Prerequisite for Preparation of Culture Media

Culture media contains several components some of which are included in already synthesized commercially available growth media, and some of which are included or added as additional growth constituents to the growth media in the course of their preparation. Some of the major components of culture media include agar or agar-agar (the major solidifying agent), peptone (the source of proteins or amino acids), yeast and meat extract (the source of vitamins and other minerals), sodium chloride (the source of sodium and chloride ions), glucose (the source of carbon and energy), water and other important chemical constituents such as indicators and dyes that aid in the characterization and identification of some microorganisms during microbial cultivation.

Most commercially available culture media exist in powdered form as powdered agar base that must be properly weighed out and dissolved in the correct amount of distilled water for the preparation of growth media. And it is critical that the investigator or students acquaints themselves with the directives of preparing each of the culture media as stipulated by the manufacturers of the different growth media. Also, some culture media such as blood agar and Sabouraud dextrose agar (SDA) require the addition of extra growth nutrients and inhibitory substances such as blood and antibiotics in the course of their preparation; and the correct amount of these substances must be added during the preparation of such culture media in order to get optimum result.

For optimum result, culture media required for microbiological investigations should always be prepared or compound in such a way that it support the growth of the microorganisms being sought for, either from an environmental sample or from a nosocomial or hospital sample. Most culture media are prepared from their powdered agar base (containing all the necessary growth nutrients in their correct amount); and it is critical for microbiologist to stick to the manufacturer’s instructions when preparing them because the instruction varies from one culture media to another. Preparation of culture media in the microbiology laboratory is not too cumbersome; and knowledge of the basic techniques involved is important so that the growth media prepared will be competent enough to support the development of microbes (which have been removed from their natural environment, and must be provided with all necessary nutrients and environmental conditions for optimum growth).

The steps involved in the preparation of culture or growth media shall be succinctly explained in this section. Note: The procedure for the preparation of culture media (especially for bacteriological and mycological studies) in the microbiology laboratory is often the same. Though there may be some variations in the different media available for microbiological investigations, it is recommended that students and users of culture media always follow the manufacturer’s instruction of each growth media (usually written on the body of the media container) in order to be properly guided in the preparation.

  • Weighing: Powdered agar should be properly weighed (in grams) using the weighing balance.
  • Dissolving & heating: Weighed powdered agar should be dissolved in the correct volume of distilled water and then heated for some minutes prior to sterilization.
  • pH testing: The pH of the culture media should be adjusted after sterilization when the need arises using the pH meter. The final pH of most microbiological culture media is usually between 7.2 – 7.4 ± 0.2.
  • Sterilization: Culture media solution is sterilized in the autoclave at 121oC for 15 mins.
  • Dispensing or plate pouring: Sterilized culture media solution should be poured into clean Petri dishes where they will gel, and be used for further studies. Broth or liquid culture media do not gel.
  • Sterility testing: The sterility of poured culture media plates should be determined by leaving the poured plates in the incubator overnight or for 18-24 hrs after gelling. Presence of microbial growth shows that the poured plates are not sterile for microbiological studies.
  • Storage: Poured culture media plates should be stored in the refrigerator until use.

REFERENCES

Atlas R.M (2010). Handbook of Microbiological Media. Fourth edition. American Society of Microbiology Press, USA.

Balows A, Hausler W, Herrmann K.L, Isenberg H.D and Shadomy H.J (1991). Manual of clinical microbiology. 5th ed. American Society of Microbiology Press, USA.

Garcia L.S (2010). Clinical Microbiology Procedures Handbook. Third edition. American Society of Microbiology Press, USA.

Madigan M.T., Martinko J.M., Dunlap P.V and Clark D.P (2009). Brock Biology of Microorganisms, 12th edition. Pearson Benjamin Cummings Inc, USA.

 

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