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Benson: Microbiological
Applications Lab Manual,
Eighth Edition
Front Matter
Preface
© The McGraw−Hill
Companies, 2001
This eighth edition of
Microbiological Applications
differs from the previous edition in that it has acquired
four new exercises and dropped three experiments. It
retains essentially the same format throughout, how-
ever. In response to requests for more emphasis on lab-
oratory safety, three new features have been incorpo-
rated into the text. In addition, several experiments
have been altered to improve simplicity and reliability.
The three exercises that were dropped pertain to fla-
gellar staining, bacterial conjugation, and nitrification in
soil. All of these exercises were either difficult to per-
form, unreliable, or of minimal pedagogical value.
To provide greater safety awareness in the labora-
tory, the following three features were added: (1) an
introductory laboratory protocol, (2) many cautionary
boxes dispersed throughout the text, and (3) a new ex-
ercise pertaining to aseptic technique.
The three-page laboratory protocol, which fol-
lows this preface, replaces the former introduction. It
provides terminology, safety measures, an introduc-
tion to aseptic technique, and other rules that apply to
laboratory safety.
To alert students to potential hazards in performing
certain experiments, caution boxes have been incorpo-
rated wherever they are needed. Although most of these
cautionary statements existed in previous editions, they
were not emphasized as much as they are in this edition.
Exercise 8 (Aseptic Technique) has been struc-
tured to provide further emphasis on culture tube han-
dling. In previous editions it was assumed that students
would learn these important skills as experiments were
performed. With the risk of being redundant, six pages
have been devoted to the proper handling of culture
tubes when making inoculation transfers.
Although most experiments remain unchanged,
there are a few that have been considerably altered.
Exercise 27 (Isolation of Anaerobic Phototrophic
Bacteria), in particular, is completely new. By using
the Winogradsky column for isolating and identifying
the phototrophic sulfur bacteria, it has been possible
to greatly enrich the scope of this experiment. Another
exercise that has been altered somewhat is Exercise
48, which pertains to oxidation and fermentation tests
that are used for identifying unknown bacteria.
The section that has undergone the greatest reor-
ganization is Part 10 (Microbiology of Soil). In the
previous edition it consisted of five exercises. In this
edition it has been expanded to seven exercises. A
more complete presentation of the nitrogen cycle is of-
fered in Exercise 58, and two new exercises (Exercises
61 and 62) are included that pertain to the isolation of
denitrifiers.
In addition to the above changes there has been
considerable upgrading of graphics throughout the
book. Approximately thirty-five illustrations have been
replaced. Several critical color photographs pertaining
to molds and physiological tests were also replaced to
bring about more faithful color representation.
I am greatly indebted to my editors, Jean Fornango
and Jim Smith, who made the necessary contacts for
critical reviews. As a result of their efforts the following
individuals have provided me with excellent sugges-
tions for improvement of this manual: Barbara Collins
at California Lutheran University, Thousand Oaks, CA;
Alfred Brown of Auburn University, Auburn, AL;
Lester A. Scharlin at El Camino College, Torrance, CA;
and Hershell Hanks at Collin County Community
College, Plano, TX.
vii
Preface
Benson: Microbiological
Applications Lab Manual,
Eighth Edition
Front Matter
Laboratory Protocol
© The McGraw−Hill
Companies, 2001
Welcome to the exciting field of microbiology! The
intent of this laboratory manual is to provide you with
basic skills and tools that will enable you to explore a
vast microbial world. Its scope is incredibly broad in
that it includes a multitude of viruses, bacteria, proto-
zoans, yeasts, and molds. Both beneficial and harmful
ones will be studied. Although an in-depth study of
any single one of these groups could constitute a full
course by itself, we will be able to barely get ac-
quainted with them.
To embark on this study it will be necessary for
you to learn how to handle cultures in such a way that
they are not contaminated or inadvertently dispersed
throughout the classroom. This involves learning
aseptic techniques and practicing preventive safety
measures. The procedures outlined here address these
two aspects.
It is of paramount importance that you
know all the regulations that are laid down here as
Laboratory Protocol.
Scheduling
During the first week of this course
your instructor will provide you with a schedule of
laboratory exercises arranged in the order of their per-
formance. Before attending laboratory each day,
check the schedule to see what experiment or experi-
ments will be performed and prepare yourself so that
you understand what will be done.
Each laboratory session will begin with a short
discussion to brief you on the availability of materials
and procedures.
Since the preliminary instructions
start promptly at the beginning of the period,it is
ex-
tremely important that you are not late to class.
Personal Items
When you first enter the lab, place
all personal items such as jackets, bags, and books in
some out of the way place for storage. Don’t stack
them on your desktop. Desk space is minimal and
must be reserved for essential equipment and your
laboratory manual. The storage place may be a
drawer, locker, coatrack, or perimeter counter. Your
instructor will indicate where they should be placed.
Attire
A lab coat or apron must be worn at all times
in the laboratory. It will protect your clothing from ac-
cidental contamination and stains in the lab. When
leaving the laboratory, remove the coat or apron. In
addition, long hair must be secured in a ponytail to
prevent injury from Bunsen burners and contamina-
tion of culture material.
T
ERMINOLOGY
Various terms such as sterilization, disinfection, ger-
micides, sepsis, and aseptic techniques will be used
here. To be sure that you understand exactly what they
mean, the following definitions are provided.
