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Exercise 3. Culture Media Preparation
Laboratory Techniques in Biological Sciences
Biology Unit-Philippine Science High School-Main Campus
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.
2.
3.
4.
5.
Nitrogen sources
Carbon sources
Energy sources
Minerals
Growth factors
Other substances may be added as needed; for example, buffer salts, indicator
dye, reducing substances and selective agents. The culture medium can be prepared from
basic ingredients or from commercially available digests.
The most commonly used liquid medium is nutrient broth. However, coconut water
can be used as substitute. It contains sugars, amino acids, vitamins and other growth factors
needed by the organisms. With fastidious organisms, other compounds may be added to the
nutrient broth or coconut water in order to meet the needs of the specific bacteria.
One of the most common media used in microbiology is nutrient agar/broth. It
consists of beef extract and peptone. Beef extract, a hydrolyzed extract of meat at a
concentration of 0.3-0.5%, will support the growth of microorganisms when combined with
peptone. It contains peptides and amino acids, nucleotide fractions, organic acids, minerals and
some vitamins. Its function is to complement the nutritive properties of peptone by contributing
minerals, phosphates, energy sources and those essential factors missing in peptone.
Peptones are hydrolyzed products of proteins brought about by the action of the
enzyme protease. Peptones are commonly used as nitrogen source but may also supply other
requirements such as Na+, CI-, PO4, and trace metals. Peptones used in carbohydrate
fermentation should not contain fermentable carbohydrates. Meat peptones may contain traces
of glycogen but are normally high in utilizable carbon compounds such as lactate. Yeast
extracts and yeast peptones are considered to be substantial sources of water-soluble vitamins
in culture media.
Materials:
Phenolphthalein
Agar
Coconut water
Gulaman bar
Cheesecloth
Bromthymol blue
Bromcresol green
1 M KH2PO4
0.05M and 1M NaOH
0.05M and 1M HCI
Flask for media
Test Tubes
Cotton
3 “lapad bottles”
Exercise 3. Culture Media Preparation
Laboratory Techniques in Biological Sciences
Biology Unit-Philippine Science High School-Main Campus
Procedure:
I. Preparation of Nutrient Broth
1. Mix 7.5g of Ready Mix Nutrient Broth Powder to a 250 ml of distilled water in a 500 ml flask.
2. Hasten the solution of these materials by mixing and warming. Cover the flask with a cotton plug.
Label your flask.
II. Preparation of Nutrient Agar
1.
2.
3.
4.
To every 250 ml of nutrient broth in a 500 ml flask, add 5.75 g of agar powder.
Dissolve the agar in a double boiler or in a boiling water bath.
After the agar is dissolved, restore to original volume by adding hot water.
Cover the flask with a cotton plug. Label your flask.
Agar is a component used for solidifying bacteriological media. It is a gel-forming
polysaccharide, which is extractable from several species of red seaweeds (agarophytes). It is a mixture
of at least two polysaccharides: agarose (ca 70%) and agaropectin (ca 30%) components. Agar should
be capable of remaining molten and fluid at 40-45°C after cooling from boiling point. Different grades of
agar are available and depending upon the brand, the required amount of agar will vary.
III. Preparation of Coconut Water Medium
1. Obtain coconut water from the market or factory and strain 4x through several layers of gauze or
cheesecloth to remove extraneous materials.
2. Adjust pH to 7, following instructions in Section V.
3. Dispense 250 ml coconut water in a 500 ml flask or in tubes and submit to instructor for
sterilization.
4. The resulting medium is cloudy due to precipitates. Allow precipitates to settle and decant
aseptically to sterile containers. Some colloidal materials may remain in solution. (If you are not
yet adept with aseptic techniques, your instructor will do the transfer). The medium can be used
even with the precipitates but to avoid confusion of the precipitates with microbial growth, it is
best to decant. Always compare turbidity of the inoculated broth with an uninoculated control
tube.
IV. Preparation of Coconut Water Agar (CWA)
1. Filter coconut water as in Section III.
2. Obtain 10 g local agar (gulaman) and soak in the 250 ml coconut water for 20 minutes.
3. Dissolve in boiling water bath. If extraneous materials are present, filter medium through few
layers of gauze or cheesecloth.
4. Dispense as required and sterilize as above.
For plating, fill tubes with 15-20 ml; for slanting, use 8 ml. Agar slopes or slants are made by
allowing liquefied agar to solidify in a slanting position. Media should always be stored in a cool moist
environment to prevent evaporation.
V. Adjusting the pH of Culture Media
For bacteriology studies, culture media must be sterile and must have suitable reaction.
Sterilization may be accomplished in a number of ways
Exercise 3. Culture Media Preparation
Laboratory Techniques in Biological Sciences
Biology Unit-Philippine Science High School-Main Campus
To adjust the pH of nutrient broth or coconut water medium to 7.0 or neutral:
1. Prepare a 50 ml 10M HCl and 50 ml 10M NaOH.
2. Adjust the pH by adding the necessary reagent. Use the digital pH meter to determine the pH
value.
This lab. exercise was partially adapted from:
FERNANDEZ, W.L., I.F. DALMACIO, A.K. RAYMUNDO, AND A.F. ZAMORA. 1995. Laboratory Manual:
General Microbiology. 2nd ed. University of the Philippines Los Baños (UPLB). Microbiology Laboratory,
IBS, CAS, UPLB. 115 pp.
LIST OF REFERENCES (or any microbiology book or microbiology manual)
ANONYMOUS. 1984 Difco Manual, 10th Ed.
Detroit, Michigan. Difco Laboratories Inc.1155 p.
BOOTH, C. 1971. “Fungal culture media” In: C. Booth (ed.) Methods in Microbiology.
Vol. 4 London: Academic Press pp. 49-94.
BRIDSON, E.Y. and A. BRECKER. 1970. Designs and Formulation of Microbial Culture Media. In:
J.R. Norris & D.W. Ribbons (eds.) Methods in Microbiology. Vol. 3A. London: Academic Press,
pp.229-296.
ISAWARAN, V. 1976. A Treatise on Media and Methods Used in Bacteriological Techniques.
Manila: Information Section, Bureau of Plant Industry, 175 p.
LAPAGE, S.P., J.E. SHELTON and T.G. MITCHELL. 1970. Media For the Maintenance and
Preservation of Bacteria In: J.R. Norris and D.W. Ribbons (eds.). Methods in Microbiology. Vol. 3A.
London: Academic Press, pp. 11-134.
MARANAN, R., A. KARGANILLA and R. ALICBUSAN.
(gulaman) for Microbiological Use. Phil. Agric. 55: 1-10.
1971. Purification of Local Agar
PADUA, L.S. and B.P. GABRIEL. 1975. Coconut Water as Culture Medium for Entomophthora
coronata. Kalikasan, Philipp. J. Biol. 4: 17-22.