Procurement and maintenance of Microbial cultures.

1.0 OBJECTIVE

To describe the procedure procurement and maintenance of Microbial cultures.

2.0 SCOPE

This procedure is applicable the procedure for procurement and maintenance of Microbial cultures in Microbiology Lab.

3.0 RESPONSIBILITY

3.1 Microbiologist /Officer is responsible for the execution. 

3.2 HOD or designee is responsible for review, effective implementation and compliance to the procurement and maintenance of culture for execution.         

4.0 PROCEDURE 

4.1 PROCUREMENT

4.1.1 All new and existing cultures procured from American Type Culture Collection(ATCC) or National Collection of Industrial Microorganism(NCIM)  or from any of the authorized sources 

4.1.2 Cultures shall be procured once in a year or as and when required.

4.1.3 Upon receipt, the culture tube shall be checked physically for its intactness and then immediately stored in refrigerator between 2 - 8ºC

6.1.4 Damaged tube shall be discarded immediately as per the current version of ref SOP.

4.2 INITIATION

4.2.1 Prepare the media required, as per the current version of SOP.

4.2.2 Aseptically open the container containing the culture [usually in the form of a bead/ slants] and transfer it to the nutrient medium.

4.2.3 Label the container / test tube with the name of the culture, strain no. and date of   transfer. Incubate the above tubes at 30-35ºC for 48 hrs for bacterial cultures and 20-25ºC for 120 hrs for fungal cultures.

4.2.4 After incubation check for the purity of the culture through simple gram staining techniques.

4.2.5 Record the date of initiation of culture in attachment #1

4.3 PREPARATION OF STOCK (seed lot) CULTURE

4.3.1 Aseptically, transfer sterile normal saline in the respective culture slants and harvest it to get the culture suspension.

4.3.2 Prepare 20% of glycerol, take 80 ml of water and 20 ml of glycerol and sterilize it. 

4.3.3 Add 1:1 ratio of culture suspension to 20% glycerol to get final suspension with 10% of glycerol for each culture.  

4.3.4 Make a seed lots like A, B, C and so on to get 12 lots, according to the requirement. 

4.3.5 Label the tubes with: Name of culture, Strain no., Culture Lot No., Date of transfer, transferred by and valid up to.  

4.3.6 Preserve this suspension in Deep freezer for preservation and use them whenever required.

4.4 MAINTENANCE OF CULTURE

4.4.1 Subculture the individual seed lot culture for further use as A1L and A1S on respective nutritive agar slants.

4.4.2 Where, A1L is made available for Laboratory use and A1S is kept under refrigeration for storage to be used for subsequent transfers. Incubate the culture tube at the appropriate temperature and for the requisite time period. 

4.4.3 Proceed with subcultures monthly until four transfers are made from the original seed lot A as shown in flow chart as per attachment # 1

4.4.4 Start the entire procedure using seed lot B and so on; until seed lot culture stock have been used.

4.4.5 Record the date of preparation of stock culture in Culture maintenance record as per Attachment #2

4.4.6 Whenever required, or once a month until four transfers made from the original seed lot culture.

4.4.7 Culture Maintenance flow chart as per the attachment # 3