Laboratory Exercise 2
Introduction to Microbiological Media Preparation
Expectations of this lab:
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Understand what a culture medium is and the two main types of media: defined and
undefined (complex).
Understand that microbiological media are categorized based on the purpose of use: allpurpose, selective, differential, selective and differential.
Understand why media are categorized in the above manner and what chemical
compounds or reagents are responsible for the designation.
At the end of the lab be able to prepare a culture medium (plates, broths, deeps, or slants)
to grow a bacterial culture.
Have a thorough understanding of what sterilization means and the methods that can be
implemented to achieve sterilization for various materials.
Introduction
Robert Koch, a founding father in microbiology, introduced the concept of pure cultures and
growing microorganisms in culture media during the late 19th century. A basic growth medium
will provide important macronutrients as well as vitamins and growth factors necessary for
microorganisms to grow. Agar was first used in culture media in Robert Koch’s lab. Agar is
extracted from red algae and it can be in a solid state up to 100oC. Once it starts to partially
dissolve and form a clear transparent solution, it will stay a liquid until the temperature drops to
around 37- 40oC. At this point the agar will start to gel and once it reaches the room temperature
around 25oC the agar sol will be solid and white. Very few non-marine bacteria hydrolyze agar
and this is one main advantages of using agar as a solidifying agent in microbiological media.
There are many categories of culture media and sometimes the same medium can be named
differently by various companies. In this laboratory, we will primarily use an all-purpose
medium called Tryptic Soy Agar (TSA). This same medium is sometimes called Trypticase Soy
Agar or Tryptone Soya Agar. All these media have an identical composition although the brand
name is slightly different. The liquid medium used in this laboratory is called Tryptic Soy Broth
(TSB).
Types of microbiological media
There are two main categories of culture media, complex (undefined) or defined.
A defined medium will contain measured amounts of salts and nutrients of known composition
while a complex medium (also called an undefined medium) will have components that are not
nutritionally quantified. A few examples of defined media and a complex medium are given
below.
Complex medium: Tryptic Soy Agar (1)
Composition per liter:
Pancreatic digest of casein
17.0g (composition of this digest is not known)
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Agar
15.0g
NaCl
5.0g
Papaic digest of soybean meal 3.0g (composition of this digest is not known)
K2HPO4
2.5g
Glucose
2.5g
Defined medium: Waksman’s Sulfur Medium (1)
Composition per liter:
KH2PO4
3.0g
MgSO4.7H2O
0.5g
(NH4)2SO4
0.2g
CaCl2.2H2O
0.2g
Fe2 (SO4)3
0.1mg
Defined medium for Rhodopseudomonas (1)
Composition per liter:
Malic acid
4.0g
(NH4)2SO4
1.0g
K2HPO4
0.9g
KH2PO4
0.6g
MgSO4.7H2O
0.2g
CaCl2.2H2O
0.075g
EDTA
0.020g
FeSO4.7H2O
0.012g
Thiamine
1.0mg
Biotin
0.015mg
Trace elements
1.0mL
pH 6.8 +/- 0.2 at 25oC
Trace Elements:
Composition per 250mL:
H3BO3
MnSO4.H2O
Na2MoO.2H2O
ZnSO4.7H2O
CoCl2.6H2O
Cu (NO3)2.3H2O
0.7g
0.4g
0.19g
0.060g
0.050g
0.010g
Culture media can be characterized in different ways. An all-purpose medium will provide the
nutritional requirements of a wide range of bacteria. Certain compounds can be added to a
medium to make the medium selective. These compounds help promote the growth of a selected
few types of bacteria. The most common compounds added to a medium to make it selective are
NaCl, antibiotics, dyes, and specific inhibitors. Differential media will have certain compounds
in the medium that allows them to differentiate between closely related organisms.
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Figure 2-1 Tryptic soy agar (general or an all-purpose medium)
TSA plate divided into six sections and spot inoculated with Escherichia coli, Staphylococcus aureus, Staphylococcus
epidermidis, Enterobacter aerogenes, Pseudomonas fluorescens, and incubated at 30oC for 48 hours.
Figure 2-2 MacConkey agar (selective and differential)
MA plate divided into six sections and spot inoculated with Escherichia coli, Staphylococcus aureus, Staphylococcus
epidermidis, Enterobacter aerogenes, Pseudomonas fluorescens, and incubated at 30oC for 48 hours.
2015 ASM plate: Citrobacter freundii on MacConkey agar.
Figure 2-3 Mannitol salt agar (selective and differential)
MSA plate divided into six sections and spot inoculated with Escherichia coli, Staphylococcus aureus, Staphylococcus
epidermidis, Enterobacter aerogenes, Pseudomonas fluorescens, and incubated at 30oC for 48 hours.
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Different forms of media
A bacterial culture medium can be used in various forms according to the need of the laboratory
work or research performed. Broth cultures (liquid cultures) can be used to grow big batches of
bacteria in Erlenmeyer flasks or in small test tubes, depending in the purpose of the exercise.
Semi-solid and solid media will contain necessary nutrients for the growth of microbes as well
as a solidifying agent like granulose agar. Solid bacterial media will generally have 1.5% agar
while a semi-solid medium used in motility studies will have only about 0.4% agar.
