Lab 2 Microbiological culture

1. Aseptic technique
When working in a microbiology laboratory, aseptic technique is fundamental to the success and safety of an
experiment. Generally, aseptic techniques are procedures or processes performed by scientists under sterile
conditions to ensure that microbial contaminants do not harm colleagues and are not introduced into sterile
solutions, supplies, or experimental cultures. To minimize the possibility of microbial contamination, the below
rules and recommendations are encouraged. Additional information on risk assessment and precautions can be
found in the Center for Disease Control (CDC) publication “Biosafety in Microbiological and Biomedical
Laboratories, (BMBL)” at http://www.cdc.gov1.
1.1 Personal Protection and Cleanliness
In a Microbiology laboratory, it is important to maintain the health and safety of all personnel. To avoid health
risks, laboratory scientists should wear protective gear and be aware of any potential hazards nearby. Generally,
protective gear serves two purposes; to protect the scientist from laboratory hazards and to shield the experiment
from unintentional contamination. Below, we describe several mechanisms to maintain laboratory safety.
 Upon entering and exiting a lab, be sure to thoroughly wash your hands with antimicrobial soap.
 While in the lab, avoid touching your face or eyes. Further, do not handle make-up or contacts as they may
become contaminated.
 Do not store or handle food or beverages in the laboratory.
 Only enter the lab wearing closed-foot shoes. Open-toed shoes should never be worn in a laboratory as it leaves
you susceptible to potential cuts or infection from broken glassware or sharp equipment.
 While performing an experiment, wear a clean, fitted laboratory coat. For additional protection, use a closed-
front laboratory coat. Laboratory coats should never leave the lab, and should be cleaned frequently.
Additionally, ensure that the sleeves of your coat are fitted so that they will not catch fire near an open flame,
nor become entangled.
 When working with microorganisms, wear fitted, disposable gloves. Flakes of dry skin harbor bacteria, which
may provide a source of contamination; wearing gloves will mitigate the risk. Additionally, disposable gloves
decrease the risk of infecting any hand wounds.
 Pull back long hair or use a hair cover. Long hair is notorious for attracting dust, a potential source of
contamination. Additionally, long hair could be a health hazard when working near an open flame.
 Use protective eye-wear or a face shield when working with hazardous materials or cultures. If your eyes come
in contact with microorganisms or harmful chemicals, wash your eyes for 15 minutes in an eye-wash and seek
medical attention immediately.
1.2 Handling Microbial Cultures and Media
When working with any microbial strain, propagation success depends heavily on the prevention of cross-
contamination by other microorganisms. Sources of contamination can include non-sterile supplies, media,
reagents, unclean work surfaces and incubators, airborne particles, and unclean gloves. Below, we describe some
tips for handling microbial cultures and media.
Maintain a Sterile Work Area
 Before and after use, disinfect all work surfaces with 70% ethanol or an appropriate disinfectant. This is
especially important after any spills.
 Maintain an uncluttered work space; all work surfaces should only contain equipment that is required for your
experiment.
 Ensure that you have all necessary supplies before beginning an experiment. Being prepared will reduce the
likelihood of careless contamination.
 Work may be performed in a thoroughly sterilized biosafety cabinet. Biosafety cabinets can be sterilized via
ultraviolet light in conjunction with 70% ethanol or an appropriate disinfectant.
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 Do not open windows or use fans that circulate outside air. If possible, work in laboratory settings that have
air vents covered with filters. This will prevent the contamination of cultures by airborne particles.
 Frequently clean water baths used for thawing or warming media or solutions.
 Routinely sterilize incubators used for microbial propagation.
Handling Media
 Before and after use, sterilize the outside container of all media and reagents with 70% ethanol. Also, do not
leave containers of media open longer than necessary.
 Aliquot sterile solutions into smaller volumes whenever possible. If you are unsure of the sterility of your
media, it is best to discard it immediately.
 Avoid pouring sterile liquids from one container to another, this increases the likelihood of media
contamination. Rather, use sterile pipettes for the aseptic transfer of media.
 Never mouth pipette. This poses a health risk for personnel as well as increases the risk of contamination.
 Always use sterile glass or disposable plastic pipettes to work with liquid media. Use each pipette only once
to avoid cross contamination.
 Do not open sterile media, petri dishes, or pipette containers until you are ready to use them.
Handling Microbial Cultures
 Before working with media and microbial cultures, wipe your work area with 70% ethanol or an appropriate
disinfectant.
 Ensure you are wearing appropriate protective clothing. This will protect you from the culture as well as reduce
accidental culture contamination.
 Only use sterile glassware, equipment, media, and reagents. Check media for contamination by observing for
turbidity.
 Handle only one microbial culture at a time. The risk of cross contamination or misidentification increases
when more than one strain is handled at a time.
 When working with test tube cultures, hold cultures at an angle after you remove the lid to avoid airborne
particles from falling into the culture. Sterilize the outside of the culture tube using a Bunsen burner flame.
 When working with plated cultures, hold the petri dish lid at an angle after you remove the lid to avoid airborne
particles falling into the culture dish.
 When handling a microbial culture, work quickly and carefully in an environment that has minimal
distractions. Do not leave the lid off your culture for extended periods of time.
 Never take cultures outside of the laboratory.
 Notify the laboratory supervisor immediately of any spills.
1.3 Biological Safety Cabinets
When working with hazardous or sterile materials, it is recommended that all associated procedures are
performed in a biosafety cabinet. These apparatuses are enclosed, ventilated workspaces designed to protect
laboratory personnel and materials from cross contamination during routine procedures. Generally, work spaces
within the biosafety cabinet are protected through the use of a high-efficiency particulate air (HEPA) filtration
method, which removes harmful microbes from the air.
Biological Safety Cabinet Classes
Class I: These biological safety cabinets are open-front negative pressure systems with HEPA filtration systems.
Class I biosafety cabinets provide protection for laboratory personnel and the environment, but will not provide
product protection. These cabinets are often used to enclose specific equipment or procedures that may generate
potentially hazardous aerosols.
Class II: These biological safety cabinets are open-front, ventilated, laminar-flow cabinets. These provide HEPA-
filtered, recirculated airflow within the work space. Class II cabinets provide protection to laboratory personnel,
the environment, and to products by drawing a curtain of sterile air over the products that are being handled.
These cabinets are commonly used in microbiology laboratories working with potentially infectious agents
(Biosafety Level 1 or 2) as they protect the contained materials from extraneous airborne contaminants.

