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Laboratory Exercise 4
CULTURE MEDIA PREPARATION

The survival and continuous growth of microorganisms depend on adequate supply of nutrient
and favorable environment. A culture medium is any nutrient material prepared for the growth and
cultivation of microorganisms. It must satisfactorily supply all the factors required by the
microorganisms for growth. The chemical compounds may be grouped as:
1. Nitrogen sources
2. Carbon sources
3. Energy sources
4. Minerals
5. Growth factors

The medium that is used to culture the microorganisms depends on the microorganism that one
is trying to isolate or identify. Different nutrients may be added to the medium, making it higher in protein
or in sugar. Various pH indicators are often added for differentiation of microbes based on their
biochemical reactions; the indicators may turn one color when slightly acidic, another color when
slightly basic. Other added ingredients may be growth factors, NaCl, and pH buffers which keep the
medium from straying too far from neutral as the microbes metabolize. The culture medium can be
prepared from basic ingredients or from commercially available digests.
It can exist in three consistencies: liquid, solid, and semisolid. A liquid medium lacks solidifying
agent and is called broth medium. Agar is a component used for solidifying bacteriological media. It is
a gel-forming polysaccharide which is extractable from several species of red species (agarophytes). It
is a mixture of at least two polysaccharides: agarose (ca. 70%) and agaropectin (ca. 30%), with D-
galactose and 3,6-anhydro-L-galactose as chief components. Agar liquefies at 100°C and solidifies at
40°C. Agar should be capable of remaining molten and fluid at 40-45°C after cooling from boiling point.
Different grade of agar are available and depending upon the brand, the required amount of agar will
vary. A completely solid medium requires an agar concentration of 1.5% to 1.8%. A concentration of
less than 1% agar results in semi-solid medium.
In this experiment, each student will:
1. Compare the different forms of culture media in terms of function and composition;
2. Prepare different culture media.

MATERIALS / EQUIPMENT:
Used bond papers 10mL pipettes
Petri dishes (2 per person) cotton, gauze, and white thread
10ml culture tubes (4 per person) Autoclave
alcohol lamp hot plate
Media bottles (2 per group) Nutrient Agar (NA)
2 Test tube racks (metal and wooden) Nutrient Broth (NB)
masking tapes/autoclave tapes Eosin-Methylene Blue (EMB) Agar
150-200 ml Erlenmeyer flasks (2 per group)
Labelling tapes

PROCEDURE:

Preparation of cotton plugs

Plugs made of non-absorbent cotton wool are used in test tubes, flasks, and pipettes to prevent
micro-organisms from passing in or out and contaminating either the culture or the environment. The
necessary movements of air in and gaseous products out are not prevented and the gaps between the cotton
wool fibers are even wide enough for micro-organisms to pass through. However, this does not happen
because micro-organisms (negatively charged) are “filtered” out by being attracted to and adsorbed on the
oppositely charged cotton wool. The cotton wool must remain dry because this filtration property is lost if the
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cotton wool becomes moist – hence the use of non-absorbent cotton wool.
1. Place the gauze on the mouth or edge of a flask or test tube. Use your finger to press into the center
of the gauze to allow space for the cotton wool. Make about 1 cm space allowance for test tubes and
about 3 cm for flasks.
2. Tightly stuff the gauze with cotton wool until it reaches the edge of the tube/flask.
3. Use a thread to knot and tighten the gauze. Leave 3 cm of gauze as a handle.
4. Test the cotton plug by pulling out the handle.
5. The gauze should be re-used twice

Preparation of the NA and EMB

1. Most of the basic microbiological media is available in powdered or dehydrated form. Since most
of the stock media are in the dehydrated form, avoid too much exposure of the media to the
environmental moisture. Keep the media container tightly closed. Always use a dry spatula in
obtaining the media.
2. Calculations for the amount of media powder in proportion to distilled water will be demonstrated
in class by your Instructor. One may also check for the instruction label of microbiological media
for media preparation.
3. Culture media is prepared by weighing out ____ grams of NA powder or EMB powder and
dissolved to ____ ml of distilled water. Be careful not to get this powder on the balance.
4. Before turning on the thermal stirrer, one should have hand towels or potholders handy. Using
the towels to protect your hand, grasp the neck of the flask tightly and remove it from the stir
plate.
5. The next step will require application of heat to the mixture. Before doing this, however, one
should be aware that agar has a strong tendency to boil when it reaches 100°C. DO NOT USE
EXCESSIVE AND SUDDEN RISE IN TEMPERATURE TO AVOID CHARRING OF THE
MEDIUM. Use minimum amount of heat during sterilization. To avoid over boiling during
sterilization, do not fill the vessel with more than 2/3 full of liquid.
6. At first sign that the mixture is boiling and the agar is dissolved, it will turn clear, deeper tan for
NA. For EMB, it will turn reddish purple with a greenish cast.
7. Turn off the thermal stirrer, take the flask off the heat and allow it to cool for a few minutes.
8. Label the flask with the media name, date, and Family Names of the members of the group.
9. After preparation of the media, place the flask in the autoclave with those of the rest of the class.
Follow Procedure B for sterilization.
10. After sterilization, allow the culture media to cool down. Store the media in a cool, dark place and
in a tightly stoppered container (to avoid contamination) in the refrigerator and not near the
freezer area. Plate-dispensed media are only good for 1 week provided they are free of
contamination.

Preparation of the Agar Slants and Agar Deeps/Stabs

1. Dissolve the dehydrated medium in distilled water (Check label instructions).
2. Heat to boiling on a medium temperature to facilitate proper dissolution and dispersion of the
medium particles.
3. Allow the medium to cool a bit and dispense or pipe out 5 ml into test tubes.
4. Cover the SLANT tubes and the DEEP tubes with clean cotton plugs. Sterilize the medium in an
autoclave.
5. Place all of the tubes which have been pipetted out in the test tube rack.
6. SLANTS are prepared by solidifying the agar with the tube in a slanting position (the size and the
angle of the slant will determine whether it is a regular or a special slant).
7. BUTTS are prepared by solidifying the tube in an upright position.
8. If the media are not used immediately; store them in a ziplock or plastic container and place in
the refrigerator. Always label the containers with the name of the media and the date of
preparation.
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Preparation of the Broth

1. Dissolve the dehydrated broth medium in distilled water (Check label instructions).
2. Heat to boiling on a medium temperature to facilitate proper dissolution and dispersion of the
medium particles.
3. Allow the medium to cool a bit and dispense 5ml into clean and dry 10 ml culture tubes.
4. Cover the tubes with cotton plugs and sterilize the medium in an autoclave. Tighten the plugs
after sterilization.
5. If the media are not used immediately; store them in a ziplock or plastic container and place in
the refrigerator. Always label the containers with the name of the media and the date of
preparation. No heat need be applied at this stage.

Study Questions:
Short but concise answers must be placed after every question. Copy each question first before writing
your answer. Maximum of 2 sentences.
1. List the factors that will guide you in choosing what media to prepare.
2. Why do we always use distilled water instead of tap water in dissolving our media?
3. What are the problems encountered in media sterilization? What could have been the cause of
such problem?
4. Classify the different culture media according to:
a. Composition
b. Consistency
c. Manner of preparation
d. Function