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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.