Microbial Limit Testing of Non-Sterile Pharmaceuticals: Stepwise Procedure, Methods, and Acceptance Criteria

Microbial Limit Testing (MLT) is a fundamental quality control test in pharmaceutical microbiology that determines the number and types of microorganisms present in non-sterile products. It ensures that microbial contamination remains within acceptable limits and that the product is safe for patient use.

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🔬 What is Microbial Limit Testing?

Microbial Limit Testing evaluates the bioburden (microbial load) of pharmaceutical products such as tablets, syrups, ointments, and creams. It checks for both:

• Quantitative limits – total number of viable microorganisms (bacteria, yeast, and mold)
• Qualitative limits – absence of specified pathogenic microorganisms (e.g., Escherichia coli, Salmonella, Staphylococcus aureus)

📘 Reference Guidelines:

• United States Pharmacopeia (USP <61> & <62>)
• European Pharmacopoeia (EP 2.6.12 & 2.6.13)
• Indian Pharmacopoeia (IP 2.2.9 & 2.2.13)

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⚗️ Objectives of Microbial Limit Testing

• To determine the Total Aerobic Microbial Count (TAMC)
• To determine the Total Yeast and Mold Count (TYMC)
• To check for the absence of specified microorganisms
• To ensure compliance with pharmacopeial microbiological standards

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🧫 Test Methods Used in MLT

1. Plate Count Method (for TAMC and TYMC)

This method involves culturing a diluted sample on agar plates and counting the number of colonies formed after incubation.

2. Membrane Filtration Method

Used when the product can be filtered. The sample is passed through a sterile 0.45 µm filter that traps microorganisms. The filter is then placed on appropriate agar media for incubation.

3. Most Probable Number (MPN) Method

Used for liquid samples where microbial counts are low or when direct plating is not suitable.

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🧍 Stepwise Procedure for Microbial Limit Testing

Step 1: Preparation of Test Environment

• Perform all testing inside a Grade A laminar airflow cabinet located in a Grade B cleanroom.
• Ensure all glassware, media, and diluents are sterile and validated.
• Conduct environmental monitoring before and after the test.

Step 2: Sample Preparation

• Weigh or measure the required amount of product as per pharmacopeial recommendation.
• Use sterile Buffered Sodium Chloride Peptone Solution (pH 7.0) or other diluents.
• For oily or viscous products, add sterile polysorbate 80 or appropriate neutralizing agents.

Step 3: Detection of Total Aerobic Microbial Count (TAMC)

1. Prepare a suitable dilution of the sample (usually 1:10).
2. Transfer 1 mL of the diluted sample into Plate Count Agar (PCA).
3. Incubate at 30–35°C for 3–5 days.
4. Count the number of colonies and calculate CFU/g or CFU/mL.

Step 4: Detection of Total Yeast and Mold Count (TYMC)

1. Transfer 1 mL of the diluted sample into Sabouraud Dextrose Agar (SDA).
2. Incubate at 20–25°C for 5–7 days.
3. Count yeast and mold colonies separately.

Step 5: Test for Specified Microorganisms

Performed as per USP <62> to check the absence of specific pathogens.

• Escherichia coli
• Staphylococcus aureus
• Pseudomonas aeruginosa
• Salmonella spp.
• Clostridium spp. (if applicable)
• Candida albicans (for certain dosage forms)

Enrich the sample in selective media and confirm using differential identification tests.

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🧪 Acceptance Criteria as per USP

Product Type	TAMC (CFU/g or mL)	TYMC (CFU/g or mL)	Specified Microorganisms
Oral preparations	Not more than 103	Not more than 102	Absence of E. coli
Topical products	Not more than 102	Not more than 101	Absence of P. aeruginosa, S. aureus, C. albicans
Rectal/vaginal preparations	Not more than 102	Not more than 101	Absence of P. aeruginosa, S. aureus, C. albicans

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🧠 Validation of Microbial Limit Test

Before testing, perform method suitability (neutralization) validation to ensure the product does not inhibit microbial growth.

• Add known inoculum (10–100 CFU) of test organisms into the product.
• Ensure recovery within ±0.5 log of the control count.
• Use neutralizing agents if antimicrobial activity is observed.

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⚠️ Common Issues and Troubleshooting

• High microbial counts: Check sample handling, environment, and diluent sterility.
• No growth in validation: Product may have inhibitory properties—use neutralizers.
• False positives: Contamination during plating—perform tests in Grade A area.

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💡 Best Practices for Reliable MLT Results

• Use freshly prepared and validated media.
• Ensure aseptic technique during all manipulations.
• Perform environmental and personnel monitoring during testing.
• Keep detailed documentation and traceability for each test.
• Regularly calibrate instruments (pipettes, incubators, balances).

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📈 Conclusion

Microbial Limit Testing (MLT) is a critical microbiological quality control test for non-sterile pharmaceutical products. It ensures that the total microbial load and specified microorganisms are within acceptable limits defined by pharmacopeial standards. Accurate testing, proper validation, and adherence to GMP and aseptic techniques guarantee product safety and regulatory compliance.

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💬 About the Author

Siva Sankar is a Pharmaceutical Microbiology Consultant and Auditor with extensive experience in sterility testing, validation, and GMP compliance. He provides consultancy, training, and documentation services for pharmaceutical microbiology and cleanroom practices.

📧 Contact: siva17092@gmail.com
📱 Mobile: 09505626106

Disclaimer: This article is for educational purposes and does not replace your laboratory’s SOPs or regulatory guidance. Always follow validated methods and manufacturer instructions.