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Class III: These biological safety cabinets are totally enclosed, ventilated systems with gas-tight construction.
These cabinets are used through attached rubber gloves. Generally, the air supply is drawn into the cabinet through
HEPA filters, and the exhaust air is filtered by two HEPA filters installed in a series. This biosafety cabinet system
is commonly used with high-risk infectious agents (Biosafety Level 3 or 4) to prevent the escape of aerosols.
Below, we describe some tips on how to properly use a Class I or Class II biosafety cabinet when working with
non-infectious materials.
Keeping a Biosafety Cabinet Safe
 Certify all biosafety cabinets upon installation.
 Routinely test the quality of airflow.
 Ensure the integrity of filters.
 Biosafety cabinets should be approved by the resident Biosafety officer.
 Ensure that all biosafety cabinets are routinely recertified
Biosafety Cabinet Sanitation
 Clean work surfaces with an appropriate disinfectant before and after use.
 If the biosafety cabinet is equipped with germicidal UV lights, decontaminate work surfaces before and after
use by turning on the UV light for at least 15 minutes. Never use the UV light while the biosafety cabinet is in
use.
 Routinely remove any biohazard waste from the biosafety cabinet.
 Using an appropriate disinfectant, wipe down the outer surface of all pipettes, pipette tip boxes, media,
materials, etc. prior to placing them in the biosafety cabinet.
 Always wear a clean lab coat and sterile gloves when working in a biosafety cabinet.
Proper use of a Biosafety Cabinet
 Turn on the biosafety cabinet 15 minutes prior to use.
 Only raise the biosafety cabinet sash to the recommended level, this will reduce disruption to the air flow as
well as assist in the prevention of airborne contaminant entry.
 When using a biosafety cabinet, limit the amount of movement in the cabinet and do not remove your arms.
Additionally, limit the access to the area around the biosafety cabinet. This will reduce disruption to the airflow.
 Do not use open flames within a biosafety cabinet. The resulting heat from the flame can disrupt the air flow
provided by the biosafety cabinet, increasing the risk for contamination. Additionally, gas leaks associated
with Bunsen burners or the use of an alcohol-based disinfectant near an open flame can result in fire or injury.

