Questions reinforce the conclusions and ask the student to interpret results. The range of questions requires students to think about their results, recall facts, and then use this information to answer the questions. Critical Thinking ques-tions are designed to help students use their new knowledge and practice analytical skills. Clinical Application questions have been collected from the literature. They are designed to encourage stu-dents to synthesize their new information to relate concepts and techniques to clinical applications. This lab manual and the course textbook should provide sufficient background to enable students to answer Critical Thinking and Clinical Application questions.

PREPARATION GUIDEThe comprehensive Preparation Guide for Labora-tory Experiments in Microbiology, Twelfth Edition (ISBN 0-134-60520-9) provides all the information

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the instructor needs to set up and teach a laboratory course with this manual. It includes the following:

• General instructions for setting up the lab• Information on obtaining and preparing cultures,

media, and reagents, and expected results for eachof the biochemical tests and cultures used

• A master table showing the techniques and bio-safety level required for each exercise

• Cross-references for each exercise to specific pagesin Tortora/Funke/Case Microbiology: An Introduc-tion, Thirteenth Edition.

• For each exercise: helpful suggestions, detailedlists of materials needed, and answers to all thequestions in the student manual

To make Laboratory Experiments in Microbiol-ogy, Twelfth Edition, easy to use, we have carefully designed the experiments to use inexpensive, read-ily available, nonhazardous materials. Moreover, the exercises have been thoroughly tested in our classes in Minnesota and California by students with a wide variety of talents and interests. Our students have enjoyed their microbiology laboratory experiences; we hope yours will, too!

ACKNOWLEDGMENTSWe are most grateful to the following individuals for their time, talent, and interest in our work. Each person care-fully read and edited critical parts of the manuscript.

Chuck Hoover of the University of California, San Francisco, for making us aware of the new techniques used in dental microbiology for Exercise 47.

Anne Jayne of the University of San Francisco for offering suggestions and a capsule stain.

Kylin Johnson of Skyline College for her invalu-able assistance in preparing materials.

We are indebted to St. Olaf College and Skyline College for providing the facilities and resources in which innovative laboratory exercises can be developed. We are grateful for the wonderful students who have inspired us and have made teaching microbiology a joy.

We would like to commend the staff at Pearson Education for their support. In particular, we thank Jennifer McGill-Walker, our Acquisitions Editor; Ami Sampat and Katrina Taylor for their editorial skill and conscientiousness; and Nancy Tabor for expertly guid-ing this manual through the production process.

Special thanks go to Clifton Franklund of Ferris State University for his expert work on the pre-lab questions that accompany this manual.

Last, but not least, our gratitude goes to Michelle Johnson, who gave her professional insights and was

a sustaining presence; and Don Biederman, for their encouragement and support.

REVIEWERSWe are deeply grateful to the following reviewers who helped shape the direction of the Twelfth Edition:

Veronica Amaku, Lone Star College–CyFair CampusJennifer Bess, Hillsborough Community College–Dale

Mabry CampusLaurie Bradley, Hudson Valley Community CollegeLynn B. DeSanto, Lackawanna CollegeRegina D. Foster, Oklahoma State University Insti-

tute of TechnologyJennifer Hatchel, College of Coastal GeorgiaSuzanne Kempke, St. Johns River State CollegeMustapha Lahrach, Hillsborough Community Col-

lege–SouthShore CampusLuis A. Materon, University of Texas–Pan AmericanStacy Pfluger, Angelina CollegeInes Rauschenbach, Rutgers University and Union

County CollegeJason Rothman, California State Polytechnic Univer-

sity, PomonaMisty D. Wehling, Southeast Community CollegeDaniel Westholm, The College of St. Scholastica

ASM RECOMMENDED LABORATORY SKILLS

ASM: Laboratory skills

A student successfully completing basic microbiology will demonstrate ability in

1. Using a brightfield light microscope to view andinterpret slides, includinga. Correctly setting up and focusing the

microscopeb. Proper handling, cleaning, and storage of the

microscopec. Correctly using all lensesd. Recording microscopic observations

2. Properly preparing slides for microbiological ex-amination, includinga. Cleaning and disposing of slidesb. Preparing smears from solid and liquid

culturesc. Performing wet mount and/or hanging drop

preparationsd. Performing Gram stains

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d. Extrapolating plate counts to obtain the cor-rect CFU or PFU in the starting sample

6. Using standard microbiology laboratory equip-ment correctly, includinga. Using the standard metric system for

weights, lengths, diameters, and volumesb. Lighting and adjusting a laboratory burnerc. Using an incubator

A SPECIAL NOTE TO STUDENTSThis book is for you. The study of microbiology is dynamic because of the diversity of microbes and the variability inherent in every living organism. Out-side the laboratory—on a forest walk or tasting a fine cheese—we experience the activities of microbes. We want to share our excitement for studying these small organisms. Enjoy!

