Benson: Microbiological Front Matter Preface © The McGraw−Hill
Applications Lab Manual, Companies, 2001
Eighth Edition
Preface
This eighth edition of Microbiological Applications Although most experiments remain unchanged,
differs from the previous edition in that it has acquired there are a few that have been considerably altered.
four new exercises and dropped three experiments. It Exercise 27 (Isolation of Anaerobic Phototrophic
retains essentially the same format throughout, how- Bacteria), in particular, is completely new. By using
ever. In response to requests for more emphasis on lab- the Winogradsky column for isolating and identifying
oratory safety, three new features have been incorpo- the phototrophic sulfur bacteria, it has been possible
rated into the text. In addition, several experiments to greatly enrich the scope of this experiment. Another
have been altered to improve simplicity and reliability. exercise that has been altered somewhat is Exercise
The three exercises that were dropped pertain to fla- 48, which pertains to oxidation and fermentation tests
gellar staining, bacterial conjugation, and nitrification in that are used for identifying unknown bacteria.
soil. All of these exercises were either difficult to per- The section that has undergone the greatest reor-
form, unreliable, or of minimal pedagogical value. ganization is Part 10 (Microbiology of Soil). In the
To provide greater safety awareness in the labora- previous edition it consisted of five exercises. In this
tory, the following three features were added: (1) an edition it has been expanded to seven exercises. A
introductory laboratory protocol, (2) many cautionary more complete presentation of the nitrogen cycle is of-
boxes dispersed throughout the text, and (3) a new ex- fered in Exercise 58, and two new exercises (Exercises
ercise pertaining to aseptic technique. 61 and 62) are included that pertain to the isolation of
The three-page laboratory protocol, which fol- denitrifiers.
lows this preface, replaces the former introduction. It In addition to the above changes there has been
provides terminology, safety measures, an introduc- considerable upgrading of graphics throughout the
tion to aseptic technique, and other rules that apply to book. Approximately thirty-five illustrations have been
laboratory safety. replaced. Several critical color photographs pertaining
To alert students to potential hazards in performing to molds and physiological tests were also replaced to
certain experiments, caution boxes have been incorpo- bring about more faithful color representation.
rated wherever they are needed. Although most of these I am greatly indebted to my editors, Jean Fornango
cautionary statements existed in previous editions, they and Jim Smith, who made the necessary contacts for
were not emphasized as much as they are in this edition. critical reviews. As a result of their efforts the following
Exercise 8 (Aseptic Technique) has been struc- individuals have provided me with excellent sugges-
tured to provide further emphasis on culture tube han- tions for improvement of this manual: Barbara Collins
dling. In previous editions it was assumed that students at California Lutheran University, Thousand Oaks, CA;
would learn these important skills as experiments were Alfred Brown of Auburn University, Auburn, AL;
performed. With the risk of being redundant, six pages Lester A. Scharlin at El Camino College, Torrance, CA;
have been devoted to the proper handling of culture and Hershell Hanks at Collin County Community
tubes when making inoculation transfers. College, Plano, TX.
vii
,Benson: Microbiological Front Matter Laboratory Protocol © The McGraw−Hill Benson: Microbiological Front Matter Laboratory Protocol © The McGraw−Hill
Applications Lab Manual, Companies, 2001 Applications Lab Manual, Companies, 2001
Eighth Edition Eighth Edition
Laboratory Protocol
Hand Washing Before you start working in the lab, glass. Don’t try to pick up the glass fragments
Laboratory Protocol wash your hands with a liquid detergent and dry them
with paper toweling. At the end of the period, before
with your fingers.
5. Contaminated material must never be placed in a
leaving the laboratory, wash them again. wastebasket.
