Maintenance Of Cultures

• PROCEDURE

Ø      Bacterial 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.

Ø      Prepare 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.

Ø      Boil 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.

Ø      After autoclaving, prepare the solutions by slanting the test tubes to desirable angle.

Ø      After the medium get solidified, keep the prepared slants into the incubators (37°C for 48 hrs.) for pre incubation.

Ø      Prior to sub culturing incubation is necessary because to check for the contamination of slants occurs in process.

Ø      After incubation, clean the exterior surface of test tube with IPA 70% solution.

Ø      Mark each test tube with the name of organism & date of subculture.

Ø      At 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.

Ø      Subculture 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

Ø      Inoculate 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).

Ø      After incubation check the cultures visibly for any contamination.

Ø      Mark the Newly prepared lot of cultures tubes as WORKING CULTURES and one as STOCK CULTURE.

Ø      After 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.

Ø      Prepare new slants from the STOCK CULTURE of previous month ( at each month ,as a new passage) and should proceed from more than four passage.

Ø      After completion of two passages from a single STOCK CULTURE, take the passage from MOTHER    

Ø      CULTURE slant, proceed for the next passage , by preparing STOCK CULTURE and WORKING  CULTURE.

Ø      Enter the date of sub-culturing and passage number in the format as mentioned in Annexure I for sub-culturing of micro-organism. 

Ø      After every one year procure the new culture from any national recognized institution with certificate for   authenticity of the cultures.

Ø      The working cultures shall be used for preparing suspensions for positive control; for Growth Promotion    test etc.

Ø      The STOCK CULTURE shall be used only for sub culturing.

Ø      After one month when the new cultures are ready for use destroy the old cultures.

• DISPOSAL OF CULTURES  :

Ø      Aseptically pipette 10ml of disinfectant solution in the culture tubes.

Ø      Sterilise at 121 °C for 20 mins.

Ø      After sterilization collect the media in the double polybag, pour sufficient disinfectant solution and tie it

Ø      properly. Dispose this bag in Incinerator.

• MEDIA USED FOR PREPARING SLANTS :

Ø      For Bacteria : Soyabean Casein digest Agar.

For Fungi  : Sabaroud Dextrose Agar.