Ted R. Johnson and Christine L. Case

3. Using properly aseptic techniques for the transferand handling of microorganisms and instruments,includinga. Sterilizing and maintaining sterility of trans-

fer instrumentsb. Performing aseptic transferc. Obtaining microbial samples

4. Using appropriate microbiological media and testsystems, includinga. Isolating colonies and/or plaquesb. Maintaining pure culturesc. Using biochemical test mediad. Recording accurately macroscopic observations

5. Estimating the number of microbes in a sampleusing serial dilution techniques, includinga. Choosing and using correctly pipettes and

pipetting devicesb. Spreading correctly diluted samples for

countingc. Estimating appropriate dilutions

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Contents

Preface vi Introduction 1Student Safety Contract 7

PART 1 Microscopy 9 1 Use and Care of the Microscope 11 2 Examination of Living Microorganisms 21

PART 2 Handling Bacteria 29 3 Microbes in the Environment 314 Transfer of Bacteria: Aseptic Technique 41

PART 3 Staining Methods 51 5 Preparation of Smears and Simple Staining 53 6 Negative Staining 59 7 Gram Staining 63 8 Acid-Fast Staining 71 9 Structural Stains (Endospore, Capsule, and Flagella) 7710 Morphologic Unknown 87

PART 4 Cultivation of Bacteria 9511 Isolation of Bacteria by Dilution Techniques 9712 Special Media for Isolating Bacteria 105

PART 5 Microbial Metabolism 10913 Carbohydrate Catabolism 11114 Fermentation 11715 Protein Catabolism, Part 1 12516 Protein Catabolism, Part 2 13117 Respiration 13918 Unknown Identification and Bergey’s Manual 147

PART 6 Microbial Growth 15319 Oxygen and the Growth of Bacteria 15520 Determination of a Bacterial Growth Curve: The Role of

Temperature 16321 Biofilms 171

PART 7 Control of Microbial Growth 17722 Physical Methods of Control: Heat 17923 Physical Methods of Control: Ultraviolet Radiation 18724 Chemical Methods of Control: Disinfectants and

Antiseptics 19525 Chemical Methods of Control: Antimicrobial Drugs 20126 Effectiveness of Hand Scrubbing 209

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PART 8 Microbial Genetics 21327 Regulation of Gene Expression 21528 Isolation of Bacterial Mutants 22329 Transformation of Bacteria 22930 DNA Fingerprinting 23531 Genetic Engineering 24332 Ames Test for Detecting Possible Chemical

Carcinogens 253

PART 9 The Microbial World 26133 Fungi: Yeasts and Molds 26334 Phototrophs: Algae and Cyanobacteria 27335 Protozoa 28136 Parasitic Helminths 287

PART 10 Viruses 29737 Isolation and Titration of Bacteriophages 29938 Plant Viruses 307

PART 11 Interaction of Microbe and Host 31139 Epidemiology 31340 Koch’s Postulates 321

PART 12 Immunology 33141 Innate Immunity 33342 Agglutination Reactions: Slide Agglutination 34143 Agglutination Reactions: Microtiter Agglutination 34944 ELISA Technique 353

PART 13 Microorganisms and Disease 35945 Bacteria of the Skin 36146 Bacteria of the Respiratory Tract 36747 Bacteria of the Mouth 37548 Bacteria of the Gastrointestinal Tract 38149 Bacteria of the Genitourinary Tract 38950 Identification of an Unknown from a Clinical Sample 39751 Rapid Identification Methods 403

PART 14 Microbiology and the Environment 41352 Microbes in Water: Multiple-Tube Technique 41553 Microbes in Water: Membrane Filter Technique 42354 Microbes in Food: Contamination 43155 Microbes Used in the Production of Foods 43956 Microbes in Soil: The Nitrogen and Sulfur Cycles 44357 Microbes in Soil: Bioremediation 455

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Appendices 463A Pipetting 463B Dilution Techniques and Calculations 466C Use of the Spectrophotometer 470D Graphing 472E Use of the Dissecting Microscope 474F Use of the Membrane Filter 475G Electrophoresis 477H Keys to Bacteria 479

Art and Photography Credits CR-1

Index 000