Tabletop Disinfection. The first chore of the day
Welcome to the exciting field of microbiology! The addition, long hair must be secured in a ponytail to
will be to sponge down your desktop with a disinfec- ACCIDENTAL SPILLS
tant. This process removes any dust that may be pre- All accidental spills, whether chemical or biological,
intent of this laboratory manual is to provide you with prevent injury from Bunsen burners and contamina- sent and minimizes the chances of bacterial contami-
basic skills and tools that will enable you to explore a tion of culture material. must be reported immediately to your instructor.
nation of cultures that you are about to handle. Although the majority of microorganisms used in
vast microbial world. Its scope is incredibly broad in Your instructor will indicate where the bottles of
that it includes a multitude of viruses, bacteria, proto- this laboratory are nonpathogens, some pathogens
disinfectant and sponges are located. At the end of the
zoans, yeasts, and molds. Both beneficial and harmful TERMINOLOGY period before leaving the laboratory, perform the same
will be encountered. It is for this reason that we must
ones will be studied. Although an in-depth study of treat all accidental biological spills as if pathogens
Various terms such as sterilization, disinfection, ger- procedure to protect students that may occupy your desk
any single one of these groups could constitute a full were involved.
micides, sepsis, and aseptic techniques will be used in the next class.
course by itself, we will be able to barely get ac- Chemical spills are just as important to report be-
here. To be sure that you understand exactly what they
quainted with them. cause some agents used in this laboratory may be car-
mean, the following definitions are provided.
To embark on this study it will be necessary for Bunsen Burner Usage When using a Bunsen burner cinogenic; others are poisonous; and some can cause
Sterilization is a process in which all living mi-
you to learn how to handle cultures in such a way that to flame loops, needles, and test tubes, follow the pro- dermal damage such as blistering and depigmentation.
croorganisms, including viruses, are destroyed. The
they are not contaminated or inadvertently dispersed cedures outlined in Exercise 8. Inoculating loops and
organisms may be killed with steam, dry heat, or in-
throughout the classroom. This involves learning needles should be heated until they are red-hot. Before Decontamination Procedure Once your instructor
cineration. If we say an article is sterile, we understand
aseptic techniques and practicing preventive safety they are introduced into cultures, they must be allowed is notified of an accidental spill, the following steps
that it is completely free of all living microorganisms.
measures. The procedures outlined here address these to cool down sufficiently to prevent killing organisms will take place:
Generally speaking, when we refer to sterilization as it
two aspects. It is of paramount importance that you that are to be transferred.
pertains here to laboratory safety, we think, primarily, 1. Any clothing that is contaminated should be
know all the regulations that are laid down here as If your burner has a pilot on it and you plan to use
in terms of steam sterilization with the autoclave. The placed in an autoclavable plastic bag and auto-
Laboratory Protocol. the burner only intermittently, use it. If your burner
ultimate method of sterilization is to burn up the in- claved.
lacks a pilot, turn off the burner when it is not being
fectious agents or incinerate them. All biological 2. Paper towels, soaked in a suitable germicide, such
Scheduling During the first week of this course used. Excessive unnecessary use of Bunsen burners in
wastes must ultimately be incinerated for disposal. as 5% bleach, are placed over the spill.
your instructor will provide you with a schedule of a small laboratory can actually raise the temperature
Disinfection is a process in which vegetative, 3. Additional germicide should be poured around
laboratory exercises arranged in the order of their per- of the room. More important is the fact that unat-
nonsporing microorganisms are destroyed. Agents the edges of the spill to prevent further
formance. Before attending laboratory each day, tended burner flames are a constant hazard to hair,
that cause disinfection are called disinfectants or aerosolization.
check the schedule to see what experiment or experi- clothing, and skin.
germicides. Such agents are used only on inanimate 4. After approximately 20 minutes, the paper tow-
ments will be performed and prepare yourself so that The proper handling of test tubes, while transfer-
objects because they are toxic to human and animal els should be scraped up off the floor with an
you understand what will be done. ring bacteria from one tube to another, requires a cer-
tissues. autoclavable squeegee into an autoclavable
Each laboratory session will begin with a short tain amount of skill. Test-tube caps must never be
Sepsis is defined as the growth (multiplication) of dust pan.
discussion to brief you on the availability of materials placed down on the desktop while you are making in-
microorganisms in tissues of the body. The term asep- 5. The contents of the dust pan are transferred to an
and procedures. Since the preliminary instructions oculations. Techniques that enable you to make trans-
sis refers to any procedure that prevents the entrance autoclavable plastic bag, which may itself be
start promptly at the beginning of the period, it is ex- fers properly must be mastered. Exercise 8 pertains to
of infectious agents into sterile tissues, thus prevent- placed in a stainless steel bucket or pan for trans-
tremely important that you are not late to class. these skills.
