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USP 61
Microbial Enumeration Test
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Quick understanding of the test
USP 61 - Microbial Enumeration Test
USP 61, also known as the microbial enumeration test, is a standardized method used to quantify mesophilic bacteria and fungi that may grow under aerobic conditions in non-sterile products.
Applicable to pharmaceuticals, cosmetics, personal care products, food, medical devices and more.
- Pseudomonas aeruginosa
- Staphylococcus aureus
- Bacillus subtilis
- Aspergillus niger
- Candida albicans
- Test samples are diluted using suitable buffers.
- Growth promotion tests are performed on the media.
- Test samples suspected to contain antimicrobial agents are subjected to neutralization and then incubated.
- Microbial counts in the sample are assessed using either membrane filtration method or plate count method.
- Negative controls are carried out to ensure there is no external contamination.
- Ensures that non-sterile products are free from harmful microbial contamination.
- USP 61-certified products are more likely to meet the stringent quality requirements for market approval.
Turnaround Time
Testing typically takes 5–6 days.
Results
The number of bacterial or fungal colonies formed on the plates is counted and expressed as Colony-Forming Units (CFU) for Total Aerobic Microbial Count (TAMC) and Total Yeast and Mold Count (TYMC).
Passing criteria
- For products designed for infants, used under the eye area or mucous membranes: <200 CFU/ml or 102 CFU/ml
- For other products: <2000 CFU/ml or 103 CFU/ml
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Abstract
The United States Pharmacopeia (USP) 61 Microbial Enumeration Test is a quality test to check the presence and quantity of micro-organisms in non-sterile products including pharmaceuticals, cosmetics and in raw materials. USP 61 is also referred to as the ‘Microbial bioburden test’, ‘Microbial Limits Test’ or ‘Microbial enumeration test.’ The test is a standard method used to assess microbial load with potential risks to ensure conformity with regulations.
The microbial enumeration test determines total aerobic microbial count (TAMC) and total yeast mold count (TYMC) in the test sample.
Importance of USP 61 Microbial Enumeration Test
Microbial enumeration tests are highly essential for pharmaceuticals and cosmetics products that come into contact with the human body. Excessive number of microbes in these products can lead to infections or spoilage and pose various health risks. Therefore, periodic testing of these products keeps microbial contamination under control, significantly reducing the risk of infections from using contaminated products. Testing also ensures that the products comply with the necessary regulatory requirements, hence, reducing possible legal penalties from the sale of contaminated products.
Test Requirements for USP 61 Testing
The test requirements for microbial enumeration test are:
- A 1:10 dilution of the sample must be prepared using suitable buffers.
- No more than 100 CFU of microbial strain must be added to the sample, ensuring inoculation volume is less than 1%.
- Media should be verified to support microbial growth using growth promotion tests.
- If the sample shows antimicrobial activity, antimicrobial agents must be neutralized with neutralizing agents to avoid interference with microbial counts.
- Appropriate test methods must be used to test the sample (membrane filtration or plate count method). The choice of method depends on the nature of the product and the required microbial limit.
- Bacterial samples should be incubated at 30–35°C whereas fungal samples should be incubated at 20–25°C.
- Negative controls should be carried out to ensure that there is no external contamination.
Products tested at MIS using USP 61 Testing
The standard applies to nonsterile pharmaceutical products, cosmetics, nutraceuticals, and certain medical devices. The most commonly tested products include oral drugs, topical creams, lotions, and raw materials, among others.
USP 61 Microbial Enumeration Test Method Explained
- Test samples are inoculated on Soybean-Casein Digest Agar for the Total Aerobic Microbial Count (TAMC) and on Sabouraud Dextrose Agar for the Total Yeast and Mold Count (TYMC).
- Test samples suspected to contain antimicrobial agents which might interact with the test outcome must be subjected to neutralization and dilution techniques to ensure that the product’s antimicrobial agents will not suppress the growth of suspected microorganisms in samples.
- Test organisms used in neutralization and dilution tests are Pseudomonas aeruginosa (ATCC 15442), Escherichia coli (ATCC 8739), Staphylococcus aureus (ATCC 6538P), Enterococcus hirae (ATCC 10541), Klebsiella pneumoniae (ATCC 4352), MRSA (ATCC 33591), Salmonella enterica (ATCC 10708), Candida albicans (MTCC 3017).
