Microbiological Laboratory Techniques

19/2/22, 10:25 Microbiological Laboratory Techniques | Clinical Gate
Microbiological Laboratory
Techniques
4
Microbiological Laboratory Techniques

OUTLINE
Aseptic Technique in Laboratory Preparation and Analysis
Sterilization
Disinfection
Sanitization
Culture Techniques
Types of Culture Media
Live Media
Incubation and Isolation
Fixation and Staining
Negative and Simple Stains
Differential Stains
Special Stains
Fixation and Staining for Electron Microscopy
Identification Techniques
Morphology
Cultural Characteristics
Physiological/Biochemical Characteristics
Molecular Analysis Microbiology for the Healthcare Professional
https://clinicalgate.com/microbiological-laboratory-techniques/#s0075 1/18
LEARNING OBJECTIVES
After reading this chapter, the student will be able to:
• Describe the general concept of aseptic techniques used in laboratory

preparation and analysis
• Explain and differentiate between sterilization, disinfection, and

sanitization
• Describe the different types of culture media and their possible physical
state
• Discuss inoculation, incubation, and isolation
• Describe the various fixation and staining techniques to identify microbes

with the light microscope
• Describe the fixation and staining methods used in electron microscopy
• Classify the bacteria according to their shape for morphological

identification
• Describe the different culture characteristics of microbes used for the

purpose of identification
• Discuss the physiological, biochemical, and genetic characteristics used

for microbial identification purposes
KEY TERMS
agar
antiseptics
aseptic technique
bactericidal
bacteriostatic
chemical defined media
complex media
contaminants
cultures Microbiology for the Healthcare Professional
culture media
degermation
differential media
disinfection
enriched media
filamentous
fomite
fusiform
germicide
hemolysins
incubated
inoculation
inoculum
isolation
liquid media
nonsynthetic media
pellicle
peptone
pleomorphic
pour plate
sanitization
sediment
selective media
semisolid media
solid media Microbiology for the Healthcare Professional
spread plate
sterile
sterilization
streak plate
synthetic media
the five “I’s”
turbid
WHY YOU NEED TO KNOW
HISTORY
To understand microbiology is to understand the laboratory; its basic

equipment, how its equipment is used, the procedures carried out, and—
importantly—the preparation of and meaning of test results.
In the 1600s, the newly created microscope yielded images of samples

previously too small to be seen with the naked eye. Reports of
interpretation of these early microbiological laboratory observations led to
the realization that life could exist in a single cell, followed in the 1800s by
the cell doctrine.
In addition, laboratory findings of Pasteur and Koch revealed pathogenic

microorganisms to be causes and carriers of disease, from which evolved
the germ theory of disease. In 1877, Pasteur and Joubert described
microbial antagonism among bacteria and in 1899 the term “antibiotic” was
coined. Present-day biotechnological methodologies have grown from
laboratory findings that prompted the development of new technologies to
drive further advances in microbiology as a scientific discipline.
IMPACT
Verification of the successes and failures of putative treatments for

pathogenic diseases is performed under laboratory conditions in the
search for new drug and antibiotic therapy. Moreover, results from the
laboratory give basic understanding of microorganisms, their mechanisms
of action, and their relationship with the environment.
The addition of clinical and laboratory experience permits a rational

approach to the question of “what happens if?” and suggests pertinent
experiments that can be carried out safely under carefully controlled
laboratory conditions. Microbiology for the Healthcare Professional
One of the beneficial outgrowths of microbiological investigations in the

laboratory has been the need to use aseptic techniques for the growth and
identification of specific microorganisms. Culture techniques were derived
from the necessity to rapidly grow and accurately identify potential
pathogens in order to treat individuals or take appropriate steps to prevent
outbreaks of disease, epidemics, or pandemics.
FUTURE
The acknowledgment of threats to outbreaks of pathogenic disease has

brought a public awareness to the importance of compliance with proven
preventative procedures and therapies.
Aside from these ever-present battles against commonly confronted

pathogens, another deadly issue has arisen—bacteriological warfare.
Again, our most effective weapon against this potential disaster comes
from the laboratory. Recently, the use of anthrax as a bioterrorism weapon
was attempted in Washington, D.C. Through the prompt use of laboratory
procedures, it was identified and safely disposed of, although
weaponization of pathogens is and continues to be a bioterrorism threat.
Threats to human health via pathogenic organisms or via bioterrorism