ing infection. Aseptic techniques refer to those prac- port to an autoclave.
tices that are used by microbiologists to exclude all 6. All materials, including the squeegee and dust-
Personal Items When you first enter the lab, place organisms from contaminating media or contacting Pipetting Transferring solutions or cultures by pan, are autoclaved.
all personal items such as jackets, bags, and books in living tissues. Antiseptics are chemical agents (often pipette must always be performed with a mechanical
some out of the way place for storage. Don’t stack dilute disinfectants) that can be safely applied exter- suction device. Under no circumstances is pipetting
them on your desktop. Desk space is minimal and nally to human tissues to destroy or inhibit vegetative by mouth allowed in this laboratory.
must be reserved for essential equipment and your bacteria. ADDITIONAL IMPORTANT
laboratory manual. The storage place may be a REGULATIONS
drawer, locker, coatrack, or perimeter counter. Your Disposal of Cultures and Broken Glass The fol-
instructor will indicate where they should be placed. lowing rules apply to culture and broken glass disposal: Here are a few additional laboratory rules:
ASEPTIC TECHNIQUES 1. Petri dishes must be placed in a plastic bag to be 1. Don’t remove cultures, reagents, or other materi-
Attire A lab coat or apron must be worn at all times When you start handling bacterial cultures as in autoclaved. als from the laboratory unless you have been
in the laboratory. It will protect your clothing from ac- Exercises 9 and 10, you will learn the specifics of 2. Unneeded test-tube cultures must be placed in a granted specific permission.
cidental contamination and stains in the lab. When aseptic techniques. Some of the basic things you will wire basket to be autoclaved. 2. Don’t smoke or eat food in the laboratory.
leaving the laboratory, remove the coat or apron. In do are as follows: 3. Used pipettes must be placed in a plastic bag for 3. Make it a habit to keep your hands away from your
autoclaving. mouth. Obviously, labels are never moistened
4. Broken glass should be swept up into a dustpan with the tongue; use tap water or self-adhesive la-
and placed in a container reserved for broken bels instead.
ix x
,Benson: Microbiological Front Matter Laboratory Protocol © The McGraw−Hill Benson: Microbiological I. Microscopy Introduction © The McGraw−Hill
Applications Lab Manual, Companies, 2001 Applications Lab Manual, Companies, 2001
Eighth Edition Eighth Edition
Laboratory Protocol
1
4. Always clean up after yourself. Gram-stained
slides that have no further use to you should be
7. If you have borrowed something from someone,
return it. PART Microscopy
washed and dried and returned to a slide box. 8. Do not leave any items on your desk at the end of
Coverslips should be cleaned, dried, and returned. the period.
Staining trays should be rinsed out and returned to 9. Do not disturb another class at any time. Wait un-
their storage place. til the class is dismissed.
5. Return all bulk reagent bottles to places of storage. 10. Treat all instruments, especially microscopes,
6. Return inoculating loops and needles to your stor- with extreme care. If you don’t understand how a
age container. Be sure that they are not upside piece of equipment functions, ask your instructor.
down. 11. Work cooperatively with other students in group- Although there are many kinds of microscopes available to the mi-
assigned experiments, but do your own analyses
crobiologist today, only four types will be described here for our
of experimental results.
use: the brightfield, darkfield, phase-contrast, and fluorescence
microscopes. If you have had extensive exposure to microscopy in
previous courses, this unit may not be of great value to you; how-
ever, if the study of microorganisms is a new field of study for you,
there is a great deal of information that you need to acquire about
the proper use of these instruments.
Microscopes in a college laboratory represent a considerable
investment and require special care to prevent damage to the
lenses and mechanicals. The fact that a laboratory microscope
may be used by several different individuals during the day and
moved around from one place to another results in a much greater
chance for damage and wear to occur than if the instrument were
used by only one individual.
The complexity of some of the more expensive microscopes
also requires that certain adjustments be made periodically.
