microbial isolates from pharmaceutical environments or product samples.

2. Scope

This procedure applies to microbial isolates obtained during preservative efficacy testing, environmental monitoring, water testing, and product sterility assessments within the Analytical Method Development (AMD) department.

3. Responsibilities

• Microbiologist: Prepares inoculum, performs biochemical identification tests, and interprets results.
• Lab Assistant: Assists in media preparation, incubation, and maintenance of microbial stock cultures.
• Group Leader: Reviews and approves identification reports.
• QA Executive: Verifies adherence to procedures and ensures test results are documented in line with regulatory expectations.

4. Accountability

The Head of Analytical Method Development is accountable for ensuring accurate and validated identification of microbial contaminants or isolates using approved biochemical test procedures.

5. Procedure

5.1 Culture Preparation

1. Revive microbial isolate on appropriate solid media (e.g., Nutrient Agar, Sabouraud Dextrose Agar).
2. Incubate at 30–35°C for bacteria and 20–25°C for fungi for 24–72 hours as appropriate.
3. Confirm purity of isolate by streaking on fresh media before identification.

5.2 Biochemical Identification

1. Use commercial biochemical test kits (e.g., HiBac™, API™, or equivalent).
   • For Gram-negative bacteria: Use enteric identification systems.
   • For Gram-positive cocci: Use catalase, coagulase, and salt tolerance tests.
   • For yeasts: Use sugar assimilation and urease tests.
2. Follow manufacturer’s protocol for:
   • Inoculum preparation (0.5 McFarland standard)
   • Inoculation and incubation
   • Reading color reactions or turbidity results
3. Enter results into software or coding charts provided by the kit manufacturer.

5.3 Manual Confirmatory Tests (if required)

1. Perform manual tests such as:
   • Indole, Methyl Red, Voges-Proskauer, Citrate (IMViC)
   • Triple Sugar Iron (TSI) slants
   • Oxidase, Catalase, Urease tests
2. Use positive and negative control organisms for each test.
3. Record observations with interpretation in Annexure-1.

5.4 Data Recording and Interpretation

1. Record biochemical profiles in the template provided (Annexure-2).
2. Report identification up to genus and species level where possible.
3. Document uncertainty in identification and recommend re-confirmation if required.

6. Abbreviations

• SOP: Standard Operating Procedure
• API™: Analytical Profile Index
• CFU: Colony Forming Units
• IMViC: Indole, Methyl Red, Voges-Proskauer, Citrate

7. Documents

1. Manual Biochemical Test Result Log – Annexure-1
2. Identification Result Report Template – Annexure-2
3. Biochemical Kit Lot and Expiry Tracker – Annexure-3

8. References

• USP <62> Microbiological Examination of Nonsterile Products: Tests for Specified Microorganisms
• Indian Pharmacopoeia: Microbiological Tests
• Manufacturer protocols for HiMedia™, bioMérieux™, or equivalent kits

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name	Meena Joshi	Deepak Goyal	Sunita Reddy
Designation	Microbiologist	QA Reviewer	Head – AMD
Department	Analytical Method Development	QA	Analytical Method Development

11. Annexures

Annexure-1: Manual Biochemical Test Result Log

Test	Result	Interpretation	Controls Used
Indole	Positive	Escherichia coli	E. coli (Positive), S. aureus (Negative)

Annexure-2: Identification Result Report

Sample ID	Biochemical Kit Used	Profile Code	Organism Identified	Confidence Level
ISO-23-001	API 20E	5144772	Enterobacter cloacae	99.5%

Annexure-3: Kit Lot and Expiry Record

Kit Name	Manufacturer	Lot No.	Expiry Date	Status
HiBac™ GN ID	HiMedia	HB2345	31/08/2025	Active

Revision History:

Revision Date	Revision No.	Details	Reason	Approved By
21/05/2025	2.0	Integrated biochemical test kits and added confirmatory test annexures	Annual Review and Audit Update	Sunita Reddy
10/05/2022	1.0	Initial SOP Release	New SOP	QA Head