SOP on Reference culture Maintenance

Work Instruction
Reference  Culture  Maintenance

(Analytical)

Lab  Code

Version No.:                                    

Effective Date:                                

Review Date:                                  

Supersedes:                                   

Author(s):                                        

	Technical Approver	Authorized By
Name
Designation
Signature
Date

1.            Scope

This instruction defines the measures that should be applied for maintaining reference cultures used in the microbiological laboratory.

2.            Procedure

Enter details in Subculture Record (Appendix 1) for every new reference culture procured.

(Note: Reference cultures shall be obtained from recognized national/ international culture collection facilities such as ATCC, MTCC, NCIM, NCDC etc.)

Reference strains in lyophilized form should be revived as per protocol provided by the agency which includes recommended media, incubation temperature etc.

The culture received in lyophilized form is revived and verified for its purity by appropriate tests given in test method.

In case, reference culture is obtained in a slant, it should be subcultured as soon as received.

Revival of cultures

Culture vial/ pellet should be revived by a suitable methodology to get the mother culture (Tier-1). This is further used to prepare reference stock cultures. Specific supplier instruction should be strictly followed for opening freeze dried vials. After revival, the mother culture should be stored as cryogenic beads in a vial inside deep freezer (-20 deg C, for longer storage, typically 2-3 years) or on slants under sterile paraffin (2-8 deg C, for a shorter storage, typically 6-8 months).

Cryogenic beads preparation shall be as follows:

Prepare a suspension of cells or spores of the isolate to be stored, preferably using an 18 to 24 hour old culture grown on solid medium. The colonial growth is suspended in a recommended cryoprotectant liquid, in a cryogenic vial containing about 10-20 of the porous beads.

The suspension is mixed by inversion and then allowed to stand at room temperature for 15-20 minutes. During this period a layer of cells will adhere to the porous beads. The excess cryoprotectant liquid is then removed by aspiration and the vial is immediately transferred to a freezer, at -20^oC.
To resuscitate a culture, remove a single bead quickly and use it to inoculate a solid, non-selective medium. The vial should be resealed and returned to the freezer immediately so that the remainder of the culture is not allowed to thaw.

Stock culture and Working culture

Transfer a loopful from the mother culture in non-selective media slants to obtain the stock cultures (Tier-2). Typically, four stock slants are prepared on which a loopful of culture is transferred and incubated. Transfer from mother culture to stock slant constitutes passage-1. Stock slants should be stored under a layer of sterile paraffin in refrigerator until next use.

Prepare a working culture (Tier-3) from stock slant as and when required (Passage-2). Every subsequent transfer will result in next passage. Maximum of 5 passages are permissible for a working culture derived from a particular stock culture. All cultures including stock shall be disposed immediately after 5^th passage, as per SOP on R&D waste disposal.

Morphological, biochemical, serological verification

Perform morphological, biochemical and serological tests (as per methodology for individual organism) to verify that the culture meets required characteristics as per the test methodology. This should be done at the time of reviving the new culture. Culture purity shall also be checked during every transfer by Gram staining. In the event of non-satisfactory results, the reference culture shall be replaced with the new one.

            Records

Certificates and instruction/product insert from the reference culture collection should be filed in a   ‘Reference Culture File’.

All verification data (biochemical, serological and morphological tests) should be entered in LNB.

Subculture details should be entered in Subculture Record (Appendix 1)

3.            Special Instructions

Biohazard cleanup

In case of accidental leakage or spilling following procedure should be followed. Do not leave the area unattended and divert personnel away from the spillage area. Wear disposable gloves and pick up as much material as possible and place in biohazard bag. Saturate the spill area with germicidal solution for appropriate time. Wipe the area with paper towel and place them in biohazard bag, seal it and dispose them through R&D waste disposal.

Store reference cultures in a dedicated refrigerator only. Never mix the cultures with test samples.

Culture slants & plates should be labeled clearly. Slants/ vials should be tightly closed and sealed with parafilm tape for longer storage.

All sorts of live culture remains should be autoclaved at 121 deg C for 30 minutes before disposal. 

     R&D microbiologist/ DI is responsible for maintenance, storage, and disposal of reference cultures.       

4.            Appendices

Appendix I

Subculture Record: <Culture Name & Strain Number>

Tier & Culture No. *	Date of Transfer	Passage #	Purity / LNB page #	Storage Condition	Signature

(* Cultures are serially numbered with prefix MC for mother culture; SC + sr. no. for stock culture transferred from mother culture and WC + sr. no. for  working culture transferred from stock culture)

5.        Revision History

WI No.	Supersedes No. & Date	Changes made (in brief)

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