PREREQUISITE FOR PREPARATION OF CULTURE MEDIA

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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. The factors to be considered and taken into consideration in the preparation of culture media are as follows:  

  1. Weighing: Powdered agar should be properly weighed (in grams) using the weighing balance.
  2. Dissolving & heating: Weighed powdered agar should be dissolved in the correct volume of distilled water and then heated for some minutes prior to sterilization. 
  3. 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.
  4. Sterilization: Culture media solution is sterilized in the autoclave at 121oC for 15 mins.
  5. 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. 
  6. 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.  
  7. Storage: Poured culture media plates should be stored in the refrigerator until use.  

GENERAL GUIDELINE FOR THE PREPARATION OF CULTURE MEDIA IN THE MICROBIOLOGY LABORATORY

The protocol involved in culture media preparation is as follows:  

  1. Measure out the correct amount of distilled water required for the culture media preparation using a measuring cylinder. The water should be dispensed into a conical flask, beaker, tubes or bottles depending on the choice of the microbiologist. For example, 400 ml of distilled water is required to prepare 400 ml of nutrient agar solution; and this should be measured and dispensed into a clean conical flask or beaker.
  2. Measure out the appropriate amount of the powdered agar (in grams) using a weighing balance. Most culture media powder is hygroscopic in nature, and they absorb moisture from the atmosphere when left open to form a solid mass. The container of culture media powder should always be covered to prevent this from happening.   
  3. Dispense the measured powdered agar into the correct amount of distilled water in a conical flask. Shake the conical flask properly after adding the powdered media to the distilled water. Caution: Powdered agar should always be added to water and not the other way round. The reason for this is to ensure proper dissolution of the powdered agar and to avoid burning when heating.
  4. The dissolved powdered agar solution should be boiled under a Bunsen burner flame for complete dissolution; and boiling or heating should not last too long (3-5 minutes is allowable) in order to avoid burning or charring of the agar. 
  5. Twenty mill (20 ml) each of the dissolved heated agar solution should be dissolved into clean capped bottles (e.g. McCartney bottles). In some cases, heated agar solution in conical flask can be sterilized directly without dispensing into capped bottles.
  6. Sterilize the agar solution in the autoclave as recommended. Sterilization of culture media solution is usually carried out at 121oC for 15 mins and at 15 psi (psi=pounds per square inch). Caution: Allow the indicator of the autoclave to fall back to zero before opening the equipment.
  7. Sterilized culture media solution should be allowed to cool to about 45-50oC before pouring plate. At this temperature, the prepared agar solution can be poured into clean Petri dishes as the case may be; and this should be done around Bunsen burner flame which provides a sterile environment for pouring plates. 
  8. Poured plates should be allowed on the bench for solidification or gelling of the medium. Broth or liquid culture medium does not gel because they lack agar (the solidifying agent in culture media). 
  9. Poured culture media plates should be macroscopically observed for the presence of microbial growth after gelling. A sterile plate is one that is free of any form of microbial growth after pouring plate; and such plates are ready for further microbiological analysis. Caution: Prepared culture media plates should be properly labeled and stored in the refrigerator until they are ready for use.

Further reading

Brooks G.F., Butel J.S and Morse S.A (2004). Medical Microbiology, 23rd edition. McGraw Hill Publishers. USA.

Goldman E and Green L.H (2008). Practical Handbook of Microbiology, Second Edition. CRC Press, Taylor and Francis Group, 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.

Mahon C. R, Lehman D.C and Manuselis G (2011). Textbook of Diagnostic Microbiology. Fourth edition. Saunders Publishers, USA.

Patrick R. Murray, Ellen Jo Baron, James H. Jorgensen, Marie Louise Landry, Michael A. Pfaller (2007). Manual of Clinical Microbiology, 9th ed.: American Society for Microbiology.

Wilson B. A, Salyers A.A, Whitt D.D and Winkler M.E (2011). Bacterial Pathogenesis: A molecular Approach. Third edition. American Society of Microbiology Press, USA.

Woods GL and Washington JA (1995). The Clinician and the Microbiology Laboratory. Mandell GL, Bennett JE, Dolin R (eds): Principles and Practice of Infectious Diseases. 4th ed. Churchill Livingstone, New York.

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