SOP for Microbial Culture Management in a QC Laboratory

In short: This example SOP shows how a pharmaceutical microbiology laboratory can buy, revive, store and subculture reference cultures so that the strains used in growth promotion, method suitability and other tests stay traceable and close to the original reference strain. Adapt the values to your own quality system.

Reference microbial cultures (ATCC, NCTC, NCYC, MTCC and similar collections) are used to check culture media, validate methods and run positive controls. If a strain is subcultured too often, or its identity and purity are not checked, test results lose their meaning. A written culture management procedure prevents this.

This is an illustrative template. Values marked “example in-house criterion” are site practices, not regulatory requirements.

Purpose

To describe the management of microbial cultures in the microbiology laboratory, from procurement to disposal.

Scope

This example SOP applies to the microbiology laboratory and to all reference cultures and their derived seed lot and working cultures.

What the guidance expects

WHO Good practices for pharmaceutical microbiology laboratories (WHO TRS 961, Annex 2, 2011), section 6.2 on reference cultures says, in summary:

• Reference cultures are needed to show acceptable media performance, to validate methods and to check that test methods remain suitable.
• Reference strains may be subcultured once to make reference stocks, stored as deep-frozen or lyophilised aliquots; thawed stocks must not be refrozen.
• Working cultures for routine use should be primary subcultures of the reference stock and should not normally be subcultured further.
• Usually not more than five generations (passages) from the original reference strain can be subcultured, and only if a standard method defines this or the laboratory has documentary evidence that no relevant property has changed.
• Commercial derivatives of reference strains may only be used as working cultures.

The United States Pharmacopeia general chapter <1117> Microbiological Best Laboratory Practices and the chapters on microbial tests also discuss culture handling; this article does not reproduce them, so check the current edition you hold.

Responsibilities

• Microbiologist: procures, revives, subcultures, labels, checks and discards cultures and keeps the records.
• Head of Microbiology: approves the culture list and the subculture schedule.
• Quality Assurance: reviews the SOP and the records.

Procedure

1. Procurement of cultures

1. List the cultures needed (collection name, strain number, use) in the list of microbial cultures (Annexure 1).
2. Raise a purchase requisition. Buy only from a recognised culture collection or authorised distributor, with a certificate that states the strain, passage number and permissible subculturing period.
3. Example in-house criterion: procure cultures once a year.
4. Example in-house criterion: accept a culture only if it is not more than two passages away from the reference strain, as stated on the certificate.
5. On receipt, record the details and an in-house identification number in the culture inward record (Annexure 2). Example: E. coli received on 01/10/20 is numbered “E.coli 011020”.
6. Store the culture at 2–8 °C, or as the supplier recommends, until it is revived.

2. Reconstitution of freeze-dried cultures

1. Disinfect the outside of the ampoule, vial, slant or loop with 70% IPA.
2. Open the culture aseptically in a laminar air flow unit or biosafety cabinet.
3. Reconstitute with 0.5–1.0 ml of sterile water or as the supplier instructs. This is the mother culture.
4. Record the details in the culture maintenance record (Annexure 3).

3. Revival and preparation of the seed lot

1. Streak the mother culture on an agar plate to confirm purity (see the isolation and identification procedure, Annexure 4). Where an automated identification system is used, identification may be done that way.
2. At the same time, inoculate the whole content of the vial or ampoule into about 100 ml of sterile soybean casein digest medium. The plate is for the purity check; the broth is for the seed lot.
3. Discard the remaining vial contents under the laboratory’s culture disposal procedure and record disposal (Annexure 8).
4. Incubate according to the culture type (example in-house criteria, to be aligned with your pharmacopoeial method):

5. After incubation, confirm purity and identity by colony characteristics, microscopy, staining and biochemical tests, or by an automated identification system.
6. Mix the culture with sterile 10% glycerol (1:1) and dispense 2–3 ml into sterile cryovials. Example in-house criterion: prepare 14 vials. These form the seed lot culture (SLC).
7. Number the vials SLC-1, SLC-2 and so on, and store at −30 °C or colder (example in-house criterion).
8. Label each vial (Annexure 6) with: name of culture, strain number, passage number, culture ID, date of subculturing, name of the person who subcultured, and use-before date.
9. Example in-house criterion: use 12 vials, one per month, over 12 months, and keep 2 as spare stock in case of breakage or spillage. Do not use vials after one year. Discard the spare vials at the end of the year.