Fig. 1 Biological Safety Cabinet Classes

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2. Culturing Techniques
A culture medium is a solution of nutrients that is required for microbial growth. Depending on their
composition or use, culture media can be categorized into several groups; these include defined, complex,
selective, and enrichment medium. In a defined medium, the exact chemical composition is known. These types
of media are usually composed of pure biochemicals, and are often used to study the minimal nutrient requirement
of a microorganism. In contrast, the exact chemical composition of a complex medium is not known. This latter
medium type often contains reagents of a biological origin, such as yeast extract and peptone, where the exact
chemical composition is unknown. Complex media usually provide a large range of growth factors that assist in
the cultivation of unknown and fastidious bacterial species.
Medium may also be formulated as selective or to enrich. A selective medium is formulated to inhibit the
growth of certain bacterial species and/or promote the growth of a specific species. These media can consist of
additional selective reagents, such as high salt concentration to select for halophiles, or can be used under selective
growth conditions. An enrichment medium also allows for the growth of specific bacterial species; however,
enrichment media are supplemented with a reagent that permits, rather than inhibits, the growth of a particular
species.
Generally, bacterial culture media are mixtures of proteins, salts, trace elements, amino acids, and
carbohydrates. The presence and volume of these components can vary significantly among bacterial species
depending on the macro- and micro-nutrient requirements of each strain. The manner of which bacterial strains are
cultured also varies widely. Liquid media are often used for the growth and propagation of pure batch cultures,
while solid agar-based media are used for the isolation of pure cultures. All culture media must be sterilized prior
to its use. This is often accomplished by heating the media at high temperatures within an autoclave. This
instrument provides 15 pounds per square inch of steam pressure, allowing for temperatures to reach and be
maintained at 121°C. All media must be loosely capped prior to sterilization to equalize the pressure of the
container and to prevent contamination upon removal from the autoclave. Below, we will describe how to inoculate
various broth and agar cultures using aseptic technique.
2.1 Broth Culture
A broth culture is a liquid nutritional medium used to culture live microorganisms in either a test tube or an
Erlenmeyer flask. This form of culture allows for the rapid growth of many microorganisms, and is often used to
prepare specimens for cryopreservation or to propagate large volumes of microbial cultures. Below we describe
an aseptic protocol for the inoculation of a broth culture.
Materials
Alcohol burner Sterile broth media
Slant or broth culture Wire inoculation loop
NOTE 1: Sterile, disposable loops can be used to inoculate cultures. These loops are ready-to-use and do not
require flame sterilization.
Procedure
1. Flame a wire inoculating loop to sterilize it. The inoculating loop should be heated to a red glow using a alcohol
burner. Ensure that both the loop and the lower portion of the handle have been sterilized. Allow the loop to
cool before obtaining a culture. If the loop is too hot, it will cause the cells to burst.
2. With your other hand, pick up a culture tube and remove the cap with the fourth and fifth fingers of the hand
that is holding the loop. Never lay a cap down on the lab bench, this will result in contamination. Always hold
the open tube at an angle so that dust does not fall into the tube and contaminate the culture.
3. Pass the opening of the culture tube through the alcohol burner flame. This will kill any microorganisms on
the lip of the tube.
4. Insert the sterile wire loop into the culture tube. If you have a culture growing on an agar slant, touch the
inoculation loop onto the culture surface and gently gather a small amount of the microorganism. If you have
a broth culture, the inoculating loop can be submerged within the culture.
5. Flame the mouth of the culture tube again, replace the cap, and set it aside.
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6. With your free hand, pick up a tube of sterile media and remove the cap with the fourth and fifth fingers of the
hand that is holding the loop.
7. Pass the opening of the media tube through the alcohol burner flame. This will kill any microorganisms on the
lip of the tube.
8. Gently insert the inoculation loop into the media and move the loop back and forth several times to inoculate
the media.
9. Remove the inoculation loop and flame the mouth of the test tube again, replacing the cap. Set the tube aside.
10. Sterilize the inoculating loop as described in step 1.
11. Incubate the broth culture under the appropriate growth conditions.