Sterile petri dishes are used to make TSA plates while TSA slants and deeps are made in glass
tubes. Before any medium containing agar is dispensed into plates or tubes they will be sterilized
in an autoclave. Liquid media with or without agar can be dispensed into tubes prior to
autoclaving, but heating the medium to the boiling point is necessary if agar is present in the
medium prior to distributing the desired aliquots into tubes. When making slants from a medium,
the medium should be boiled for a few minutes for agar to go into solution, dispensed into tubes,
autoclaved, and then slanted on racks at the end of autoclaving until the agar solidifies. Special
racks with wings are available for this purpose or racks can be slanted using a stack of paper
towels.
Sterilization of microbiological media
Sterilization of microbiological media, glassware, waste, and any other necessary lab item which
is not heat sensitive is achieved through autoclaving. An autoclave is a machine similar to a big
pressure cooker that utilizes heat and steam to achieve sterilization. An autoclave can increase
the internal temperature of the chamber to about 132oC. The typical liquid cycle will be 121oC at
15 psi pressure for 20 minutes or longer depending on the total volume sterilized. Sterilization
is achieved when all living cells including bacterial endospores are dead. Bacterial endospores
are dormant structures formed by a few genera under adverse conditions. Boiling and other
methods of destroying most other living cells will not destroy endospores. Clostridium and
Bacillus are two common endospore forming genera. They are two main indicator organisms in
thermal sterilization (e.g., food industry – canning) that ensure the safety of the consumers.
There are a few other methods of sterilization. Heat labile materials like antibiotic solutions and
urea broth utilized in this lab can be sterilized by filtration. A 0.2 µ pore size filter will filter
out most bacteria and 20nm range filters will be necessary to eliminate viruses. Ethylene oxide
also can be used for sterilization of heat sensitive materials. UV radiation works best for
surfaces while dry heat sterilization is good for sterilizing glassware in the lab. Glassware is
sterilized by utilizing dry ovens at 160-170oC for about two hours.
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Figure 2-4 Autoclave (steam sterilizer)
The autoclave chamber in the middle is loaded with items to be sterilized and a pre-set liquid or
gravity cycle chosen (e.g., L45 cycle: 121oC at 15 psi pressure for 45 minutes)
Materials
Stirring hotplate
Magnetic stir bars
Sterile petri dishes
Test tubes and caps
Balance
Weigh paper/boats
Spatulas
Pipettes
Tryptic Soy Broth and agar
Deionized water (DI water)
One 2l Erlenmeyer flask per medium
3-4 500 ml Erlenmeyer flasks and aluminum foil covers
Slant rack
Procedure
Tryptic Soy Agar (TSA), 1000mL batch:
1. Weigh 30g TSB (Tryptic Soy Broth) and 15g agar using a balance and a weighing boat
and add to an Erlenmeyer flask.
2. Add 1000 mL of DI water and a stir bar to the flask.
3. Cap the flask with aluminum foil and mix on a stir plate until all the ingredients are
thoroughly mixed.
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4. Place the flask on a polypropylene bin and autoclave on setting L45.
5. Remove from the autoclave using heat resistant gloves, cool in a 50oC water bath for
approximately 20 minutes before pouring into sterile petri dishes as demonstrated by the
instructor.
Insert figures 1.2 , 1.3, 1.4, and 1.7 from MLTA 4th edition
Tryptic Soy Broth (TSB), 1000mL batch
1.
2.
3.
4.
Weigh 30g of TSB and add to an Erlenmeyer flask.
Add 1000mL of DI water, and mix until thoroughly dissolved.
Using a pipette dispense 7mL into each test tube.
Cap the test tubes, place them in a polypropylene bin, and sterilize in an autoclave on
setting L20.
5. Remove from autoclave and allow the tubes to cool to room temperature.
Tryptic Soy Agar slants (TSA), 1000mL batch
1. Weigh 30g TSB (Tryptic Soy Broth) and 15g agar using a balance and a weighing boat
and add to an Erlenmeyer flask.
2. Add 1000 mL of DI water and a stir bar to the flask.
3. Cap the flask with aluminum foil and mix on a stir plate until all the ingredients are
thoroughly mixed.
4. Gradually increase the heat until the medium is completely clear and boiling.
5. Dispense the medium into test tubes in an autoclavable slant rack (i.e., 7 mL per tube)
6. Place the rack in a poly propylene bin and autoclave on setting L20.
7. Remove from the autoclave using heat resistant gloves, and slant the rack and let it cool.
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Results
Figure 2-5 Tryptic soy agar plates (Label with name, date, and the bacterium)
Figure 2-6 Tryptic soy agar slant (L) and Tryptic soy broths (R)
Common problems and tips
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Inaccurate measurements of ingredients: weighing 1.5g of agar instead of 15g would
result in a medium that would not solidify.
Media will not gel. Not adding agar when making solid media.
Contamination due to improper aseptic technique during pouring of plates.
Under sterilization of media: a larger media volume would need a longer autoclave
setting (e.g., L45 cycle vs L20 cycle).
Capping tubes too tightly before autoclaving. This will result in under sterilization due
to poor steam circulation. Tight bottle caps may cause glass bottles to explode in the
autoclave.
Not capping tubes tightly after sterilization. This may lead to contamination of the
broths. Too loose caps will pop out during sterilization in the autoclave.
References
1. Atlas RM, Parks LC. 1993. Handbook of Microbiological Media. CRC Press Inc.
Florida.
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