Knowing how to make these adjustments to get the equipment to
perform properly is very important. An attempt is made in the five
exercises of this unit to provide the necessary assistance in getting
the most out of the equipment.
Microscopy should be as fascinating to the beginner as it is to
the professional of long standing; however, only with intelligent un-
derstanding can the beginner approach the achievement that oc-
curs with years of experience.
xi 1
, Benson: Microbiological I. Microscopy 1.Brightfield Microscopy © The McGraw−Hill Benson: Microbiological I. Microscopy 1.Brightfield Microscopy © The McGraw−Hill
Applications Lab Manual, Companies, 2001 Applications Lab Manual, Companies, 2001
Eighth Edition Eighth Edition
Brightfield Microscopy • Exercise 1
stage. Note, also, the location of the mechanical Most microscopes have some provision for reduc-
1 Brightfield Microscopy stage control in figure 1.2.
Light Source In the base of most microscopes is po-
sitioned some kind of light source. Ideally, the lamp
should have a voltage control to vary the intensity of
ing light intensity with a neutral density filter. Such a
filter is often needed to reduce the intensity of light be-
low the lower limit allowed by the voltage control. On
microscopes such as the Olympus CH-2, one can simply
place a neutral density filter over the light source in the
light. The microscope in figure 1.2 has a knurled wheel base. On some microscopes a filter is built into the base.
on the right side of its base to regulate the voltage sup-
plied to the light bulb. The microscope base in figure Lens Systems All microscopes have three lens sys-
A microscope that allows light rays to pass directly Lens Care At the beginning of each laboratory pe- 1.4 has a knob (the left one) that controls voltage. tems: the oculars, the objectives, and the condenser.
through to the eye without being deflected by an in- riod check the lenses to make sure they are clean. At
tervening opaque plate in the condenser is called a the end of each lab session be sure to wipe any im-
brightfield microscope. This is the conventional type mersion oil off the immersion lens if it has been used.
of instrument encountered by students in beginning More specifics about lens care are provided on page 5.
courses in biology; it is also the first type to be used
in this laboratory. Dust Protection In most laboratories dustcovers
All brightfield microscopes have certain things in are used to protect the instruments during storage. If
common, yet they differ somewhat in mechanical op- one is available, place it over the microscope at the
eration. An attempt will be made in this exercise to end of the period.
point out the similarities and differences of various
makes so that you will know how to use the instru-
ment that is available to you. Before attending the first
COMPONENTS
laboratory session in which the microscope will be Before we discuss the procedures for using a micro-
used, read over this exercise and answer all the ques- scope, let’s identify the principal parts of the instru-
tions on the Laboratory Report. Your instructor may ment as illustrated in figure 1.2.
require that the Laboratory Report be handed in prior
to doing any laboratory work. Framework All microscopes have a basic frame
structure, which includes the arm and base. To this
framework all other parts are attached. On many of
CARE OF THE INSTRUMENT the older microscopes the base is not rigidly attached
Microscopes represent considerable investment and to the arm as is the case in figure 1.2; instead, a pivot
can be damaged rather easily if certain precautions are point is present that enables one to tilt the arm back-
not observed. The following suggestions cover most ward to adjust the eyepoint height.
hazards.
Stage The horizontal platform that supports the mi-
Transport When carrying your microscope from croscope slide is called the stage. Note that it has a
one part of the room to another, use both hands when clamping device, the mechanical stage, which is
holding the instrument, as illustrated in figure 1.1. If used for holding and moving the slide around on the
it is carried with only one hand and allowed to dangle
at your side, there is always the danger of collision
with furniture or some other object. And, incidentally,
under no circumstances should one attempt to carry
two microscopes at one time.
Clutter Keep your workstation uncluttered while
doing microscopy. Keep unnecessary books, lunches,
and other unneeded objects away from your work
area. A clear work area promotes efficiency and re-
sults in fewer accidents.
Electric Cord Microscopes have been known to
tumble off of tabletops when students have entangled
a foot in a dangling electric cord. Don’t let the light
cord on your microscope dangle in such a way as to Figure 1.1 The microscope should be held firmly with
hazard foot entanglement. both hands while carrying it. Figure 1.2 The compound microscope Courtesy of the Olympus Corporation, Lake Success, N.Y.
2 3
Applications Lab Manual, Companies, 2001
Eighth Edition
Preface
This eighth edition of Microbiological Applications Although most experiments remain unchanged,
differs from the previous edition in that it has acquired there are a few that have been considerably altered.
four new exercises and dropped three experiments. It Exercise 27 (Isolation of Anaerobic Phototrophic
retains essentially the same format throughout, how- Bacteria), in particular, is completely new. By using
ever. In response to requests for more emphasis on lab- the Winogradsky column for isolating and identifying
oratory safety, three new features have been incorpo- the phototrophic sulfur bacteria, it has been possible
rated into the text. In addition, several experiments to greatly enrich the scope of this experiment. Another
have been altered to improve simplicity and reliability. exercise that has been altered somewhat is Exercise
The three exercises that were dropped pertain to fla- 48, which pertains to oxidation and fermentation tests
gellar staining, bacterial conjugation, and nitrification in that are used for identifying unknown bacteria.
soil. All of these exercises were either difficult to per- The section that has undergone the greatest reor-
form, unreliable, or of minimal pedagogical value. ganization is Part 10 (Microbiology of Soil). In the
To provide greater safety awareness in the labora- previous edition it consisted of five exercises. In this
tory, the following three features were added: (1) an edition it has been expanded to seven exercises. A
introductory laboratory protocol, (2) many cautionary more complete presentation of the nitrogen cycle is of-
boxes dispersed throughout the text, and (3) a new ex- fered in Exercise 58, and two new exercises (Exercises
ercise pertaining to aseptic technique. 61 and 62) are included that pertain to the isolation of
The three-page laboratory protocol, which fol- denitrifiers.
lows this preface, replaces the former introduction. It In addition to the above changes there has been
provides terminology, safety measures, an introduc- considerable upgrading of graphics throughout the
tion to aseptic technique, and other rules that apply to book. Approximately thirty-five illustrations have been
laboratory safety. replaced. Several critical color photographs pertaining
To alert students to potential hazards in performing to molds and physiological tests were also replaced to
certain experiments, caution boxes have been incorpo- bring about more faithful color representation.
rated wherever they are needed. Although most of these I am greatly indebted to my editors, Jean Fornango
cautionary statements existed in previous editions, they and Jim Smith, who made the necessary contacts for
were not emphasized as much as they are in this edition. critical reviews. As a result of their efforts the following
Exercise 8 (Aseptic Technique) has been struc- individuals have provided me with excellent sugges-
tured to provide further emphasis on culture tube han- tions for improvement of this manual: Barbara Collins
dling. In previous editions it was assumed that students at California Lutheran University, Thousand Oaks, CA;
would learn these important skills as experiments were Alfred Brown of Auburn University, Auburn, AL;
performed. With the risk of being redundant, six pages Lester A. Scharlin at El Camino College, Torrance, CA;
have been devoted to the proper handling of culture and Hershell Hanks at Collin County Community
tubes when making inoculation transfers. College, Plano, TX.
vii
,Benson: Microbiological Front Matter Laboratory Protocol © The McGraw−Hill Benson: Microbiological Front Matter Laboratory Protocol © The McGraw−Hill
Applications Lab Manual, Companies, 2001 Applications Lab Manual, Companies, 2001
Eighth Edition Eighth Edition
Laboratory Protocol
Hand Washing Before you start working in the lab, glass. Don’t try to pick up the glass fragments
Laboratory Protocol wash your hands with a liquid detergent and dry them
with paper toweling. At the end of the period, before
with your fingers.
5. Contaminated material must never be placed in a
leaving the laboratory, wash them again. wastebasket.