Fig. 2 procedure for inoculating a nutrient broth

2.2 Agar Slant

Agar slants are a form of solid media generated from the addition of a gelling agent, such as agar, to a broth
culture. To prepare an agar slant, agar is added to the broth culture then heated within an autoclave to dissolve the
agar and sterilize the media. This medium will then solidify at temperature which is lower than 40°C. Agar slants
provide a large surface area on which to grow microorganisms and are frequently used as a method to temporarily
store actively growing cultures.
Materials
Slant or broth culture Wire inoculation loop
Alcohol burner Sterile agar slant

Procedure
1. Flame a wire inoculating loop to sterilize it. The inoculating loop should be heated to a red glow using an
alcohol burner. Ensure that both the loop and the lower portion of the handle have been sterilized. Allow the
loop to cool before obtaining a culture. If the loop is too hot, it will cause the cells to burst (NOTE 1).

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2. With your other hand, pick up a culture tube and remove the cap with the fourth and fifth fingers of the hand
that is holding the loop. Never lay a cap down on the lab bench, this will result in contamination. Always hold
the open tube at an angle so that dust does not fall into the tube and contaminate the culture.
3. Pass the opening of the culture tube through the alcohol burner flame. This will kill any microorganisms on
the lip of the tube.
4. Insert the sterile wire loop into the culture tube. If you have a culture growing on an agar slant, touch the
inoculation loop onto the culture surface and gently gather a small amount of the microorganism. If you have
a broth culture, the inoculating loop can be submerged within the culture.
5. Flame the mouth of the culture tube again, replace the cap, and set it aside.
6. With your free hand, pick up the sterile agar slant and remove the cap with the fourth and fifth fingers of the
hand that is holding the loop (See step 2).
7. Pass the opening of the media tube through the alcohol burner flame. This will kill any microorganisms on the
lip of the tube.
8. Gently insert the inoculation loop into the test tube and gently streak the microbial culture onto the surface of
the agar slant. Do not puncture the agar.
9. Remove the inoculation loop and flame the mouth of the test tube again, replacing the cap. Set the tube aside.
10. Sterilize the inoculating loop as described in step 1.
11. Incubate the agar slant under the appropriate growth conditions.

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Fig. 3 Procedure for inoculating a nutrient agar slant from a slant culture