agents will be met by work done in laboratories. Laboratory work has been
and continues to be the cornerstone of protection against disease, of
microbiology, and of world health.
Aseptic Technique in Laboratory Preparation

and Analysis
Medical and clinical laboratories test biological specimens to determine the
health status of a patient and to identify the disease-causing pathogen for
the purpose of appropriate treatment. Research laboratories include basic,
clinical, and pharmaceutical laboratories, each dealing with a specific
aspect of microbiological research.
Microorganisms are everywhere in the environment. In order to selectively

identify specific microbes they must be grown in controlled laboratory
environments. Beginning with pure sterile cultures, the key is to control the
factors to which the cultures are subjected. In other words, when working
with microbial cultures, it is necessary to ensure that organisms are
selectively introduced into the culture and that other environmental
organisms do not contaminate it. Aseptic technique is a procedure that is
performed under sterile conditions, a method that prevents the introduction
of unwanted organisms or contaminants into an environment. This
process is characterized Microbiology
by strict adherence to details. The
for the Healthcare use of aseptic
Professional
technique controls, limits, or prevents contamination by fomites. A fomite
is any inanimate object or substance capable of transporting pathogens

from one medium or individual to another. Aseptic technique is essential in
the microbiology laboratory to prevent any contamination of laboratory
personnel (see Chapter 5, Safety Issues), cultures, supplies, and
equipment.
Air currents must be controlled by closing laboratory doors and windows to

prevent microbes on surfaces from becoming airborne and entering the
cultures. When handling cultures to prepare slides or to transfer organisms
to another medium, the transfer loops and needles need to be sterilized by
flame or incinerator before and after use (Figure 4.1). Furthermore, culture
plates are held in a position that minimizes exposure of the medium to the
environment. When removing lids/stoppers from test tubes, the lids should
remain held in the hand and not placed on other surfaces such as
countertops during the transfer of materials from one tube to another.
Flaming is one of the physical methods of sterilization and must always be
applied to the lips of test tubes and also of flasks whenever culture liquid is
poured from one container to another, as in the case of pouring culture
plates (Figure 4.2).
Microbiology for the Healthcare Professional
FIGURE 4.1 Sterilization of a loop.One of the primary

inoculation tools in the microbiology laboratory is the loop.
Metal loops must be sterilized in the flame of a Bunsen burner
by heating the wire until it glows. Presterilized, disposable
plastic loops are also available for inoculation.
FIGURE 4.2 Aseptic technique and media containers.Bacteria

and fungi are everywhere in the environment, including the air.
The mouth of a tube or bottle opening is a potential point of
contamination and flaming of the opening in direct flame
immediately after opening and before closing is an effective
technique to prevent potential contamination on this exposed
surface.
Sterilization and disinfection procedures are daily routines in

microbiological laboratories. They are essential to ensure that cultures,
containers, media, and equipment are handled in such a way that only the
desired organism will grow and others will be eliminated or excluded.
Sterilization
Sterilization is the destruction or removal of all microorganisms, including
bacteria and their endospores, viruses, fungi, and prions. This can be
accomplished by physical methods such as heat, radiation, and filtration, or
by chemical methods. The wide application of sterilization processes
makes it necessary to impose strict control measures to validate the
results. When using dry heat or moist heat sterilization, physical, chemical,
or biological indicators can be used to validate the desired results (Table
4.1). The general resistance of microbes to methods of sterilization ranges
from bacterial endospores, with the highest resistance to sterilization, to
vegetative cells, with moderate to least resistance.
TABLE 4.1 Microbiology for the Healthcare Professional
Methods for Validating Dry or Moist Heat Sterilization
Chemical Biological Test

Physical Methods
Methods Organism
Dry Temperature-recording Color change Bacillus subtilis var.
heat charts indicator niger
Moist Temperature-recording Color change Bacillus
heat charts indicator stearothermophilus
Physical and chemical methods of sterilization are applied in the

microbiological laboratory to ensure that equipment and materials are free
of microorganisms. Aseptic technique is the first and most important step in
ensuring that manipulation of specimens during investigative procedures
does not infect laboratory personnel or contaminate cultures or the
laboratory environment (see Chapter 5, Safety Issues). Bacteria are found
practically everywhere including fingertips and bench tops and therefore it
is essential to minimize contact with these surfaces. Only sterile items are
free of potentially contaminating microorganisms and once a sterile object
comes in contact with a nonsterile surface, the object can no longer be
considered sterile. The most commonly used instrument in the
microbiology laboratory for the sterilization of media and glassware is the
autoclave (Figure 4.3). A detailed discussion of the different types of
physical and chemical methods of sterilization is provided in Chapter 19
(Physical and Chemical Methods of Control; for overviews see Tables 19.3
and 19.5).
FIGURE 4.3 Autoclave.The autoclave is one of the primary