Tabletop Disinfection. The first chore of the day
Welcome to the exciting field of microbiology! The addition, long hair must be secured in a ponytail to
will be to sponge down your desktop with a disinfec- ACCIDENTAL SPILLS
tant. This process removes any dust that may be pre- All accidental spills, whether chemical or biological,
intent of this laboratory manual is to provide you with prevent injury from Bunsen burners and contamina- sent and minimizes the chances of bacterial contami-
basic skills and tools that will enable you to explore a tion of culture material. must be reported immediately to your instructor.
nation of cultures that you are about to handle. Although the majority of microorganisms used in
vast microbial world. Its scope is incredibly broad in Your instructor will indicate where the bottles of
that it includes a multitude of viruses, bacteria, proto- this laboratory are nonpathogens, some pathogens
disinfectant and sponges are located. At the end of the
zoans, yeasts, and molds. Both beneficial and harmful TERMINOLOGY period before leaving the laboratory, perform the same
will be encountered. It is for this reason that we must
ones will be studied. Although an in-depth study of treat all accidental biological spills as if pathogens
Various terms such as sterilization, disinfection, ger- procedure to protect students that may occupy your desk
any single one of these groups could constitute a full were involved.
micides, sepsis, and aseptic techniques will be used in the next class.
course by itself, we will be able to barely get ac- Chemical spills are just as important to report be-
here. To be sure that you understand exactly what they
quainted with them. cause some agents used in this laboratory may be car-
mean, the following definitions are provided.
To embark on this study it will be necessary for Bunsen Burner Usage When using a Bunsen burner cinogenic; others are poisonous; and some can cause
Sterilization is a process in which all living mi-
you to learn how to handle cultures in such a way that to flame loops, needles, and test tubes, follow the pro- dermal damage such as blistering and depigmentation.
croorganisms, including viruses, are destroyed. The
they are not contaminated or inadvertently dispersed cedures outlined in Exercise 8. Inoculating loops and
organisms may be killed with steam, dry heat, or in-
throughout the classroom. This involves learning needles should be heated until they are red-hot. Before Decontamination Procedure Once your instructor
cineration. If we say an article is sterile, we understand
aseptic techniques and practicing preventive safety they are introduced into cultures, they must be allowed is notified of an accidental spill, the following steps
that it is completely free of all living microorganisms.
measures. The procedures outlined here address these to cool down sufficiently to prevent killing organisms will take place:
Generally speaking, when we refer to sterilization as it
two aspects. It is of paramount importance that you that are to be transferred.
pertains here to laboratory safety, we think, primarily, 1. Any clothing that is contaminated should be
know all the regulations that are laid down here as If your burner has a pilot on it and you plan to use
in terms of steam sterilization with the autoclave. The placed in an autoclavable plastic bag and auto-
Laboratory Protocol. the burner only intermittently, use it. If your burner
ultimate method of sterilization is to burn up the in- claved.
lacks a pilot, turn off the burner when it is not being
fectious agents or incinerate them. All biological 2. Paper towels, soaked in a suitable germicide, such
Scheduling During the first week of this course used. Excessive unnecessary use of Bunsen burners in
wastes must ultimately be incinerated for disposal. as 5% bleach, are placed over the spill.
your instructor will provide you with a schedule of a small laboratory can actually raise the temperature
Disinfection is a process in which vegetative, 3. Additional germicide should be poured around
laboratory exercises arranged in the order of their per- of the room. More important is the fact that unat-
nonsporing microorganisms are destroyed. Agents the edges of the spill to prevent further
formance. Before attending laboratory each day, tended burner flames are a constant hazard to hair,
that cause disinfection are called disinfectants or aerosolization.
check the schedule to see what experiment or experi- clothing, and skin.
germicides. Such agents are used only on inanimate 4. After approximately 20 minutes, the paper tow-
ments will be performed and prepare yourself so that The proper handling of test tubes, while transfer-
objects because they are toxic to human and animal els should be scraped up off the floor with an
you understand what will be done. ring bacteria from one tube to another, requires a cer-
tissues. autoclavable squeegee into an autoclavable
Each laboratory session will begin with a short tain amount of skill. Test-tube caps must never be
Sepsis is defined as the growth (multiplication) of dust pan.
discussion to brief you on the availability of materials placed down on the desktop while you are making in-
microorganisms in tissues of the body. The term asep- 5. The contents of the dust pan are transferred to an
and procedures. Since the preliminary instructions oculations. Techniques that enable you to make trans-
sis refers to any procedure that prevents the entrance autoclavable plastic bag, which may itself be
start promptly at the beginning of the period, it is ex- fers properly must be mastered. Exercise 8 pertains to
of infectious agents into sterile tissues, thus prevent- placed in a stainless steel bucket or pan for trans-
tremely important that you are not late to class. these skills.
