Introduction
The cultivation of microorganisms in vitro, apart from its substrate in nature, is possible by providing substances that the organisms can use as food.
The requirements of microorganisms are very diverse. For their isolation a culture medium must be prepared that provides a balanced mixture of these components. They have to be added in a relation to each other that guarantees good growth. The basic media for cultivation should contain a mineral base that provides all those nutrients that can be supplied to any organisms in inorganic form. This can be supplemented with carbon, nitrogen and phosphorus source and an extra energy source or electron acceptor if necessary-relating in the special requirements
A culture medium is an aqueous solution of various nutrients required by microorganisms. It is impossible to prepare a single culture medium that can support the growth of all bacteria although many complex media can support a wide array of microbial forms. Synthetic media are composed entirely of chemically defined nutrients,meeting only the minimal requirements of the organisms.Complex media is a rich medium with chemically undefined ingredients providing optimal conditions for growth. Selective media are culture media containing at least one ingredient that inhibits the reproduction of unwanted organisms, but permits the reproduction of specific microorganisms. Enrichment cultures are used to increase the relative concentration of a desired microorganism with respect to others that may be also present in the sample. Differential media are special formulations designed to differentiate among microorganisms or group of microorganisms. Differential media usually contain a chemical that is utilized or altered by some microorganism but not by others.
Different agar tubes from left to right a) slant b) slant-butt c) butt
OBJECTIVES:
1. To prepare simple bacteriological media
2. To emphasize basic bacteriological technique in preserving sterility and preventing contamination of culture media.
3. To put aseptic technique into practice.
METHODOLOGY:
1. Seawater Agar. Calculate the total amount of medium needed to prepare 9 plates. About 15ml of medium is required to make one plate. Compute and weigh out the necessary amount of agar and broth based on manufacturer’s specification on the label. Compute for the amount of sodium chloride to be incorporated in the medium depending on the prevailing salinity of the sample. Dilute to required volume of distilled water and heat in water bath until agar is in solution. Plug flasks with cotton and cover with aluminum foil and sterilize at 121oC for 15 min. let the medium cool to 50oC before pouring in plates. All manipulations are done aseptically inside the laminar flow.
2. Cetrimide Agar. Compute and weigh out the necessary amount of Cetrimide agar, glycerol to prepare nine plates based on manufacturer’s specification. Incorporate sodium chloride based on salinity requirement. Dilute to required volume of distilled water and heat in water bath until agar is in solution. Plug flasks with cotton and cover with aluminum foil and sterilize at 121oC for 15 min. let the medium cool to 50oC before pouring in plates. All manipulations are done aseptically.
3. Potato Dextrose Agar. Compute and weigh out the necessary amount of potato starch, dextrose and agar to prepare ten plates based on manufacturer’s specification. Incorporate sodium chloride based on salinity requirement. Dilute to required volume of distilled water and heat in water bath until agar is in solution. Plug flasks with cotton and cover with aluminum foil and sterilize at 121oC for 15 min. let the medium cool to 50oC before pouring in plates. All manipulations are done aseptically.
4. Thiosulfate Bile Sucrose Agar. Compute and weigh out the necessary amount of medium to prepare ten plates based on manufacturer’s specification. Incorporate sodium chloride based on salinity requirement. Dilute to required volume of distilled water and heat in water bath until agar is in solution. Do not autoclave. Let the medium cool to 50oC before pouring in plates. All manipulations are done aseptically.
5. Seawater indole-nitrate broth. Compute and weigh out the necessary amount of indole –nitrate broth to prepare thirty tubes based on manufacturer’s specification. About 9ml is required per tube. Incorporate sodium chloride based on salinity requirement. Dilute to required volume of distilled water and heat in water bath until medium is in solution. Dispense in tubes and to each tube insert fermentation tube, bottom up to capture the gas. Plug with cotton, cover with aluminum foil and sterilize at 121oC for 15 min.
6. Triple sugar iron agar. Compute and weigh out the necessary amount of triple sugar iron agar to prepare nine plates based on manufacturer’s specification. Incorporate sodium chloride based on salinity requirement. Dilute to required volume of distilled water and heat in water bath until agar is in solution. Plug flasks with cotton and cover with aluminum foil and sterilize at 121oC for 15 min let the medium cool to 50oC before pouring in plates. All manipulations are done aseptically
7. NOTE: SAVE ALL PREPARED MEDIA FOR THE NEXT EXERCISE.
RESULTS AND DISCUSSION
1. What are the aseptic techniques to be observed in preparing media?
2. How are media classified? Give examples.
3. When making an agar plate, the nutrient agar is cooled first to about 450C. Why?
4. What makes agar a good support for microbial growth?
5. What are the different methods of maintaining cultures? Give the advantages and disadvantages of each method
Preparation of Artificial Media
Name: Date Performed:
Rating
RESULTS:
Medium | Recommended Preparation | Volume to be prepared | Amount of Medium Needed |
Seawater Agar | |||
Nutrient agar | |||
Sodium chloride | |||
Cetrimide Agar | |||
Glycerol | |||
Potato dextrose agar | |||
Thiosulfate bile sucrose agar | |||
Nitrate -indole Broth | |||
Triple sugar iron agar |
REFERENCES:
