Laboratory Exercises in Microbiology.pdf

Harley−Prescott:

Laboratory Exercises in

Microbiology, Fifth Edition

Front Matter

Preface

Companies, 2002

PREFACE

Take interest, I implore you, in those sacred dwellings which one designates

by the expressive term: laboratories. Demand that they be multiplied, that

they be adorned. These are the temples of the future—temples of well-being

and of happiness. There it is that humanity grows greater, stronger, better.

Louis Pasteur

(French chemist, founder of microbiology, 1822–1895)

There are many excellent microbiology laboratory

manuals on the market and many others that are

called “in-house” productions because they are writ-

ten for a microbiology course at a particular school.

Why another microbiology manual? The answer is

straightforward. Many instructors want a manual

that is directly correlated with a specific textbook.

As a result, this laboratory manual was designed

and written to be used in conjunction with the text-

book Microbiology, fifth edition, by Lansing M.

Prescott, John P. Harley, and Donald A. Klein; how-

ever, it can be used with other textbooks with slight

adaptation.

Since this manual correlates many of the micro-

biological concepts in the textbook with the various

exercises, comprehensive introductory material is

not given at the beginning of each exercise. Instead,

just enough specific explanation is given to com-

plement, augment, reinforce, and enhance what is

in the textbook. We feel that time allocation is an

important aspect of any microbiology course. Stu-

dents should not be required to reread in the labora-

tory manual an in-depth presentation of material

that has already been covered satisfactorily in

the textbook.

Each exercise has been designed to be modular

and short. This will allow the instructor to pick and

choose only those exercises or parts of exercises

that are applicable to a specific course. Several ex-

ercises usually can be completed in a two- or three-

hour laboratory period. The exercises have also

been designed to use commonly available equip-

ment, with the least expense involved, and to be

completed in the shortest possible time period.

Considering the above parameters, the purpose of

this laboratory manual is to guide students through a

process of development of microbiological technique,

experimentation, interpretation of data, and discovery

in a manner that will complement the textbook and

make the study of microbiology both exciting and

challenging. According to an old Chinese proverb:

Tell me and I will forget.

Show me and I might remember.

Involve me and I will understand.

These words convey our basic philosophy that it is ex-

periences in the microbiology laboratory and the sci-

entific method that help develop students’ critical

thinking and creativity and that increase their appreci-

ation of the mechanisms by which microbiologists an-

alyze information. The laboratory accomplishes this

by having students become intensely and personally

involved in the knowledge they acquire.

The array of exercises was chosen to illustrate the

basic concepts of general microbiology as a whole

and of the individual applied fields. The protocols

vary in content and complexity, providing the instruc-

tor with flexibility to mold the laboratory syllabus to

the particular needs of the students, available time and

equipment, and confines and scope of the course. Fur-

thermore, it provides a wide spectrum of individual

exercises suitable for students in elementary and ad-

vanced general microbiology as well as those in vari-

ous allied health programs.

In 1997, the American Society for Microbiology,

through its Office of Education and Training, adopted

a Laboratory Core Curriculum representing themes

and topics considered essential to teach in every intro-

ductory microbiology laboratory, regardless of its em-

phasis. An instructor might add items appropriate to

allied health, applied, environmental, or majors mi-

crobiology courses.

The Laboratory Core is not meant to be a syllabus

or outline. The core themes and topics are meant to

frame objectives to be met somewhere within the in-

troductory microbiology laboratory. Depending on the

Harley−Prescott:

Laboratory Exercises in

Microbiology, Fifth Edition

Front Matter

Preface

Companies, 2002

specific emphasis of the course, a single lab session

could meet multiple core objectives, focus on one ob-

jective, or emphasize a topic that is not in the lab core

but is important to that particular course.