Sterilization
is a process in which all living mi-
croorganisms, including viruses, are destroyed. The
organisms may be killed with steam, dry heat, or in-
cineration. If we say an article is sterile, we understand
that it is completely free of all living microorganisms.
Generally speaking, when we refer to sterilization as it
pertains here to laboratory safety, we think, primarily,
in terms of steam sterilization with the autoclave. The
ultimate method of sterilization is to burn up the in-
fectious agents or
incinerate
them. All biological
wastes must ultimately be incinerated for disposal.
Disinfection
is a process in which vegetative,
nonsporing microorganisms are destroyed. Agents
that cause disinfection are called
disinfectants
or
germicides.
Such agents are used only on inanimate
objects because they are toxic to human and animal
tissues.
Sepsis
is defined as the growth (multiplication) of
microorganisms in tissues of the body. The term
asep-
sis
refers to any procedure that prevents the entrance
of infectious agents into sterile tissues, thus prevent-
ing infection.
Aseptic techniques
refer to those prac-
tices that are used by microbiologists to exclude all
organisms from contaminating media or contacting
living tissues.
Antiseptics
are chemical agents (often
dilute disinfectants) that can be safely applied exter-
nally to human tissues to destroy or inhibit vegetative
bacteria.
A
SEPTIC
T
ECHNIQUES
When you start handling bacterial cultures as in
Exercises 9 and 10, you will learn the specifics of
aseptic techniques. Some of the basic things you will
do are as follows:
ix
Laboratory Protocol
Benson: Microbiological
Applications Lab Manual,
Eighth Edition
Front Matter
Laboratory Protocol
© The McGraw
−
Hill
Companies, 2001
Hand Washing
Before you start working in the lab,
wash your hands with a liquid detergent and dry them
with paper toweling. At the end of the period, before
leaving the laboratory, wash them again.
Tabletop Disinfection.
The first chore of the day
will be to sponge down your desktop with a disinfec-
tant. This process removes any dust that may be pre-
sent and minimizes the chances of bacterial contami-
nation of cultures that you are about to handle.
Your instructor will indicate where the bottles of
disinfectant and sponges are located. At the end of the
period before leaving the laboratory, perform the same
procedure to protect students that may occupy your desk
in the next class.
Bunsen Burner Usage
When using a Bunsen burner
to flame loops, needles, and test tubes, follow the pro-
cedures outlined in Exercise 8. Inoculating loops and
needles should be heated until they are red-hot. Before
they are introduced into cultures, they must be allowed
to cool down sufficiently to prevent killing organisms
that are to be transferred.
If your burner has a pilot on it and you plan to use
the burner only intermittently, use it. If your burner
lacks a pilot, turn off the burner when it is not being
used. Excessive unnecessary use of Bunsen burners in
a small laboratory can actually raise the temperature
of the room. More important is the fact that unat-
tended burner flames are a constant hazard to hair,
clothing, and skin.
The proper handling of test tubes, while transfer-
ring bacteria from one tube to another, requires a cer-
tain amount of skill. Test-tube caps must never be
placed down on the desktop while you are making in-
oculations. Techniques that enable you to make trans-
fers properly must be mastered. Exercise 8 pertains to
these skills.
Pipetting
Transferring solutions or cultures by
pipette must always be performed with a mechanical
suction device. Under no circumstances is pipetting
by mouth allowed in this laboratory.
Disposal of Cultures and Broken Glass
The fol-
lowing rules apply to culture and broken glass disposal:
1. Petri dishes must be placed in a plastic bag to be
autoclaved.
2. Unneeded test-tube cultures must be placed in a
wire basket to be autoclaved.
3. Used pipettes must be placed in a plastic bag for
autoclaving.
4. Broken glass should be swept up into a dustpan
and placed in a container reserved for broken
glass. Don’t try to pick up the glass fragments
with your fingers.
5. Contaminated material must never be placed in a
wastebasket.
A
CCIDENTAL
S
PILLS
All accidental spills, whether chemical or biological,
must be reported immediately to your instructor.
Although the majority of microorganisms used in
this laboratory are nonpathogens, some pathogens
will be encountered. It is for this reason that we must
treat all accidental biological spills as if pathogens
were involved.
Chemical spills are just as important to report be-
cause some agents used in this laboratory may be car-
cinogenic; others are poisonous; and some can cause
dermal damage such as blistering and depigmentation.
Decontamination Procedure
Once your instructor
is notified of an accidental spill, the following steps
will take place:
1. Any clothing that is contaminated should be
placed in an autoclavable plastic bag and auto-
claved.
2. Paper towels, soaked in a suitable germicide, such
as 5% bleach, are placed over the spill.
3. Additional germicide should be poured around
the edges of the spill to prevent further
aerosolization.
4. After approximately 20 minutes, the paper tow-
els should be scraped up off the floor with an
autoclavable squeegee into an autoclavable
dust pan.
5. The contents of the dust pan are transferred to an
autoclavable plastic bag, which may itself be
placed in a stainless steel bucket or pan for trans-
port to an autoclave.
6. All materials, including the squeegee and dust-
pan, are autoclaved.
A
DDITIONAL
I
MPORTANT
R
EGULATIONS
Here are a few additional laboratory rules:
1. Don’t remove cultures, reagents, or other materi-
als from the laboratory unless you have been
granted specific permission.
2. Don’t smoke or eat food in the laboratory.
3. Make it a habit to keep your hands away from your
mouth. Obviously, labels are never moistened
with the tongue; use tap water or self-adhesive la-
bels instead