ing infection. Aseptic techniques refer to those prac- port to an autoclave.
tices that are used by microbiologists to exclude all 6. All materials, including the squeegee and dust-
Personal Items When you first enter the lab, place organisms from contaminating media or contacting Pipetting Transferring solutions or cultures by pan, are autoclaved.
all personal items such as jackets, bags, and books in living tissues. Antiseptics are chemical agents (often pipette must always be performed with a mechanical
some out of the way place for storage. Don’t stack dilute disinfectants) that can be safely applied exter- suction device. Under no circumstances is pipetting
them on your desktop. Desk space is minimal and nally to human tissues to destroy or inhibit vegetative by mouth allowed in this laboratory.
must be reserved for essential equipment and your bacteria. ADDITIONAL IMPORTANT
laboratory manual. The storage place may be a REGULATIONS
drawer, locker, coatrack, or perimeter counter. Your Disposal of Cultures and Broken Glass The fol-
instructor will indicate where they should be placed. lowing rules apply to culture and broken glass disposal: Here are a few additional laboratory rules:
ASEPTIC TECHNIQUES 1. Petri dishes must be placed in a plastic bag to be 1. Don’t remove cultures, reagents, or other materi-
Attire A lab coat or apron must be worn at all times When you start handling bacterial cultures as in autoclaved. als from the laboratory unless you have been
in the laboratory. It will protect your clothing from ac- Exercises 9 and 10, you will learn the specifics of 2. Unneeded test-tube cultures must be placed in a granted specific permission.
cidental contamination and stains in the lab. When aseptic techniques. Some of the basic things you will wire basket to be autoclaved. 2. Don’t smoke or eat food in the laboratory.
leaving the laboratory, remove the coat or apron. In do are as follows: 3. Used pipettes must be placed in a plastic bag for 3. Make it a habit to keep your hands away from your
autoclaving. mouth. Obviously, labels are never moistened
4. Broken glass should be swept up into a dustpan with the tongue; use tap water or self-adhesive la-
and placed in a container reserved for broken bels instead.
ix x
,Benson: Microbiological Front Matter Laboratory Protocol © The McGraw−Hill Benson: Microbiological I. Microscopy Introduction © The McGraw−Hill
Applications Lab Manual, Companies, 2001 Applications Lab Manual, Companies, 2001
Eighth Edition Eighth Edition
Laboratory Protocol
1
4. Always clean up after yourself. Gram-stained
slides that have no further use to you should be
7. If you have borrowed something from someone,
return it. PART Microscopy
washed and dried and returned to a slide box. 8. Do not leave any items on your desk at the end of
Coverslips should be cleaned, dried, and returned. the period.
Staining trays should be rinsed out and returned to 9. Do not disturb another class at any time. Wait un-
their storage place. til the class is dismissed.
5. Return all bulk reagent bottles to places of storage. 10. Treat all instruments, especially microscopes,
6. Return inoculating loops and needles to your stor- with extreme care. If you don’t understand how a
age container. Be sure that they are not upside piece of equipment functions, ask your instructor.
down. 11. Work cooperatively with other students in group- Although there are many kinds of microscopes available to the mi-
assigned experiments, but do your own analyses
crobiologist today, only four types will be described here for our
of experimental results.
use: the brightfield, darkfield, phase-contrast, and fluorescence
microscopes. If you have had extensive exposure to microscopy in
previous courses, this unit may not be of great value to you; how-
ever, if the study of microorganisms is a new field of study for you,
there is a great deal of information that you need to acquire about
the proper use of these instruments.
Microscopes in a college laboratory represent a considerable
investment and require special care to prevent damage to the
lenses and mechanicals. The fact that a laboratory microscope
may be used by several different individuals during the day and
moved around from one place to another results in a much greater
chance for damage and wear to occur than if the instrument were
used by only one individual.
The complexity of some of the more expensive microscopes
also requires that certain adjustments be made periodically.
Knowing how to make these adjustments to get the equipment to
perform properly is very important. An attempt is made in the five
exercises of this unit to provide the necessary assistance in getting
the most out of the equipment.