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 Fig. 4 Procedure for inoculating a nutrient agar slant from an agar plate
2.3 Streak Plate
Similar to the agar slant, streak plates are a form of solid media generated from the addition of a gelling agent,
such as agar, to a broth culture. This medium is then added to a petri dish, rather than a test tube, and allowed to
solidify. The term “streak plate” refers to how the culture is inoculated on the agar surface. This particular
inoculation technique is used to isolate a pure colony from a single microbial species. Samples obtained from the
resulting pure colony can then be grown on a new plate so that the organism can be identified, studied, or the
colony morphology can be analyzed. For more information on the analysis of colony morphology, see the section
entitled “Colony Morpholgy.” Below we describe an aseptic protocol for the inoculation of a streak plate.
Materials
Slant or broth culture Wire inoculation loop
Alcohol burner Sterile petri dish with agar
Procedure
1. Place a petri dish agar-side-up onto the bench top. Using a permanent marker, draw a “T” on your plate to
separate your plate into 3 sections.
2. Flame a wire inoculating loop to sterilize it. The inoculating loop should be heated to a red glow using a Bunsen
burner. Ensure that both the loop and the lower portion of the handle have been sterilized. Allow the loop to
cool before obtaining a culture. If the loop is too hot, it will cause the cells to burst.
3. With your other hand, pick up a culture tube and remove the cap with the fourth and fifth fingers of the hand
that is holding the loop. Never lay a cap down on the lab bench, this will result in contamination. Always hold
the open tube at an angle so that dust does not fall into the tube and contaminate the culture.
4. Pass the opening of the culture tube through the Bunsen burner flame. This will kill any microorganisms on
the lip of the tube.
5. Insert the sterile wire loop into the culture tube. If you have an agar slant, touch the inoculation loop onto the
culture surface and gently gather a small amount of the microorganism. If you have a broth culture, the
inoculating loop can be submerged within the culture.
6. Flame the mouth of the culture tube again, replace the cap, and set it aside.
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7. With your free hand, pick up the agar-half of the petri dish, leaving the lid face up on your bench.
8. Streak out the culture onto section 1 of your plate. Return the petri dish to its lid.
9. Sterilize the inoculating loop as described in step 1.
10. Once cool, pick up the agar-half of the petri dish and pass the inoculation loop 1-3 times through section 1,
dragging the culture through section 2 multiple times. Be sure your streaks do not overlap. Return the petri
dish to its lid.
11. Sterilize the inoculating loop as described in step 1.
12. Once cool, pick up the agar-half of the petri dish and pass the inoculation loop 1-3 times through section 2,
dragging the culture through section 3 multiple times. Be sure your streaks do not overlap. Return the petri
dish to its lid.
13. Sterilize the inoculating loop as described in step 1.
14. Incubate the petri dish under the appropriate growth conditions. Be sure to incubate the plate agar-side-up, this
will prevent condensation from dropping into the culture. With appropriate streak plate technique, you will
obtain isolated colonies in section 3 of your plate.

Fig. 5 (A) Streak plate set-up; (B) How to inoculate a streak plate; (C) Example of a streak plate exhibiting
isolated colonies following incubation.

A B
Fig. 6 (A) Quadrant streaking for isolation into pure culture. (B) Quadrant Streaking showing the colonies
thinning as the streaking moves clockwise.
2.4 Spread Plate
Similar to the streak plate technique, a spread plate is another form of inoculation that used to isolate pure
colonies from a single microbial species. This particular technique is primarily used to quantify the number of
microorganisms within an actively growing broth culture. For more information on the enumeration of a
microorganism, view the section entitled “Bacterial Enumeration”. Below we describe an aseptic protocol for the
inoculation of a spread plate.
Materials
Broth culture Sterile 1 mL pipette bulb Alcohol burner
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Glass or metal streaking “hockey” stick 70% Ethanol Sterile petri dish with agar
NOTE 2: Pre-drying the plates will aid in spreading the culture media evenly and quickly.
Procedure
1. Place a petri dish agar-side-down onto the bench top.
2. Select a sterile 1 mL pipette and gently attach it to the pipette bulb. Do not touch the tip of the pipette, this will
contaminate it.
3. With your other hand, pick up the culture tube and remove the cap with the fourth and fifth fingers of your
opposite hand. Never lay a cap down on the lab bench, this will result in contamination. Always hold the open
tube at an angle so that dust does not fall into the tube and contaminate the culture.
4. Pass the opening of the culture tube through the Bunsen burner flame. This will kill any microorganisms on
the lip of the tube.
5. Insert the sterile pipette tip into the culture tube and aspirate 0.1 mL of growing culture.
6. Remove the pipette from the culture tube, flame the mouth of the culture tube again, replace the cap, and set it
aside. Carefully hold the pipette, do not touch the tip or lay the pipette on the bench top.
7. With your free hand, pick up the lid to the petri dish and hold it at an angle over the plate. This will prevent
dust from falling into your culture while you inoculate.
8. Slowly dispense the culture media from the pipette onto the center of the plate. Replace the petri dish lid, and
properly dispose of the pipette.
9. Sterilize the hockey stick by submerging the short, flat portion of the hockey stick into the 70% ethanol. Pass
the hockey stick quickly through the Bunsen burner flame to light it on fire. Do not leave the hockey stick in
the flame. Once the flame has burned out, allow the hockey stick to cool for a few seconds.
10. With your free hand, lift up the lid of the petri dish. Place the sterile portion of your hockey stick onto the agar
to dissipate the heat. Then spread the inoculum over the plate, evenly spreading the culture media on the surface
of the agar. Gently turn the plate while spreading the media to ensure the surface of the agar is evenly covered.
Do not puncture the agar (See: NOTE 2).
11. Continue to spread the culture media until it has dried onto surface of the plate. Replace the petri dish lid and
re-sterilize the hockey stick.
12. Incubate the petri dish under the appropriate growth conditions. Be sure to incubate the plate agar-side-up, this
will prevent condensation from dropping into the culture. With appropriate spread plate technique, you will
obtain isolated colonies on your plate.