4. Subculturing to working cultures

1. Follow the subculturing flow chart (Annexure 5).
2. In the first month, take one SLC-1 vial and streak five agar slants, labelled SLC-1WC-1 to SLC-1WC-5. Streak a plate in parallel for the purity check.
3. Incubate slants and plates as in the table above.
4. When growth is satisfactory, discard the SLC vial and record it (Annexure 8).
5. Label each working culture slant with the name of the culture, strain number, passage number, seed lot number, culture ID, date of subculturing, name of the person who subcultured and use-before date (Annexure 7). Store at 2–8 °C.
6. Example in-house criterion: use one working culture per week for routine work and discard it at the end of that week. Discard the fifth slant at month end if unused, and record it.
7. Start the next seed lot vial (SLC-2, then SLC-3 and so on) before the current cycle ends, so working cultures are always available.
8. Check purity of the seed lot and working cultures when used (Annexure 4).
9. Record every subculturing step (Annexure 3) and prepare the maintenance schedule when a new culture is first revived (Annexure 9).

5. Passage control

Count passages from the original reference strain. The mother culture, seed lot and working cultures are each one step further away. In line with WHO TRS 961 Annex 2 (section 6.2.3), the laboratory should usually not go beyond five passages from the reference strain, and should use that many only if a standard method defines it or documentary evidence shows that no relevant property has changed. Never subculture from a working culture.

6. Handling live cultures

• Wear gloves.
• Keep live cultures away from the areas used for sample testing, preferably in a separate area and inside a biosafety cabinet.
• Do positive control dilutions and inoculations in a biosafety cabinet.
• Seal agar plates with fungal growth with paraffin film to stop spores spreading.
• Disinfect surfaces immediately after any live culture plate, tube or bottle has been opened there, using an approved disinfectant and its stated contact time.
• Treat used media and cultures as biohazardous waste and decontaminate (for example by autoclaving) before disposal.

7. Records

Culture inward records, maintenance records, purity checks, labels and disposal records are GMP records. Keep them at least 1 year after the expiry date of the respective product batch they support (EU GMP Part I, Chapter 4, section 4.11; 21 CFR 211.180(a)), or longer if local law or the marketing authorisation requires.

Annexures

1. List of microbial cultures
2. Microbial culture inward record
3. Culture maintenance record
4. Purity check of microbial culture
5. Schematic flow for subculturing
6. Seed lot culture label
7. Working culture label
8. Culture disposal record
9. Schedule for maintenance of microbial cultures

Abbreviations

Frequently asked questions

Why are passages from the reference strain limited?

Each subculture gives a chance for genetic drift, contamination or loss of properties such as growth characteristics. Limiting passages keeps test strains representative of the reference strain.

What is the difference between a seed lot and a working culture?

A seed lot is a frozen stock made from the revived mother culture, kept to supply working cultures over time. A working culture is a slant or plate made from the seed lot for routine use and discarded after a short period.

Can a working culture be subcultured again?

Normally no. WHO guidance says working stocks should not normally be subcultured; make a fresh working culture from the seed lot instead.

Where should live cultures be handled?

Away from product testing areas, in a biosafety cabinet, by trained staff wearing gloves. See Good Microbiological Laboratory Practice and Preparation of Culture Dilution.

See also

• SOP for Good Microbiological Laboratory Practice
• SOP on Preparation of Culture Dilution
• SOP on Cleaning of Incubators

References

1. World Health Organization. Good practices for pharmaceutical microbiology laboratories, WHO Technical Report Series No. 961, Annex 2 (2011), sections 6.2 and Appendix 5.
2. EU GMP Part I, Chapter 4 (Documentation), section 4.11 — record retention.
3. US FDA, 21 CFR 211.180(a) — record retention.
4. United States Pharmacopeia, general chapter <1117> Microbiological Best Laboratory Practices (paywalled; not reproduced here).