regulatory compliance and quality control objectives.

2. Scope

This SOP applies to the Analytical Method Development (AMD) department for the performance, development, and optimization of TVC tests on raw materials, in-process samples, finished dosage forms, and environmental monitoring samples.

3. Responsibilities

• Microbiologist: Conducts method optimization, executes sample testing, and maintains microbiological data records.
• AMD Analyst: Coordinates method integration with formulation studies and documents procedural validations.
• QA Officer: Verifies method accuracy, calculation records, and adherence to GMP.
• Head – AMD: Ensures all optimized procedures are approved and scientifically justified.

4. Accountability

The Head of Analytical Method Development is accountable for the integrity and regulatory compliance of the Total Viable Count method and its performance outcomes.

5. Procedure

5.1 Reference Standards and Guidelines

1. Refer to:
   • USP <61> – Microbial Enumeration Tests
   • EP 2.6.12 and IP Chapter 16 – Microbial Contamination Limits
2. Establish acceptance criteria based on dosage form as per pharmacopeial limits.

5.2 Preparation of Growth Media

1. Prepare and sterilize:
   • Plate Count Agar (PCA) for bacteria
   • Sabouraud Dextrose Agar (SDA) for fungi
2. Label media with batch number, preparation date, and expiry.
3. Record in Annexure-1: Media Preparation Log.

5.3 Sample Handling and Dilution

1. Use aseptic techniques in a laminar air flow (LAF) unit.
2. Dilute the sample using 0.1% peptone water or sodium chloride solution.
3. Prepare a series of decimal dilutions (e.g., 10^-1 to 10^-4) to reach an appropriate colony range.
4. If antimicrobial substances are present, use validated neutralizers.
5. Document dilutions in Annexure-2: Sample Dilution Log.

5.4 Inoculation and Incubation

1. Transfer 1 mL of each dilution into sterile Petri dishes (in duplicate).
2. Pour ~20 mL of cooled agar (45–50°C), swirl gently to mix.
3. Allow plates to solidify and invert for incubation.
4. Incubate under the following conditions:
   • PCA: 30–35°C for 48–72 hours
   • SDA: 20–25°C for 5–7 days
5. Record incubation conditions in Annexure-3: Inoculation and Incubation Record.

5.5 Enumeration and Data Recording

1. Count colonies manually or using an automated colony counter.
2. Include plates with 30–300 CFU for calculation of mean CFU/g or CFU/mL.
3. Use formula: CFU = Average Count × Dilution Factor
4. Enter all counts in Annexure-4: TVC Result Sheet.

5.6 Method Optimization Strategy

1. Evaluate different:
   • Neutralizers (e.g., lecithin, polysorbate)
   • Diluents (e.g., peptone water, phosphate buffer)
   • Volume and incubation variations for reproducibility
2. Perform spike-recovery studies to demonstrate neutralizer efficacy.
3. Assess method precision (%RSD) and accuracy (recovery % of inoculated CFU).
4. Summarize optimization in Annexure-5: Method Optimization Report.

6. Abbreviations

• TVC: Total Viable Count
• CFU: Colony Forming Unit
• PCA: Plate Count Agar
• SDA: Sabouraud Dextrose Agar
• LAF: Laminar Air Flow
• SOP: Standard Operating Procedure

7. Documents

1. Media Preparation Log – Annexure-1
2. Sample Dilution Log – Annexure-2
3. Inoculation and Incubation Record – Annexure-3
4. TVC Result Sheet – Annexure-4
5. Method Optimization Report – Annexure-5

8. References

• USP <61> – Microbial Enumeration Tests
• EP 2.6.12 – Microbial Contamination
• IP Chapter 16 – Microbiological Quality
• ICH Q6A – Specifications: Test 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: Media Preparation Log

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

Annexure-2: Sample Dilution Log

Sample ID	Diluent	Dilution Series	Neutralizer
TVC-120	0.1% Peptone Water	10⁻¹ to 10⁻³	Polysorbate 80

Annexure-3: Inoculation and Incubation Record

Plate	Temperature	Duration	Operator
SDA	22°C	5 days	Rajesh Kumar

Annexure-4: TVC Result Sheet

Dilution	Replicate 1	Replicate 2	Average	CFU/mL
10⁻²	45	48	46.5	4650

Annexure-5: Method Optimization Report

Neutralizer efficiency: 98.2%, %RSD across replicates: 2.1%, recovery of spiked CFUs within 95–105%. Method deemed robust and suitable for validation.

Revision History:

Revision Date	Revision No.	Details	Reason	Approved By
04/05/2025	2.0	Expanded method optimization and neutralizer validation steps	Annual SOP Review