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ØBacterial cultures are very sensitive and if not sub-cultured they can change their morphological & biochemical characteristics. Sub-culturing or periodic transfer is a very delicate technique which has to be carried out by avoiding chances of contamination.
ØPrepare the media Prepare slants in clean 18 mm diameter rimless test tubes and pre-incubate for 48 hrs at 30-35°C to check any contamination.
ØBoil the media till get completely dissolve. Cool to about 42 - 45°C and adjust the required pH. Fill each test tube with about 9 to 10ml of the medium & plug the tubes with non absorbent cotton & sterilize by autoclaving.
ØAfter autoclaving, prepare the solutions by slanting the test tubes to desirable angle.
ØAfter the medium get solidified, keep the prepared slants into the incubators (37°C for 48 hrs.) for pre incubation.
ØPrior to sub culturing incubation is necessary because to check for the contamination of slants occurs in process.
ØAfter incubation, clean the exterior surface of test tube with IPA 70% solution.
ØMark each test tube with the name of organism & date of subculture.
ØAt every passage from mother culture check the Gram character and morphology of the culture. In any case the passage from mother culture shall not exceed 5.
ØSubculture should be done in the Laminar flow clean air station & in between the two gas burners to avoid the chances of cross contamination. Take a loop full of culture from previous stock cultures or mother culture which ever is applicable, and inoculate in freshly prepared sterile slant
ØInoculate each organism in two slants ( prepare two slants of each organism). After inoculating ,incubate all the tubes at required temperature(i.e. 30-35°C for bacteria and 20-25°C for fungus).
ØAfter incubation check the cultures visibly for any contamination.
ØMark the Newly prepared lot of cultures tubes as WORKING CULTURES and one as STOCK CULTURE.
ØAfter observing the growth, keep the newly prepared cultured tubes in clean double plain polythene bags and preserved the tubes in the refrigerator at 2 – 8 °C.
ØPrepare new slants from the STOCK CULTURE of previous month ( at each month ,as a new passage) and should proceed from more than four passage.
ØAfter completion of two passages from a single STOCK CULTURE, take the passage from MOTHER
ØCULTURE slant, proceed for the next passage , by preparing STOCK CULTURE and WORKING CULTURE.
ØEnter the date of sub-culturing and passage number in the format as mentioned in Annexure I for sub-culturing of micro-organism.
ØAfter every one year procure the new culture from any national recognized institution with certificate for authenticity of the cultures.
ØThe working cultures shall be used for preparing suspensions for positive control; for Growth Promotion test etc.
ØThe STOCK CULTURE shall be used only for sub culturing.
ØAfter one month when the new cultures are ready for use destroy the old cultures.
DISPOSAL OF CULTURES :
ØAseptically pipette 10ml of disinfectant solution in the culture tubes.
ØSterilise at 121 °C for 20 mins.
ØAfter sterilization collect the media in the double polybag, pour sufficient disinfectant solution and tie it