in non-sterile pharmaceutical products, in accordance with pharmacopeial guidelines (USP, IP, EP).

2. Scope

This SOP applies to the Analytical Method Development (AMD) department for all activities related to establishing and validating methods for Total Aerobic Microbial Count (TAMC), Total Yeast and Mold Count (TYMC), and specified microorganism testing.

3. Responsibilities

• Microbiologist: Develops and executes microbial limit test methods, analyzes results, and prepares reports.
• Analytical Scientist: Coordinates with microbiologist and supports method development integration.
• QA Officer: Ensures method development records comply with GMP and regulatory expectations.
• Head – AMD: Reviews and approves final test methods and protocols for validation or transfer.

4. Accountability

The Head of Analytical Method Development is accountable for ensuring compliance with microbiological testing standards and the integrity of method development outcomes.

5. Procedure

5.1 Reference Guidelines

1. Review USP <61> and <62>, IP, and EP requirements for:
   • TAMC and TYMC limits
   • Specified pathogens: E. coli, Salmonella spp., S. aureus, P. aeruginosa, C. albicans
2. Determine sample-specific test requirements based on dosage form and excipient type.

5.2 Selection of Test Method

1. Choose one or both of the following methods based on sample characteristics:
   • Membrane Filtration: For aqueous solutions and large volumes
   • Plate Count (Pour/Spread): For solid, viscous, or non-filterable products
2. Record method selection rationale in Annexure-1: Method Selection Log.

5.3 Growth Medium Preparation

1. Prepare and sterilize the following media per manufacturer’s instructions:
   • Plate Count Agar (PCA) – for TAMC
   • Sabouraud Dextrose Agar (SDA) – for TYMC
   • MacConkey Agar, XLD Agar, Mannitol Salt Agar, Cetrimide Agar – for pathogen screening
2. Document batch number and expiry date of media in Annexure-2: Media Preparation Record.

5.4 Sample Preparation and Dilution

1. Prepare a 1:10 dilution of the test sample using sterile diluent (0.1% peptone water or sodium chloride).
2. Use neutralizers (e.g., lecithin, polysorbate 80) if the sample contains antimicrobial agents.
3. Further dilute as required to ensure countable colonies (30–300 CFU/plate).
4. Record preparation steps in Annexure-3: Sample Dilution Log.

5.5 Inoculation and Incubation

1. Inoculate media plates or filters with appropriate volumes (1 mL or 100 mL).
2. Incubate under conditions:
   • TAMC: 30–35°C for 3–5 days
   • TYMC: 20–25°C for 5–7 days
3. For specified pathogens, perform enrichment and selective plating steps as per pharmacopeia.
4. Document conditions and timings in Annexure-4: Incubation and Observation Log.

5.6 Enumeration and Interpretation

1. Count colonies manually or using a colony counter.
2. Calculate CFU/g or CFU/mL using dilution factor.
3. Confirm absence/presence of specified pathogens by colony characteristics and confirmatory tests (e.g., biochemical, gram staining).
4. Record findings in Annexure-5: MLT Result Summary Sheet.

5.7 Acceptance Criteria

1. Verify test results comply with the following pharmacopeial limits:
   • TAMC: ≤ 10³ CFU/g or mL
   • TYMC: ≤ 10² CFU/g or mL
   • Specified microorganisms: Absent in 1 g or 10 g (depending on product type)
2. Any deviation must be investigated and reported to QA.

6. Abbreviations

• MLT: Microbial Limit Test
• TAMC: Total Aerobic Microbial Count
• TYMC: Total Yeast and Mold Count
• CFU: Colony Forming Units
• SOP: Standard Operating Procedure

7. Documents

1. Method Selection Log – Annexure-1
2. Media Preparation Record – Annexure-2
3. Sample Dilution Log – Annexure-3
4. Incubation and Observation Log – Annexure-4
5. MLT Result Summary Sheet – Annexure-5

8. References

• USP <61> – Microbial Enumeration Tests
• USP <62> – Tests for Specified Microorganisms
• IP and EP Microbiological Guidelines
• ICH Q6A – Specifications

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name
Designation
Department

11. Annexures

Annexure-1: Method Selection Log

Sample ID	Dosage Form	Method Chosen	Justification
MLT-119	Tablet	Plate Count	Non-aqueous, not suitable for filtration

Annexure-2: Media Preparation Record

Media	Batch No.	Expiry Date	Prepared By
PCA	PCA0525	05/2026	Sunita Reddy

Annexure-3: Sample Dilution Log

Sample ID	Diluent Used	Dilution Factor	Neutralizer
MLT-119	0.1% Peptone Water	1:10	Polysorbate 80

Annexure-4: Incubation and Observation Log

Plate Type	Incubation Temp	Duration	Observation Date
PCA	32°C	5 days	18/05/2025

Annexure-5: MLT Result Summary Sheet

Test	Result	Limit	Status
TAMC	120 CFU/g	≤ 1000 CFU/g	Pass
TYMC	10 CFU/g	≤ 100 CFU/g	Pass
E. coli	Absent	Absent	Pass

Revision History:

Revision Date	Revision No.	Details	Reason	Approved By
04/05/2025	2.0	Incorporated USP <62> specified organism workflows	Regulatory update