d. extrapolating plate counts to obtain correct

CFU or PFU in the starting sample

6. Use standard microbiology laboratory

equipment correctly, including

a. using the standard metric system for

weights, lengths, diameters, and volumes

b. lighting and adjusting a laboratory burner

c. using an incubator

Laboratory Skills

A student successfully completing basic microbiol-

ogy will demonstrate the ability to

1. Use a bright-field light microscope to view and

interpret slides, including

a. correctly setting up and focusing the

microscope

b. proper handling, cleaning and storage of the

microscope

c. correct use of all lenses

d. recording microscopic observations

2. Properly prepare slides for microbiological

examination, including

a. cleaning and disposal of slides

b. preparing smears from solid and liquid

cultures

c. performing wet-mount and/or hanging drop

preparations

d. performing Gram stains

3. Properly use aseptic techniques for the transfer

and handling of microorganisms and instruments,

including

a. sterilizing and maintaining sterility of

transfer instruments

b. performing aseptic transfer

c. obtaining microbial samples

4. Use appropriate microbiological media and

test systems, including

a. isolating colonies and/or plaques

b. maintaining pure cultures

c. using biochemical test media

d. accurately recording macroscopic

observations

5. Estimate the number of microorganisms in a

sample using serial dilution techniques, including

a. correctly choosing and using pipettes and

pipetting devices

b. correctly spreading diluted samples for

counting

c. estimating appropriate dilutions

Laboratory Thinking Skills

A student successfully completing basic microbiol-

ogy will demonstrate an increased skill level in

1. Cognitive processes, including

a. formulating a clear, answerable question

b. developing a testable hypothesis

c. predicting expected results

d. following an experimental protocol

2. Analysis skills, including

a. collecting and organizing data in a

systematic fashion

b. presenting data in an appropriate form

(graphs, tables, figures, or descriptive

paragraphs)

c. assessing the validity of the data (including

integrity and significance)

d. drawing appropriate conclusions based on

the results

3. Communications skills, including

a. discussing and presenting laboratory results

or findings in the laboratory

4. Interpersonal and citizenry skills, including

a. working effectively in groups or teams so

that the task, results, and analysis are shared

b. effectively managing time and tasks to be

done simultaneously, by individuals and

within a group

c. integrating knowledge and making informed

judgments about microbiology in everyday

life

Laboratories typically supplement and integrate

closely with the lecture content in ways that are unique to

each instructor. Consequently, the laboratory content that

is considered essential for laboratory work by one instruc-

tor may be covered in lecture portion of the course by an-

other instructor, making it difficult to define specific top-

vi Preface

Harley−Prescott:

Laboratory Exercises in

Microbiology, Fifth Edition

Front Matter

Preface

Companies, 2002

ics that should be integral in all microbiology laborato-

ries. As a result, the ASM Laboratory Core Curriculum

Committee developed themes, which are broadly based

and will enable instructors to have the flexibility to use a

wide variety of laboratories to meet the suggested core.

Astudent successfully completing basic microbi-

ology will demonstrate mastery of the basic principles

of the following themes and complete laboratory activ-

ities that focus on one or more of the topics under each

theme.

Theme 1. Integrating themes—impact of

microorganisms on the biosphere and humans;

microbial diversity

Theme 2. Microbial cell biology, including cell

structure and function, growth and division, and

metabolism

Theme 3. Microbial genetics, including mutations

Theme 4. Interactions of microorganisms with

hosts (humans, other animals, plants), including

pathogenicity mechanisms and antimicrobial

agents

In order to meet the above themes, topics, and

skills (The American Society for Microbiology Labo-

ratory Core Curriculum), this manual consists of 66

exercises arranged into 11 parts covering the following

basic topics:

PART ONE, Microscopic Techniques, introduces

the students to the proper use and care of the

different types of microscopes used in the

microbiology laboratory for the study of

microorganisms.

PART TWO, Bacterial Morphology and Staining,

presents the basic procedures for visualization and

differentiation of microorganisms based on cell

form and various structures.

PART THREE, Basic Laboratory and Culture

Techniques, acquaints students with proper

laboratory procedures in preparing

microbiological media and in culture techniques

that are used in isolating microorganisms.

PART FOUR, Biochemical Activities of Bacteria,

introduces some of the biochemical activities

that may be used in characterizing and

identifying bacteria.

PART FIVE, Rapid Multitest Systems, acquaints

students with some of the multitest systems that

can be used to identify bacteria.

PART SIX, Unknown Identification, contains two

exercises that guide students through the use of

Bergey’s Manual of Systematic Bacteriology in

the identification of unknown bacteria.

PART SEVEN, Environmental Factors Affecting

Growth of Microorganisms, acquaints students

with some of the various physical and chemical

agents that affect microbial growth.

PART EIGHT, Environmental and Food

Microbiology, is concerned with the

environmental aspects of water, milk, and food.

PART NINE, Medical Microbiology, presents an

overview of some pathogenic microorganisms,

and acquaints students with basic procedures used

in isolation and identification of pathogens from

infected hosts, including those from the student’s

own body.

PART TEN, Survey of Selected Eucaryotic

Microorganisms, presents an overview that is

intended to help students appreciate the

morphology, taxonomy, and biology of the fungi.

