Procedure for Microbial Limit Testing of Ointments
| Department | Quality Control (QC)/Microbiology/Quality Assurance (QA) |
|---|---|
| SOP No. | SOP/Ointment/063 |
| Supersedes | V 1.0 |
| Page No. | Page X of Y |
| Issue Date | [Insert Issue Date] |
| Effective Date | [Insert Effective Date] |
| Review Date | [Insert Review Date] |
1. Purpose
The purpose of this Standard Operating Procedure (SOP) is to establish a standardized method for microbial limit testing of ointments to ensure compliance with microbiological safety standards and Good Manufacturing Practices (GMP).
2. Scope
This SOP applies to all personnel in the Quality Control (QC) and Microbiology departments responsible for conducting microbial limit testing of ointments during formulation development, in-process control, and final batch release.
3. Responsibilities
- Microbiologist: Conducts microbial limit testing and records results.
- Quality Control Analyst: Reviews and compiles test results.
- Quality Assurance Personnel: Verifies and approves microbial limit test reports.
- Production Supervisor: Ensures that the ointment batches conform to microbial safety requirements.
4. Accountability
The QC, Microbiology, and QA Managers are accountable for ensuring that microbial limit tests are conducted accurately and documented as per regulatory and GMP standards.
5. Procedure
5.1 Equipment and Materials
- Sterile sampling containers
- Sterile diluents (e.g., phosphate buffer, peptone water)
- Nutrient agar, MacConkey agar, Sabouraud dextrose agar
- Sterile pipettes and micropipettes
- Incubators set at 30°C – 35°C for bacteria and 20°C – 25°C for fungi
- Laminar airflow hood
- Sterile filtration system (if required)
- Colony counter
5.2 Sample
Preparation
- Collect the sample aseptically in a sterile container.
- Weigh 10 g of the ointment sample.
- Mix with 90 mL of sterile phosphate buffer or other suitable diluent to create a 1:10 dilution.
- Further dilutions (1:100, 1:1000) may be prepared based on the microbial limit specifications.
5.3 Test Methods
5.3.1 Total Aerobic Microbial Count (TAMC)
- Transfer 1 mL of diluted sample onto sterile nutrient agar plates.
- Spread evenly using a sterile glass spreader.
- Incubate plates at 30°C – 35°C for 48 – 72 hours.
- Count the number of colonies and record the results in CFU/g.
5.3.2 Total Yeast and Mold Count (TYMC)
- Transfer 1 mL of diluted sample onto sterile Sabouraud dextrose agar plates.
- Spread evenly and incubate at 20°C – 25°C for 5 – 7 days.
- Count the yeast and mold colonies and record the results in CFU/g.
5.3.3 Pathogen Testing
- For detection of Escherichia coli:
- Transfer 1 mL of diluted sample onto MacConkey agar plates.
- Incubate at 30°C – 35°C for 24 – 48 hours.
- Check for lactose fermenting colonies.
- For detection of Staphylococcus aureus:
- Transfer 1 mL of diluted sample onto Mannitol salt agar.
- Incubate at 30°C – 35°C for 24 – 48 hours.
- Check for yellow colonies indicating S. aureus.
- For detection of Pseudomonas aeruginosa:
- Transfer 1 mL of diluted sample onto cetrimide agar.
- Incubate at 30°C – 35°C for 24 – 48 hours.
- Check for greenish-blue colonies.
5.4 Acceptance Criteria
- Total Aerobic Microbial Count (TAMC) should not exceed 1000 CFU/g.
- Total Yeast and Mold Count (TYMC) should not exceed 100 CFU/g.
- Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa must be absent in 1 g of sample.
5.5 Documentation
- Record all microbial counts in the Microbial Limit Test Log.
- Document incubation conditions, sample preparation, and culture media used.
- QA personnel must review and approve results before batch release.
6. Abbreviations
- GMP – Good Manufacturing Practices
- QA – Quality Assurance
- QC – Quality Control
- TAMC – Total Aerobic Microbial Count
- TYMC – Total Yeast and Mold Count
- CFU – Colony-Forming Units
7. Documents
- Microbial Limit Test Log (Annexure-1)
- Ointment Batch Microbial Report (Annexure-2)
8. References
- WHO GMP Guidelines for Pharmaceutical Manufacturing
- USP <61> – Microbial Enumeration Tests
- USP <62> – Tests for Specified Microorganisms
- ICH Q2 (R1) – Validation of Analytical Procedures
9. SOP Version
Version 2.0
10. Approval Section
| Prepared By | Checked By | Approved By | |
|---|---|---|---|
| Signature | |||
| Date | |||
| Name | |||
| Designation | |||
| Department |
11. Annexures
Annexure-1: Microbial Limit Test Log
| Date | Batch Number | TAMC (CFU/g) | TYMC (CFU/g) | Pathogen Results | Verified By |
|---|---|---|---|---|---|
| 01/02/2025 | OINT-101 | 800 | 50 | Absent | QA Officer |
| 02/02/2025 | OINT-102 | 950 | 60 | Absent | QA Officer |