cleanroom environments and controlled areas in pharmaceutical manufacturing and analytical areas.

2. Scope

This SOP applies to the Analytical Method Development (AMD) and Microbiology departments responsible for microbiological monitoring of ambient environments, including cleanrooms, HVAC areas, laminar air flow cabinets, and critical production zones.

3. Responsibilities

• Microbiologist: Prepares media plates, validates their performance, and monitors area contamination levels.
• Analytical Scientist: Coordinates environmental monitoring requirements during product development and analysis.
• QA Officer: Reviews data records and ensures regulatory compliance.
• Head – AMD: Approves media development protocol and ensures periodic validation.

4. Accountability

The Head of Analytical Method Development is accountable for ensuring the quality, traceability, and readiness of environmental monitoring plates and their usage protocols.

5. Procedure

5.1 Media Selection and Preparation

1. Use the following media for preparation of monitoring plates:
   • Tryptic Soy Agar (TSA): for total aerobic microbial count
   • Sabouraud Dextrose Agar (SDA): for yeast and mold monitoring
2. Weigh media as per manufacturer’s instructions and prepare in bulk using purified water.
3. Sterilize in autoclave at 121°C for 15–20 minutes.
4. Dispense aseptically into:
   • 90 mm petri dishes for settle plates
   • 55 mm contact plates for surface monitoring
5. Label with batch number, preparation date, expiry, and type of media.
6. Document preparation in Annexure-1: Media Preparation Log.

5.2 Plate Quality and Growth Promotion Testing (GPT)

1. Perform GPT using ATCC cultures:
   • Staphylococcus aureus (ATCC 6538)
   • Bacillus subtilis (ATCC 6633)
   • Candida albicans (ATCC 10231)
2. Inoculate plates with ≤ 100 CFU and incubate:
   • TSA: 30–35°C for 3 days
   • SDA: 20–25°C for 5 days
3. Record recovery and morphology in Annexure-2: GPT Record Sheet.

5.3 Sterility Verification and Shelf-Life

1. Incubate a minimum of 5% of each lot of un-inoculated plates:
   • 20–25°C and 30–35°C for 14 days
2. Check for contamination. Discard batch if contamination is observed.
3. Assign a shelf-life of up to 4 weeks under controlled storage (2–8°C) with desiccant packs.
4. Document in Annexure-3: Sterility Verification Log.

5.4 Usage in Environmental Monitoring

1. Use settle plates by exposing for 4 hours in cleanroom areas, 1 hour in critical zones.
2. Use contact plates by pressing gently on surface areas (gloves, walls, equipment) for 10 seconds.
3. Record sample ID, location, time, and personnel in Annexure-4: Monitoring Usage Log.
4. Incubate as per media type and inspect for microbial growth.

5.5 Acceptance Criteria

1. Compare microbial counts to ISO 14644, WHO TRS 961 Annex 6, and internal alert/action limits:

2. Grade	Settle Plate (cfu/4hr)	Contact Plate (cfu/plate)
   A	<1	<1
   B	5	5
   C	50	25
   D	100	50

3. Any values exceeding alert/action limits must trigger deviation investigation and CAPA.

6. Abbreviations

• TSA: Tryptic Soy Agar
• SDA: Sabouraud Dextrose Agar
• GPT: Growth Promotion Test
• cfu: Colony Forming Units
• SOP: Standard Operating Procedure

7. Documents

1. Media Preparation Log – Annexure-1
2. GPT Record Sheet – Annexure-2
3. Sterility Verification Log – Annexure-3
4. Monitoring Usage Log – Annexure-4

8. References

• USP <1116> – Microbiological Control and Monitoring of Aseptic Environments
• WHO TRS 961 Annex 6 – GMP for Sterile Pharmaceutical Products
• ISO 14644-1 and 14698 – Cleanroom Standards

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	Batch No.	Preparation Date	Expiry Date	Prepared By
TSA	TSA0525	18/05/2025	18/06/2025	Sunita Reddy

Annexure-2: GPT Record Sheet

Organism	Media	CFU Inoculated	CFU Recovered	Status
B. subtilis	TSA	85	80	Pass

Annexure-3: Sterility Verification Log

Media	Lot No.	Storage Condition	Contamination Observed	Remarks
TSA	TSA0525	2–8°C	No	Acceptable

Annexure-4: Monitoring Usage Log

Date	Location	Plate Type	Exposure Time	Collected By
19/05/2025	Grade B – LAF Hood	Settle Plate	1 hr	Rajesh Kumar

Revision History:

Revision Date	Revision No.	Details	Reason	Approved By
04/05/2025	2.0	Included WHO and ISO-based alert/action limits and plate shelf-life controls	Annual SOP Review