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 Fig. 7 Procedure for Spread Plate

3. Culturing Conditions
Because bacteria can grow and thrive in a variety of environments, optimal growth temperatures may vary
significantly between species. In general, most pathogenic or commensal bacterial strains grow well at body
temperature (37°C). In contrast, many environmental strains thrive at lower temperatures, often within a range of
25°C to 30°C.
Bacterial species can be categorized based on their growth temperature; these include psychrophiles (0°C to
20°C), mesophiles (25°C to 40°C), and thermophiles (45°C to 122°C). Though bacterial strains require optimal
temperatures for growth and reproduction, most strains can withstand considerable drops in temperature and
survive several days at 4°C. At these lower temperatures, bacterial growth and metabolism are significantly
diminished.
In addition to varying requirements for optimal growth temperatures, bacteria also differ in their use of oxygen
for respiration. Aerobic organisms, such as Bacillus species, use oxygen as a terminal electron acceptor during
respiration. Similarly, microaerophiles, such as Helicobacter pylori, also require the use of oxygen, but at lower
levels than naturally occurring in the environment. In contrast, anaerobic organisms use electron acceptors such as
nitrate or sulfate, among other inorganic acceptors. These inorganic compounds, however, have a lower reduction
potential than oxygen thus resulting in less efficient respiration.
The use of oxygen and inorganic compounds by anaerobic organisms can differ greatly between species.
Obligate anaerobes, such as Clostridium species, can only survive and reproduce in the absence of oxygen; these
organisms are often killed by the presence of oxygen. Similarly, aerotolerant anaerobes, such as Lactobacillus
species, cannot use oxygen during respiration; however, unlike strict anaerobes, these microorganisms can tolerate
oxygen for short periods of time. Lastly, facultative anaerobes, such as Escherichia coli and Staphylococcus
species, are able to survive in both the presence and absence of oxygen. If given the choice, these organisms prefer
the use of oxygen during respiration as it has the greatest reduction potential as compared to other electron
acceptors.

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 When working with anaerobic cultures, it is important to avoid unnecessary exposure to oxygen. Anaerobic
conditions may be obtained for either transfer or incubation by the methods listed below. Unless specifically
mentioned, the standard anaerobic gas mixture is 80% N₂, 10% CO₂, and 10% H₂. Anaerobic conditions for transfer
may be obtained by either of the following:
1. Use of an anaerobic gas chamber.
2. Placement of test tubes under a gassing cannula system hooked to anaerobic gas.

