yeasts and molds count (TYMC), and absence of specified pathogens as per pharmacopeial requirements.

2. Scope

This SOP is applicable to the Analytical Method Development (AMD) and Quality Control (QC) departments for evaluating the microbiological quality of cream formulations during development, batch release, and stability studies.

3. Responsibilities

• Microbiologist: Prepares media, performs microbial enumeration, records and interprets results.
• QC Analyst: Collects and prepares samples for microbial analysis.
• QA Executive: Ensures compliance with regulatory standards and approves the test protocol and data.

4. Accountability

The Head of Analytical Method Development is accountable for the suitability and regulatory compliance of the microbial load testing method and its proper validation.

5. Procedure

5.1 Sample Collection and Preparation

1. Use aseptic technique to collect representative samples from at least three points (top, middle, bottom) of the batch container.
2. Weigh 10 g of cream into a sterile container.
3. Dilute with 90 mL of sterile diluent (e.g., phosphate buffer pH 7.2 or peptone water).
4. Homogenize the sample using a vortex mixer or homogenizer for 5 minutes.

5.2 Media and Reagents

• Plate Count Agar (PCA) for TAMC
• Sabouraud Dextrose Agar (SDA) for TYMC
• Bile Salt Agar, MacConkey Agar, Mannitol Salt Agar, and XLD Agar for specified pathogens

5.3 Total Aerobic Microbial Count (TAMC)

1. Transfer 1 mL of sample dilution (10^-1 to 10^-3) into sterile Petri dishes.
2. Pour 15–20 mL of sterile melted PCA and swirl to mix.
3. Incubate plates at 30–35°C for 3–5 days.
4. Count colonies and express as CFU/g.

5.4 Total Yeast and Mold Count (TYMC)

1. Repeat the procedure as above using SDA.
2. Incubate at 20–25°C for 5–7 days.
3. Count fungal colonies and express as CFU/g.

5.5 Test for Specified Pathogens

1. Perform tests for Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Salmonella spp.
2. Use selective enrichment broth followed by selective agar plating.
3. Incubate as per respective pharmacopeial methods and confirm using biochemical identification kits or test strips.

5.6 Acceptance Criteria (As per USP/Ph. Eur.)

• TAMC: NMT 10³ CFU/g
• TYMC: NMT 10² CFU/g
• Pathogens: E. coli, S. aureus, P. aeruginosa, Salmonella spp. – Absence in 1 g or 10 g as applicable

5.7 Method Validation Parameters

1. Recovery Studies: Spike known counts into placebo and test matrix. Recovery between 70–130%.
2. Specificity: Ability to detect and distinguish organisms in presence of product matrix.
3. Robustness: Evaluate under varied incubation times and temperatures.

5.8 Documentation

1. Document results in Microbial Load Test Log (Annexure-1).
2. Include media preparation, lot numbers, incubation conditions, colony count calculations, and analyst initials.

6. Abbreviations

• TAMC: Total Aerobic Microbial Count
• TYMC: Total Yeast and Mold Count
• CFU: Colony Forming Units
• USP: United States Pharmacopeia
• Ph. Eur.: European Pharmacopoeia

7. Documents

1. Microbial Load Test Log – Annexure-1
2. Pathogen Detection Record – Annexure-2
3. Validation Report Summary – Annexure-3

8. References

• USP <61>: Microbial Enumeration Tests
• USP <62>: Tests for Specified Microorganisms
• Ph. Eur. 2.6.12 and 2.6.13
• ICH Q6A: Specifications – Test Procedures and Acceptance Criteria

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name	Megha Thakur	Vivek Chavan	Sunita Reddy
Designation	Microbiologist	QA Reviewer	Head – AMD
Department	Analytical Method Development	QA	Analytical Method Development

11. Annexures

Annexure-1: Microbial Load Test Log

Sample ID	TAMC (CFU/g)	TYMC (CFU/g)	Status
CRM-260-01	85	12	Pass

Annexure-2: Pathogen Detection Record

Organism	Tested Quantity	Result	Status
Escherichia coli	1 g	Absent	Pass
Staphylococcus aureus	1 g	Absent	Pass

Annexure-3: Validation Report Summary

The microbial load test method for cream formulation CRM-260 was validated with recovery studies showing 92–104%, specificity for all target organisms confirmed, and robustness proven under varying conditions. The method is suitable for routine QC use.

Revision History:

Revision Date	Revision No.	Details	Reason	Approved By
21/05/2025	2.0	Updated validation parameters and added Annexure-3	Annual Review	Sunita Reddy
15/02/2022	1.0	Initial SOP Release	New SOP	QA Head