PART ELEVEN, Microbial Genetics and

Genomics, presents six experiments designed to

illustrate the general principles of bacterial

genetics and genomics.

The format of each exercise in this manual is in-

tended to promote learning and mastery in the shortest

possible time. To this end, each experiment is de-

signed as follows:

Safety Considerations

This laboratory manual endeavors to include many

of the safety precautionary measures established by

the Centers for Disease Control and Prevention

(CDC), Atlanta, Georgia; the Occupational Safety

and Health Administration (OSHA); and the Envi-

ronmental Protection Agency (EPA). Efforts are

made to instruct the student on safety, and all exer-

cises will contain precautionary procedures that

these agencies are enforcing in hospitals, nursing

homes, commercial laboratories, and industry. A

safety considerations box is included for each ex-

ercise to help both the instructor and student prepare

themselves for the possibility of accidents.

Both the instructor and student should keep in

mind at all times that most technical programs, such

as a microbiology laboratory, carry some measure of

associated risk. The microbiology laboratory is a

place where infectious microorganisms are handled,

examined, and studied with safety and effectiveness.

However, any of the microorganisms we work with

Preface vii

Harley−Prescott:

Laboratory Exercises in

Microbiology, Fifth Edition

Front Matter

Preface

Companies, 2002

may be pathogenic in an immunocompromised per-

son. Therefore, rather than modifying the objectives

in this laboratory manual to avoid any risk, the au-

thors propose that instructors and students imple-

ment the Centers for Disease Control and Preven-

tion (CDC) principles of biosafety throughout. One

way we propose is to simply modify the “Universal

Precautions” ( see pp. xiii–xiv ) so the wording is ap-

propriate for the classroom by simply changing

“laboratory worker” to “student.” In addition, a

written safety policy consistent with CDC guide-

lines and adopted by your institution’s governing

body will protect you, your institution, and the stu-

dents. As in any laboratory, safety should be a major

part of the curriculum. Students should be required

to demonstrate their knowledge of safety before

they begin each laboratory exercise.

Pronunciation Guide

This section contains the phonetic pronunciations for

all organisms used in the exercise. If students take the

time to sound out new and unfamiliar terms and say

them aloud several times, they will learn to use the

vocabulary of microbiologists.

Why Are the Above Bacteria, Slides, or Other

Microorganisms Used in This Experiment?

The authors have chosen specific viruses, bacteria,

fungi, protozoa, algae, and various prepared slides for

each exercise. This microbial material has been se-

lected based on cost, ease of growth, availability, reli-

ability, and most importantly, the ability to produce

the desired experimental results. In order to communi-

cate these guidelines to the student, this section ex-

plains why the authors have chosen the microbial ma-

terial being used and also gives additional

biochemical, morphological, and taxonomic informa-

tion about the microorganism(s) that the student

should find helpful when performing the experiment.

Materials per Student or Group of Students

To aid in the preparation of all exercises, each proce-

dure contains a list of the required cultures with Amer-

ican Type Culture Collection catalog numbers (Ameri-

can Type Culture Collection, 12301 Parklawn Drive,

Rockville, Maryland 29852–1776; www.ATCC.org;

703-365-2700), media, reagents, and other equipment

necessary to complete the exercise in the allocated lab

time either per student or group of students. Appen-

dixes H and I provide recipes for reagents, stains, and

culture media. Appendix J describes the maintenance

of microorganisms and supply sources.

Medical Application

Many students using this laboratory manual are either

in one of the allied health disciplines, such as nursing,

or in a preprofessional program such as premed, pre-

dent, or prevet and need to know the clinical relevance

of each exercise performed. To satisfy this need, a Med-

ical Application section is included for some of the

medically oriented exercises. Medical applications are

described for most clinical procedures as a specific ap-

plication of the purpose of the exercise. For example, a

procedure can be used for the identification of a partic-

ular microorganism or used in combination with other

exercises in a diagnosis. For these exercises, some im-

portant pathogens with their diseases and their need for

the test being performed in the exercise are listed.

Learning Objectives

Each exercise has a set of learning objectives that

define the specific goals of the laboratory session. It

is to the student’s advantage to read through this list

before coming to class. In like manner, these objec-

tives should be given special attention during the

laboratory exercise. Upon conscientious completion

of the exercise, the student should be able to meet all

of the objectives for that exercise. Before leaving the

class, students should check the objectives once

again to see that they can master them. If problems

arise, consult the instructor.

Principles

This section contains a brief discussion of the micro-

biological principles, concepts, and techniques that

underlie the experimental procedures being performed

in the exercise.

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