Microscopy should be as fascinating to the beginner as it is to
the professional of long standing; however, only with intelligent un-
derstanding can the beginner approach the achievement that oc-
curs with years of experience.
xi 1
, Benson: Microbiological I. Microscopy 1.Brightfield Microscopy © The McGraw−Hill Benson: Microbiological I. Microscopy 1.Brightfield Microscopy © The McGraw−Hill
Applications Lab Manual, Companies, 2001 Applications Lab Manual, Companies, 2001
Eighth Edition Eighth Edition
Brightfield Microscopy • Exercise 1
stage. Note, also, the location of the mechanical Most microscopes have some provision for reduc-
1 Brightfield Microscopy stage control in figure 1.2.
Light Source In the base of most microscopes is po-
sitioned some kind of light source. Ideally, the lamp
should have a voltage control to vary the intensity of
ing light intensity with a neutral density filter. Such a
filter is often needed to reduce the intensity of light be-
low the lower limit allowed by the voltage control. On
microscopes such as the Olympus CH-2, one can simply
place a neutral density filter over the light source in the
light. The microscope in figure 1.2 has a knurled wheel base. On some microscopes a filter is built into the base.
on the right side of its base to regulate the voltage sup-
plied to the light bulb. The microscope base in figure Lens Systems All microscopes have three lens sys-
A microscope that allows light rays to pass directly Lens Care At the beginning of each laboratory pe- 1.4 has a knob (the left one) that controls voltage. tems: the oculars, the objectives, and the condenser.
through to the eye without being deflected by an in- riod check the lenses to make sure they are clean. At
tervening opaque plate in the condenser is called a the end of each lab session be sure to wipe any im-
brightfield microscope. This is the conventional type mersion oil off the immersion lens if it has been used.
of instrument encountered by students in beginning More specifics about lens care are provided on page 5.
courses in biology; it is also the first type to be used
in this laboratory. Dust Protection In most laboratories dustcovers
All brightfield microscopes have certain things in are used to protect the instruments during storage. If
common, yet they differ somewhat in mechanical op- one is available, place it over the microscope at the
eration. An attempt will be made in this exercise to end of the period.
point out the similarities and differences of various
makes so that you will know how to use the instru-
ment that is available to you. Before attending the first
COMPONENTS
laboratory session in which the microscope will be Before we discuss the procedures for using a micro-
used, read over this exercise and answer all the ques- scope, let’s identify the principal parts of the instru-
tions on the Laboratory Report. Your instructor may ment as illustrated in figure 1.2.
require that the Laboratory Report be handed in prior
to doing any laboratory work. Framework All microscopes have a basic frame
structure, which includes the arm and base. To this
framework all other parts are attached. On many of
CARE OF THE INSTRUMENT the older microscopes the base is not rigidly attached
Microscopes represent considerable investment and to the arm as is the case in figure 1.2; instead, a pivot
can be damaged rather easily if certain precautions are point is present that enables one to tilt the arm back-
not observed. The following suggestions cover most ward to adjust the eyepoint height.
hazards.
Stage The horizontal platform that supports the mi-
Transport When carrying your microscope from croscope slide is called the stage. Note that it has a
one part of the room to another, use both hands when clamping device, the mechanical stage, which is
holding the instrument, as illustrated in figure 1.1. If used for holding and moving the slide around on the
it is carried with only one hand and allowed to dangle
at your side, there is always the danger of collision
with furniture or some other object. And, incidentally,
under no circumstances should one attempt to carry
two microscopes at one time.
Clutter Keep your workstation uncluttered while
doing microscopy. Keep unnecessary books, lunches,
and other unneeded objects away from your work
area. A clear work area promotes efficiency and re-
sults in fewer accidents.
Electric Cord Microscopes have been known to
tumble off of tabletops when students have entangled
a foot in a dangling electric cord. Don’t let the light
cord on your microscope dangle in such a way as to Figure 1.1 The microscope should be held firmly with
hazard foot entanglement. both hands while carrying it. Figure 1.2 The compound microscope Courtesy of the Olympus Corporation, Lake Success, N.Y.
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