Fundamentals of Microbiology (2+2)

Procedure

B. Sterilization of water (or) heat labile solutions

Sterilize all glasswares (including suction flask, glass filter assembly), seitz filter assembly, forceps etc. Set the filtration assembly by setting the rubber cork of the filter apparatus tightly set on the suction flask. Connect the suction flask to the vacuum pump. Aseptically place the sterile membrane filter over the platform in the filter assembly and tightly clamp it using holding clamp is case of glass-filter assembly or tighten the assembly in case of seitz filter. Pour known volume of water in the funnel or beaker of the filter assembly and filter under vacuum. The filtered water is sterile.

Sterility test

Aseptically transfer 10 ml of filtered water into the flask containing 90 ml of sterile TSB. Incubate for 24 h and observe for turbidity. Clear broth indicates that the filtered water is sterile. Growth of microorganisms is indicated by turbidity.

Enumeration of bacteria

Keep the media poured and dried in petri dishes. Filter 200 ml of tap water as per the procedure mentioned above. After filtration, aseptically remove the filter from the assembly and place it on EMB agar for estimating the E.coil in the water sample. Incubate for 24 to 48 h and look for growth of microorganisms. Count the number of colonies and express as the cfu/ml or 100 ml of water sample.

EMB medium is a selective medium for Escherichia coli. Colonies appearing purple with green metallic sheen are counted as E.coli.