concentration (MIC) determinations.

2. Scope

This procedure applies to the Analytical Method Development (AMD) department for evaluating antimicrobial agents in finished dosage forms, preservatives in formulations, or active ingredients with claimed antimicrobial properties.

3. Responsibilities

• Microbiologist: Designs test protocols, selects appropriate media and organisms, and executes test procedures.
• Analytical Scientist: Supports by providing formulation background and product specifications.
• QA Executive: Verifies protocol adherence and reviews test documentation.

4. Accountability

The Head of Analytical Method Development is accountable for ensuring scientifically justified and validated antimicrobial testing methods are developed and documented.

5. Procedure

5.1 Microbial Panel Selection

1. Select a panel of test organisms based on intended spectrum of the product:
   • Staphylococcus aureus (Gram-positive)
   • Escherichia coli (Gram-negative)
   • Pseudomonas aeruginosa (resistant Gram-negative)
   • Candida albicans (yeast)
   • Aspergillus brasiliensis (mold)

5.2 Method Selection Based on Sample Type

1. Agar Diffusion Method (e.g., disc diffusion): Suitable for topical formulations and solutions.
2. Broth Dilution Method (MIC/MBC): Preferred for pure API or systemic antimicrobial agents.
3. Agar Dilution: Used when disc diffusion gives inconsistent zones or for highly viscous samples.

5.3 Media and Culture Preparation

1. Prepare Mueller-Hinton Agar/Broth for bacteria and Sabouraud Dextrose Agar/Broth for fungi.
2. Standardize inoculum to 0.5 McFarland (~1–2 × 10⁸ CFU/mL).

5.4 Execution of Assay

1. Agar Diffusion:
   • Seed agar with standardized inoculum.
   • Apply discs or wells with known concentration of test sample.
   • Incubate at 30–35°C (bacteria) or 20–25°C (fungi) for 24–48 hours.
   • Measure zone of inhibition (mm) and compare with standards.
2. Broth Dilution:
   • Prepare serial dilutions of sample in sterile broth.
   • Inoculate with organism and incubate.
   • Determine MIC as the lowest concentration showing no visible growth.

5.5 Validation Parameters

1. Specificity (no interference from formulation excipients)
2. Linearity (for MIC across a concentration range)
3. Precision (zone measurement or MIC reproducibility)
4. Robustness (variation in media thickness, incubation time)

5.6 Documentation

1. Document test layout and results in Annexure-1.
2. Include digital photographs of inhibition zones (if applicable).
3. Summarize final method in validation report format (Annexure-2).

6. Abbreviations

• SOP: Standard Operating Procedure
• MIC: Minimum Inhibitory Concentration
• MBC: Minimum Bactericidal Concentration
• CFU: Colony Forming Units

7. Documents

1. Antimicrobial Test Result Log – Annexure-1
2. Method Validation Summary Report – Annexure-2
3. Media and Culture Preparation Record – Annexure-3

8. References

• USP <61> and <62> – Microbial Limit Tests
• CLSI M02/M07 – Antimicrobial Susceptibility Testing Standards
• Ph. Eur. 2.6.12 and 5.1.3

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name	Vikram Shetty	Nisha Jain	Sunita Reddy
Designation	Microbiologist	QA Reviewer	Head – AMD
Department	Analytical Method Development	QA	Analytical Method Development

11. Annexures

Annexure-1: Antimicrobial Test Result Log

Sample ID	Organism	Method	Zone (mm) / MIC (µg/mL)	Status
AMT-245	E. coli	Disc Diffusion	18 mm	Compliant

Annexure-2: Method Validation Summary

Validated disc diffusion method for AMT-245 using Mueller-Hinton agar. Linearity established from 5–25 µg/disc. Zone diameter correlates with concentration. Method deemed suitable.

Annexure-3: Media Preparation Record

Media	Batch No.	Preparation Date	QC Result
Mueller-Hinton Agar	MHA0525	18/05/2025	Growth Promotion Pass

Revision History:

Revision Date	Revision No.	Details	Reason	Approved By
21/05/2025	2.0	Incorporated MIC and validation parameters for broader application	Annual Review	Sunita Reddy
15/05/2022	1.0	Initial SOP Release	New SOP	QA Head