tools for sterilization of equipment, containers, media, and
biohazardous wastes. It is essentially a large pressure cooker
that raises the temperature of steam to about 121° C under 15–
20 psi pressure. The size and configuration of autoclaves vary
but the basic operation is same.
Disinfection
Disinfectants are applied to inanimate surfaces, medical equipment, and
other man-made objectsMicrobiology
whereas antiseptics are usedProfessional
for the Healthcare to disinfect skin.
The term disinfection refers to the use of a physical process or the use of
a chemical agent to destroy vegetative microbes and viruses. This does

not include bacterial endospores. The ideal disinfectant would result in
complete sterilization without harming other forms of life. Unfortunately,
ideal disinfectants as such do not exist and most of them only partially
sterilize. In addition to the most resistant pathogens, endospores, other
bacteria, and viruses are also highly resistant to many disinfectants.
Substances that kill bacteria are bactericidal and those that interfere with
cell growth and reproduction are bacteriostatic. Disinfectants and
antiseptics are bactericidal and bacteriostatic depending on the
concentration applied. All disinfectants are by their nature potentially
harmful, even toxic, to humans and animals. They should be handled with
appropriate care to avoid harm to the handler or recipient. The type of
disinfectant to be used depends on the surface or material to be
disinfected. Specifics on the different types of disinfectants and their
particular effectiveness are described in Chapter 19, with an overview in
Table 19.5 (Chemicals Used in the Control of Microbes).
Sanitization
Several applications in everyday life and medicine do not require
sterilization, disinfection, or antisepsis but need to reduce microorganisms
in order to control possible infections or spoilage of substances.
Sanitization achieves this by using any cleansing technique that
mechanically removes microorganisms and other debris to reduce
contamination to safe levels. Often the sanitizer used is a compound such
as soap or detergent. Restaurants, dairies, breweries, and other food
industries handle soiled utensils on a daily basis and must take appropriate
measures to sanitize them for prevention of infection, spoilage, and
contamination. This includes controlling microbes to a minimal level during
preparation and processing.
Degermation is the process by which the numbers of microbes on the

human skin are reduced by scrubbing, immersion in chemicals, or both.
Some examples of degermation include the process of presurgical
scrubbing of the hands with sterile brushes and germicidal soap before
putting on sterile surgical gloves, the application of alcohol wipes to the
skin, and the cleansing of a wound with germicidal soap and water.
Culture Techniques
Microbiologists use five basic procedures to examine and characterize
microbes: Inoculation, Incubation, Isolation, Inspection (observation), and
Identification—the five “I’s.” To culture a microorganism a small sample,
the inoculum, is introduced into a culture
Microbiology medium
for the usually
Healthcare with a platinum
Professional
wire probe streaked across its surface. This process is called inoculation
and the growth that appears on or in the medium is the culture. A culture
can be pure—containing one type of organism, or mixed—containing two
or more species.
Types of Culture Media

Nutritional requirements of particular microorganisms range from a few
simple inorganic compounds to a complex list of specific inorganic and
organic chemicals (Table 4.2). Access to carbon, the essential component
required for molecular life, is obtained in different ways by microorganisms.
Autotrophs acquire carbon from carbon dioxide in the atmosphere and
heterotrophs obtain their carbon from organic compounds. This diversity is
seen in the different types of media needed to ensure the growth of the
organism for investigation. Media vary in nutrient content and consistency
and can be classified according to their physical state, chemical
composition, and functional type.
TABLE 4.2
Nutritional Requirements for Microorganisms*
Growth Factors Micronutrients (Trace

Macronutrients
(Vitamins) Elements)
Carbon p-Aminobenzoic acid Boron
Hydrogen Folic acid Chromium
Oxygen Biotin Cobalt
Nitrogen Cobalamin (B12) Copper
Phosphorus Lipoic acid Iron
Sulfur Nicotinic acid (niacin) Manganese
Potassium Pantothenic acid Molybdenum
Magnesium Riboflavin Nickel
Sodium Thiamine Selenium
Calcium Vitamin B6 Tungsten
Iron Vitamin K group Vanadium
Hydroxamates Zinc
*Not all microbes need all of the nutrients listed; therefore, for optimal
growth environments media with specific nutrients are necessary for
specific microorganisms.
Physical State of MediaMicrobiology for the Healthcare Professional
Liquid media are water-based solutions that do not solidify at

