Preparation of media: (Thioglycollate & Sabouraud Dextrose)
Prepare as directed in the label of the manufacturer. Transfer 15-20 ml to each 50 ml flat bottom flask (about 10 flask taken) and introduce a cotton plug to each, wrapping with paper and fasten the neck with thread. Sterilize in an autoclave at 1210c/15 lb pressure for 20 minutes. After autoclaving place them in front of LAFB. After achieving the room temperature, incubate for 24-48 hours and then store for use if no evidence of contamination is found.
Filtration Method (Pore Size 0.2 µm): Prepare each membrane filter as follows. Transfer a small quantity (sufficient to moisten the membrane filter) of sterile diluent of 0.1% w/v neutral solution of meat peptone. Sample taken 2 ml in 100 ml sterile 0.1% w/v neutral solution and filter through the membrane filter and further pass through the membrane filter about 150 ml sterile diluent in such a manner that the effect of the antibiotic is neutralized in a greater magnitude. The filtration apparatus and membrane are sterilized by appropriate means. The preparation being tested is transferred through a membrane filter paper. The membrane is washed at least three times by filtering through it each time with the sterile dilute isopropyl myristate.
The membrane is cut aseptically into two equal parts and transfer one half to each of the media i.e. Thioglycollate broth & Sabouraud dextrose broth. (also perform a control test without the sample). Incubate at 30-350c for bacteria (or 20-250c for fungi) for 7 days. Observe the culture media several times during the incubation period. At intervals during the incubation period and its conclusion, examine the media for microscopic evidence of microbial growth.
When the material being tested render the media turbid so that the presence or absence of microbial growth can not be determined readily by visual examination 7 days after the incubation started, transfer suitable portions of the cultured media to fresh vessel of the same medium. Continue incubation of the original and transferred vessel for a total 14+7 = 21 days from the original incubation. If no evidence of microbial growth is found, the product being examined complies with the test for sterility. If evidence of microbial growth is found (turbid solution), the product being examined does not comply with the test for sterility.
PRECAUTION:
I. Before entering the working room wash the hands and mouth and dry in fresh air.
II. Swab the hands with sterile 70% isopropyl alcohol and put on mask & sterile hand gloves.
III. During working infront of LAFB, the UV light must be off.
I want to know from which source the above method has been taken
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