SOP on Maintenance and Preparation of Anaerobic Culture Suspension

In short: This example SOP shows how a microbiology laboratory maintains Clostridium sporogenes and prepares, by serial dilution, a culture suspension that delivers 10 to 100 CFU per 100 µL inoculum, for use in microbial limit testing. It is a template for microbiologists; adapt it to your own quality system and pharmacopoeial method.

1. Purpose and scope

To describe the maintenance of anaerobic test organism cultures and the preparation of culture suspension by the culture dilution method in the Microbiology department. The example uses Clostridium sporogenes, the organism named for the test for Clostridia in the harmonised pharmacopoeial chapters (USP <62>, Ph. Eur. 2.6.13). The harmonised chapters name C. sporogenes such as ATCC 11437 (NBRC 14293, NCIMB 12343, CIP 100651) or ATCC 19404 (NCTC 532 or CIP 79.3). Record the strain your laboratory holds.

General culture management: see SOP for microbial culture management and good microbiological laboratory practice.

2. Maintenance of the culture

1. Prepare the agar medium as per the media preparation SOP.
2. Dispense 5 mL into clean 18 mm rimless test tubes, plug with cotton, wrap the plug in paper and label with medium type, autoclave lot number and sterilisation date.
3. Sterilise by steam using the validated autoclave cycle.
4. Place the tubes under laminar airflow at about 30 degrees from the horizontal and let the medium set as slants.
5. Incubate a sample of the slants at 30 to 35 °C for 48 hours to check for contamination.
6. Streak the slant surface with the culture and incubate anaerobically at 30 to 35 °C for 72 hours.
7. Check growth and perform Gram staining.
8. Maintain the culture as per the culture maintenance annexure, using a seed-lot system. The harmonised chapters require that organisms used for inoculation are not more than five passages removed from the original master seed lot.

3. Preparation of the culture suspension

3.1 Media and diluent

1. Reconstitute Reinforced Medium for Clostridia and Columbia Agar in conical flasks with purified water, and dissolve completely (for example, in a boiling water bath).
2. Dispense 15 mL of medium into 18 mm rimless test tubes where needed, plug, wrap and label with medium type, autoclave lot and date.
3. Prepare 0.9% w/v sodium chloride solution as diluent and fill 9 mL into test tubes. Plug, wrap, label and steam sterilise by the validated cycle.
4. Remove the tubes from the autoclave and move the culture slant and dilution tubes to the microbial limit test room.

3.2 Harvest and dilution

1. Wash and scrape the slant surface with a sterile loop into 10 mL of 0.9% saline. Transfer to a sterile tube and vortex to make a uniform suspension.
2. Make ten-fold serial dilutions: transfer 1 mL into 9 mL of sterile saline (10^-1), then repeat 1 mL into 9 mL stepwise through 10^-2, 10^-3, 10^-4, 10^-5, 10^-6 and 10^-7.
3. For C. sporogenes dilutions, add 1 mL of immersion oil on top of the saline (example in-house practice to reduce air contact).
4. Plate 1 mL from each of the last three dilutions in duplicate into sterile Petri dishes by the pour plate method with Columbia Agar.
5. Incubate the plates anaerobically at 30 to 35 °C for 72 hours (within the 48 to 72 hours the harmonised chapters give for Columbia agar).
6. Keep all dilution tubes at 2 to 8 °C until counts are available.
7. Count colonies and record on the enumeration record. Identify the dilution giving 10 to 100 CFU per mL.

3.3 Selecting and using the working inoculum

• Find the dilution whose 1 mL plate gives 10 to 100 CFU. Preserve the tube one step less dilute as the working inoculum: it holds about 100 to 1000 CFU per mL.
• Use 100 microlitres of that suspension for testing, which delivers about 10 to 100 CFU, within the harmonised limit of not more than 100 CFU.
• Example: if the 10^-6 plate gives 10 to 100 CFU, preserve the 10^-5 tube and use 100 µL of it.
• As an alternative to a fresh vegetative suspension, the harmonised chapters allow a stable spore suspension of C. sporogenes, kept at 2 to 8 °C for a validated period.

3.4 Labelling and records

Label each tube with SOP number, organism name, strain number, dilution and due date. Keep an enumeration record for the anaerobic suspension, a culture maintenance record and an inoculum preparation record. Retain these records at least 1 year after the expiry date of the respective product batch tested (EU GMP Part I Chapter 4 §4.11, or 5 years after QP certification if longer; 21 CFR 211.180(a)), longer if local law or the marketing authorisation requires.

Discard used cultures and plates only after decontamination by validated autoclaving, following your site’s biosafety and waste procedure.

Frequently asked questions

Why is a layer of oil added over the saline? This example procedure adds it to reduce air contact with the anaerobic dilutions. The pharmacopoeial chapters do not require it.

Why aim for 10 to 100 CFU per inoculum? Pharmacopoeial growth promotion and method suitability tests call for a small inoculum of not more than 100 CFU, so the suspension is diluted to deliver a countable number below that limit.

Do I need to repeat the count each time? Follow your site’s validated approach: a freshly enumerated suspension is used, or a stored one within its defined validity.

References

1. USP General Chapter <62> Microbiological Examination of Nonsterile Products: Tests for Specified Microorganisms (current official version).
2. European Pharmacopoeia, 2.6.13 Microbiological Examination of Non-sterile Products: Test for Specified Micro-organisms (current edition).
3. Japanese Pharmacopoeia, 4.05 Microbiological Examination of Non-sterile Products, II. Tests for specified micro-organisms (harmonised text, published by PMDA).
4. EU GMP Part I Chapter 4 §4.11; 21 CFR 211.180(a).