EXPERIMENT 1

PREPARATION OF MEDIA AND STERILIZATION

Learning Objectives
At the end of this experiment, student must be able to
1. Perform correct practice in media preparation for microbiological use
2. Differentiate between the content of complex and defined media as well
as different forms of media prepared
3. Discuss the function of complex and defined media in relation to their
content or formulation
4. Demonstrate sterilization of media using autoclave
5. Discuss the concept and suitability of using autoclave or any other
approach for sterilization
Introduction
Any medium for cultivation of bacteria must provide certain basic
nutritional requirements, which include a carbon source that may also serve as
an energy source, water, a nitrogen source, a phosphate source and various
mineral nutrients such as iron and magnesium.
Some bacteria are capable of growth in a medium consisting of a single
carbon source, such as glucose, a simple nitrogen source, such as ammonium
salts and inorganic salts such as phosphates. This kind of medium is termed
defined or synthetic because its exact chemical composition is known.
For routine laboratory work, however, complex media are employed
where the basic nutrients are provided by complex nutrients, such as plant and
animal extracts in which the exact composition is not known. As an example,
beef extract and peptone (hydrolyzed protein) are the basic ingredients of
nutrient agar. These materials supply variety of carbon sources, nitrogen
compounds in the form of amino acids, and a mixture of cofactors, such as
vitamins. This basic medium can be further enriched to support the growth of
fastidious types of bacteria by the addition of carbohydrate sources, yeast
extract and materials such as plasma and blood, which provide a variety of
complex nutritional factors.
A broth medium is one in which the components are simply dissolved in

water.
The addition of agar (a complex carbohydrate extracted from seaweed) results
in a solid medium. Agar is an ideal solidifying agent for microbiological media
because of its melting properties and because it has no nutritive value for the
vast majority of bacteria. Solid agar melts at 90-100C; liquid agar solidifies at
about 42C.
Sterilization procedures eliminate all viable microorganisms from a
specified region. Culture dishes, test tubes, flasks, pipettes, transfer loops, and
media must be free of viable microorganisms before they can be used for

establishing pure cultures of microorganisms. There are various ways of

sterilizing the liquids, containers and instruments used in pure culture , these
include exposure to elevated temperatures or radiation levels to kill
microorganisms and filtration to remove microorganisms from solution.
Generally, the medium sterilization is carried out at 121C (achieved by using
steam at 15lb/in2) for 15 minutes by using the autoclave.

1.1 : Preparation of Complex Medium

Equipments / Consumables
Beaker (500 mL)
Schott bottle500 mL (2)
Calibrated pH meter
Universal bottles (25 non-sterile + 5 sterile)
Sterile petri plates
Autoclave
Autoclave tape
Heatproof gloves
Pasteur pipettes
Graduated cylinder, 500 mL
Graduated cylinder, 10 mL
Top pan balance
Sterile cotton swabs

Materials
Nutrient Broth powder
Agar powder

Procedures
1. Weigh out 6.5g of Nutrient Broth (NB) medium and transfer into a 500 mL
beaker. Dissolve by adding 400 mL of distilled water.
Check the pH. Adjust the pH to 7.0 (if necessary) by adding
NaOH (5.0 M) or HCl (5.0 M). Make sure to clean the pH
electrode with distilled water before and after each use.
2. Dispense the solution into graduated
cylinder and bring the volume to 500 mL by
adding distilled water.
3. Divide the solution into two flasks and
follow instruction a) and b)

a) Pour 400 mL of the solution into a

500 mL Schott bottle and add
5.0 g agar. Briefly mix the agar in
the NB solution and close the
bottle using its lid. Label the

100 mL
400 mL

bottle as NB(+agar). Note: do not

forget to write your group number

and date on the flask.

b) For another 100 mL of NB

solution (without agar); dispense
every 10 mL into 10 separate
universal bottles. Place the
bottles cap on and briefly tighten
it (DO NOT OVERTIGHTEN THE
CAP). Label the bottle as NB
4. Autoclave for 15 minutes at 121C, 15
lb/in2.
5. To demonstrate the necessity of the
sterilization step, do not autoclave one
bottle of nutrient broth.
6. After removal from the autoclave, allow
the nutrient medium (NB) to cool and
store them for use in later experiment.
7. Allow the nutrient agar (NB
+agar) to cool down until 50C.
Then, follow the instruction
below:
a) Use 50 mL nutrient agar to make
incline (slant) agar dispense
approximately 10 mL each of
nutrient agar into separate 5
sterile universal bottles. Solidify
the agar by inclining the bottles at
about 30-45C.
b) Dispense another 350 mL
nutrient agar into sterile petri
plates (to make approximately 20
agar plates).
8. Allow agar to cool and solidify. Store
these nutrient agars at a 4C refrigerator
until used for the next experiment.

1.2 : Preparation Of Defined Medium

Materials
Glucose-mineral salts agar (500 mL)
Glucose
0.25g
NH4Cl
0.5g
MgSO4.7H2O
0.1g
FeCl3.6H2O
0.0025g
Agar
10g
Nicotinic acid
0.0025g (optional)
Procedures
1. Weigh out each component above except nicotinic acid and deliver into
a 500 mL Schott bottle. Dissolve them in 400 mL distilled water.
2. Carefully adjust the pH to 7.0 with either NaOH or HCl.
3. Bring the volume to 500 mL by adding with distilled water
4. Dispense 250 mL of the medium into two 250 mL Schott bottles
5. Add nicotinic acid into one bottle only and label the bottle with GGMAN.
6. Lable another bottle containing 250 mL medium NOT added with
nicotinic acid with GGMA.
7. Autoclave the media.
8. After each of the prepared medium has been sterilized, allow it to cool
until 50C. Pour 20 mL/plate into petri plates. Let the agar solidify and
label all the plates (GGMA or GGMA-N). Store the plates in the fridge
for use in a later laboratory experiment.