temperatures above freezing and flow freely in the containers when tilted.
Most commonly, liquid media are supplied in tubes or bottles and are
called broths, milks, or infusions. A common laboratory medium is nutrient
broth, which contains beef extract and peptone (partially digested protein)
dissolved in water. Methylene blue milk and litmus milk are opaque liquids
prepared from skim milk powder and dyes. After inoculation, growth occurs
throughout the container. Enriched broths are used to grow bacteria that
are present in few numbers such as in small specimen samples obtained
from patients.
Semisolid media contain a limited amount of a solidifying agent such as

agar or gelatin, giving the medium a clotlike consistency. Semisolid media
are often used to determine motility and growth patterns of bacteria.
Solid media are dispensed in Petri plates or slanted in tubes or bottles to

provide firm and maximal surfaces for growing bacteria or fungi. By far the
most widely used and effective of these media is agar, composed of a
complex polysaccharide from the red alga Gelidium. Agar is solid at room
temperature and liquefies at the boiling temperature of water. Once in
liquid form it does not solidify until it cools to 42° C. It then can be
inoculated and poured in liquid form at temperatures that will not harm the
microbes or the handlers. Agar added to media simply gels them into a
solid form. Any medium containing 1% to 5% agar usually has the agar in
the name of the specific medium as, for example, nutrient agar, phenylethyl
alcohol agar, blood agar, and others.
LIFE APPLICATION
Raising Bacteria: A Labor of Love
Most of the bacteria raised in laboratories for testing, identification, or

experimentation grow on relatively simple media. Complex media such as
tryptic soy broth/agar, nutrient broth/agar, brain heart infusion, and blood
agar are useful for growing a wide range of bacteria. The conditions that
most organisms are grown under vary from anaerobic to microaerophilic
(5% oxygen) to capnophilic (“carbon dioxide loving”) to simply aerobic and
in temperatures ranging from 4° C to about 60° C. These medium types
and incubation conditions can be found and maintained in most
microbiology/clinical laboratories. There are, however, some bacteria that
are very picky eaters and require some extraordinary medium concoctions
to raise them in the laboratory. The normal growth conditions of other
bacteria, such as extremophiles, may be challenging to reproduce in the
average laboratory. Some pathogenic bacteria such as those in the genera
Spiroplasma and Mycoplasma need specialized media for culturing.
Spiroplasma medium is a broth that contains more than 80 ingredients
including various types of nutrients as well as antibiotics to suppress other
possible competitors. Extremophiles, whose natural growth environment is

at the vent holes at the bottom of the ocean, present some significant
challenges for culturing. The extreme pressures and temperatures required
as well as the unique nutritional requirements of these bacteria make their
cultivation possible only in laboratories with sophisticated equipment to
meet the organisms’ needs.
Chemical Classification of Media
Depending on their chemical content media can be classified as complex

or nonsynthetic media and as chemically defined or synthetic media.
• Chemically defined media or synthetic media are media with a

defined, exact chemical composition. They are prepared by means of an
exact formula, adding precise amounts of inorganic and/or organic
chemicals to distilled water. Some of these media contain minimal amounts
of chemicals such as some salts and a source of carbon; others are
special media containing a variety of precisely measured substances.
• Complex media or nonsynthetic media contain at least one component

that cannot be chemically defined and thus the medium cannot be
represented by an exact chemical formula. Complex media contain
extracts from animals, plants, or yeast. They may include blood, serum,
meat extracts, milk, yeast extracts, soybean digests, and peptone.
Functional Types of Media
General-purpose media are designed to grow a broad spectrum of

microbes that do not have any special growth requirements. Other media
are available for special growth conditions of selected organisms. These
include enriched, selective, and differential media.
• Enriched media contain complex organic substances such as blood,

serum, hemoglobin, or growth factors for the growth needs of specific
species. An example is blood agar, made by adding sterile sheep, horse,
or rabbit blood to a sterile agar base. It is widely used to grow certain
streptococci and other pathogens. Another enriched medium is chocolate
agar. Chocolate agar is enriched with heat-treated blood, which turns
brown and gives the medium the color and thus its name.
• Selective media inhibit the growth of selected organisms while allowing

