Diagnostic Laboratory Section

Preparation of medium

PREPARATION OF CULTURE MEDIUM

• Take clean sterilized conical flask of appropriate capacity to prepare Muller Hinton (MH) agar medium.
• Weigh the appropriate quantity of agar and add distilled water as per the manufacturers instructions and autoclave at 121°C at 15 pound pressure for 15 minutes.
• Allow the agar to cool in a water bath to 45-50° C.
• Pour the cooled agar (bearable heat) into petridishes on a level, horizontal surface to give a uniform depth of 4 mm.
• 60 to 70 ml of medium in 140 mm petridish.
• 25 to 30 ml of medium in 90 mm petridish.
• Allow the agar to solidly and pack the plate in an inverted position in plastic bags or in aluminum foil and store them in the refrigerator until use. Stored plates should be used within 7 days of preparation unless necessary precautions have been taken to avoid evaporation. Test each batch of plates for sterility by keeping in a few of them in an incubator for 24 hours or more.
• The pH of medium should be 7.2 to 7.4.
• Each new batch medium should be tested for presence of thymine and thymidine using Enterococcus faecalis (ATCC 29212) and the satisfactory level of divalent cations using Pseudomonas aeruginosa(ATCC 27853). The zone of inhibition must be confirmed to the control limits given in the standards.