During incubation, anaerobic conditions may be maintained by any of the following:

1. Loose screw-caps on test tubes in an anaerobic chamber.
2. Loose screw-caps on test tubes in an activated anaerobic GasPak™ jar.
3. Use of sterile butyl rubber stoppers on test tubes so that an anaerobic gas headspace is retained.
Below, we describe a general procedure to analyze the temperature and oxygen requirements of a microorganism.
Materials
Broth culture Sterile agar plates per culture
Sterile pre-reduced agar plates per culture Wire inoculation loop
Procedure
1. Inoculate the six sterile agar plates using the streak plate culturing technique.
2. Under anaerobic conditions, inoculate the three pre-reduced agar plates using the streak plate culturing
technique.
3. Incubate the plates agar-side-up at either 25°C, 30°C, or 37°C under the following conditions:
a. Aerobic – incubate plates in a standard incubator.
b. Microaerophilic – incubate plates in a candle jar. This can be any jar large enough to hold petri dishes and
a candle. Place your plate and a lit candle into the jar and seal the lid. The candle flame will consume most
of the oxygen in the jar, producing elevated levels of carbon dioxide.
c. Anaerobic – incubate pre-reduced plates in a sealed anaerobic jar, such as the GasPak™ anaerobic system
(Becton Dickinson). This system consists of a polycarbonate jar, a lid with a gasket to prevent air flow, an
indicator strip for the detection of oxygen, and a ready-to-use chemical sachet that creates an anaerobic
atmosphere.
d. Observe the cultures for signs of growth.
4. Airborne Contamination
Airborne contamination frequently occurs from aerosolized microorganisms spread by coughing and sneezing, or
by bacteria carried on the surface of microscopic dust particles. To avoid contamination by airborne particles, it is
important to always use proper aseptic technique in the laboratory. This exercise will demonstrate the extent of
contamination due to airborne and aerosolized microorganisms.
Materials Sterile petri dish with agar
Procedure
1. Place an agar plate agar-side-down on your laboratory bench top.
2. Remove the lid from the plate and set it aside.
3. Leave the agar surface exposed to the air for 60 minutes.
4. Replace the lid.
5. Incubate the plate for 48 hours at 30°C
6. Observe colony growth.

5. Colony Morphology
Colony morphology can significantly vary between species and is often used in the initial identification of a
strain. Further, morphology can vary between strains within a species due to differential gene expression. Below
are several terms commonly used to describe colony morphology.

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 Shape – This refers to the form of the colony, or its overall appearance. Common shapes include circular,
rhizoid, irregular, filamentous, and spindle.
 Margin – The margin refers to the edge of a colony, and may be an important characteristic in organism
identification. Common margins include entire, undulate (resembling waves), lobate (lobed structure), curled,
rhizoid, filamentous, or erose (irregularly notched).
 Elevation – Elevation describes the side-view of the colony. The most common are flat, raised, convex (curved
surface), pulvinate (cushion-shaped), and umbonate (having a knobby protuberance).
 Size – The size of a colony is often a useful characteristic for identification, and can be measured. Sizes are
often described as punctiform (shaped like a point), small, moderate, or large.
 Appearance – The outwards aspects of the colony surface, often depicting if the colony is glistening (glossy,
shiny) or dull (cloudy).
 Optical Property – This describes the opacity of the colonies. Colonies are frequently described as opaque
(impervious to light), translucent (lets light through diffusely), or transparent (allows light to pass through
without disruption).
 Texture – Describes the consistency of a colony. Rough colonies have a granular, flattened surface and are
often associated with loss of virulence. Smooth colonies have a glistening, rounded surface. Other terms used
to describe colony texture include mucoid (gummy, viscous),butyrous (buttery texture), or dry (brittle or
powdery colonies).
 Pigmentation – Some bacterial species produce pigments which can be either water soluble or soluble in fat.
The production of pigments may vary depending on the environmental conditions or age of the colony.

Figure 8 Colony Morphology

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 A B

Figure 9 (A) Colonies are circular, entire, convex, large, smooth, glistening, cream-colored, and translucent; (B)
Colonies are irregular, erose, flat, wrinkled, rough, dull, yellow, and opaque.
References
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Biosafety in Microbiological and Biomedical Laboratories 5edn, (U.S. Government Printing Office, HHS
Publication No. (CDC) 21-1112, 2009).
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3. Young, K. D. The Selective Value of Bacterial Shape. Microbiol Mol Biol Rev 70, 660-703 (2006).
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bacterial shape. Trends in genetics: TIG 17, 124-126 (2001).
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