the growth of others. These media are useful in isolating bacteria or fungi
from specimens that contain several different organisms. For example,
mannitol salt agar contains 7.5% NaCl, inhibitory to most human
pathogens with the exception of the genus Staphylococcus, which thrives
in mannitol salt agar and consequently its growth can be amplified in mixed
samples.
• Differential media can grow several different organisms that show visible
differences. These differences can be variations in colony size or color, a
change in medium color, or the formation of gas bubbles and precipitates.
Dyes can be used as differential agents because many of them are pH
indicators that change color in response to acid or base production by a
specific microbe. For example, MacConkey agar contains neutral red,
which is a dye that is yellow when neutral and pink or red when acidic.
Escherichia coli, a bacterium common to the intestinal tract, produces acid
when it metabolizes the lactose in the medium and develops red or pink
colonies. In contrast, Salmonella does not give off acid and therefore
remains in a natural off-white color. A comparison of general, selective,
and differential media is shown in Figure 4.4.
FIGURE 4.4 Types of media.Pictured are (A) tryptic soy agar—

a complex medium used as an all-purpose growth medium, and
(B) xylose lysine deoxycholate agar—a chemically defined agar
that is both selective and differential and used primarily in
selecting for and differentiating gram-negative enteric bacilli,
especially Shigella, Salmonella, and Providencia. Yellow
indicates the organism is utilizing the carbohydrate xylose and
the black colonies indicate the production of hydrogen sulfide.
HEALTHCARE APPLICATIONGrowth Requirements of Selected Bacteria
Bacterium Medium Atmospheric Conditions

Pseudomonas aeruginosa
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19/2/22, 10:25 Microbiological Laboratory Techniques | Clinical Gate

Aseptic Technique in Laboratory Preparation and Analysis

Types of Culture Media

Incubation and Isolation

Fixation and Staining

Negative and Simple Stains

Fixation and Staining for Electron Microscopy

Molecular Analysis Microbiology for the Healthcare Professional

After reading this chapter, the student will be able to:

• Describe the general concept of aseptic techniques used in laboratory

• Explain and differentiate between sterilization, disinfection, and

• Discuss inoculation, incubation, and isolation

• Describe the various fixation and staining techniques to identify microbes

• Describe the fixation and staining methods used in electron microscopy

• Classify the bacteria according to their shape for morphological

• Describe the different culture characteristics of microbes used for the

• Discuss the physiological, biochemical, and genetic characteristics used

chemical defined media

cultures Microbiology for the Healthcare Professional

solid media Microbiology for the Healthcare Professional

the five “I’s”

WHY YOU NEED TO KNOW

To understand microbiology is to understand the laboratory; its basic

In the 1600s, the newly created microscope yielded images of samples

In addition, laboratory findings of Pasteur and Koch revealed pathogenic

Verification of the successes and failures of putative treatments for

The addition of clinical and laboratory experience permits a rational

One of the beneficial outgrowths of microbiological investigations in the

The acknowledgment of threats to outbreaks of pathogenic disease has

Aside from these ever-present battles against commonly confronted

Threats to human health via pathogenic organisms or via bioterrorism

Aseptic Technique in Laboratory Preparation

Microorganisms are everywhere in the environment. In order to selectively

is any inanimate object or substance capable of transporting pathogens

Air currents must be controlled by closing laboratory doors and windows to

Microbiology for the Healthcare Professional

FIGURE 4.1 Sterilization of a loop.One of the primary

Microbiology for the Healthcare Professional

FIGURE 4.2 Aseptic technique and media containers.Bacteria

Sterilization and disinfection procedures are daily routines in

TABLE 4.1 Microbiology for the Healthcare Professional

Methods for Validating Dry or Moist Heat Sterilization

Chemical Biological Test

Physical and chemical methods of sterilization are applied in the

Microbiology for the Healthcare Professional

FIGURE 4.3 Autoclave.The autoclave is one of the primary

a chemical agent to destroy vegetative microbes and viruses. This does

Degermation is the process by which the numbers of microbes on the

Types of Culture Media

Nutritional Requirements for Microorganisms*

Growth Factors Micronutrients (Trace

Physical State of MediaMicrobiology for the Healthcare Professional

Liquid media are water-based solutions that do not solidify at

Semisolid media contain a limited amount of a solidifying agent such as

Solid media are dispensed in Petri plates or slanted in tubes or bottles to

Raising Bacteria: A Labor of Love

Most of the bacteria raised in laboratories for testing, identification, or

possible competitors. Extremophiles, whose natural growth environment is

Chemical Classification of Media

Depending on their chemical content media can be classified as complex

• Chemically defined media or synthetic media are media with a