- After a specified incubation period, microbial enumeration can be done using either plate count or membrane filtration test method.
Suitable Method for USP 61 Microbial Enumeration Test
Suitability testing checks if the chosen microbial enumeration method will recover microorganisms in the presence of the test product. This is done by testing if the product being examined has any inhibitory activity against the microorganisms being recovered. Should the product possess antimicrobial activity, inactivators or neutralizers are added to eliminate or neutralize such effects.
Interpretation of USP 61 Microbial Enumeration Test Results
- Total Aerobic Microbial Count (TAMC) is expressed as CFU which is equivalent to the number of colonies on Soybean-Casein Digest Agar. In addition, the presence of fungal colonies on the Soybean-Casein Digest Agar shall also be counted in the TAMC.
- Total Yeast and Mold Count (TYMC) is equivalent to the number of colonies (CFU) found on the Sabouraud Dextrose Agar. Any colonies of bacteria count toward TYMC.
Acceptable guidelines for USP 61 Microbial Enumeration Test
Non-sterile products are not required to be absolutely microorganism-free. However, it is important to ensure that these products do not harbor any harmful pathogens. This is particularly important for products applied to sensitive parts such as the eyes or the mucus membrane. The acceptable limits of microbial count as per USP standards are:
- For products designed for infants, used under the eye area or mucous membranes: <200 CFU/ml or 102 CFU/ml
- For other products: <2000 CFU/ml or 103 CFU/ml
Frequency of Conducting USP 61 Microbial Enumeration Testing
Testing frequency will vary with the product and stage of production. In the case of finished products, the test can be done on a batch basis before releasing it to the market so that the product meets the quality requirement. In the case of raw materials, testing may be required for every new lot or at regular intervals, depending on the criticality of the materials.
Benefits of USP 61 Microbial Enumeration Test
- Regulatory compliance
This test enables manufacturers to meet the highest health standards provided by regulatory bodies like the FDA and EMA.
- Market access
Adherence to this standard is mandatory to get approval for the sale of pharmaceuticals, cosmetics, and other non-sterile products.
- Quality control
The test is used as an indicator of quality assurance to help manufacturers ensure that the microbial quality of their products is checked and regulated at every stage of its processing.
- Prevention of spoilage
Testing assists in identifying the microbial contaminants that may lead to spoilage, thus allowing manufacturers to take appropriate preventive measures.
- Broad applicability
The test is applicable to nonsterile products, making it a flexible method that can be used for assessing a wide range of products.
- Consumer confidence
Products that comply with this standard can enhance the brand’s reputation and consumer trustworthiness.
Applications of USP 61 Microbial Enumeration Test
Pharmaceuticals
- Ensuring the microbiological quality of non-sterile pharmaceutical products, including tablets, capsules, syrups, and topical medications.
- Testing raw materials and excipients used in drug formulation to ensure they meet microbiological standards.
Cosmetics
- Evaluating the microbial load in cosmetic products including creams, lotions, shampoos, and makeup.
- Testing for microbial contamination in raw materials used in cosmetic formulations.
Personal care products
- Assessing the microbiological quality of personal care products such as soaps, deodorants, and toothpaste.
- Ensuring that products intended for personal hygiene do not contain harmful levels of microorganisms.
Food and beverages
- Testing non-sterile food products and beverages for microbial contamination to ensure they are safe for consumption.
- Monitoring the microbiological quality of ingredients and packaging materials used in the food industry.
Nutritional supplements
- Ensuring the microbiological safety of dietary supplements including vitamins, minerals, and herbal products.
- Testing raw materials and finished products for microbial contamination.
Medical devices
- Evaluating the bioburden on non-sterile medical devices and components prior to sterilization.
- Ensuring that medical devices meet microbiological quality standards to prevent infections in patients.
Biopharmaceuticals
- Testing biologically derived products such as vaccines, cell cultures, and tissue engineering products for microbial contamination.
- Ensuring the microbiological quality of biopharmaceutical raw materials and intermediates.
Veterinary products
- Assessing the microbiological quality of nonsterile veterinary products including medications, supplements, and care products for animals.
- Ensuring that veterinary products are free from harmful microbial contamination.
Water testing
- Monitoring the microbiological quality of water used in pharmaceutical and cosmetic manufacturing processes.
- Ensuring that water used in product formulations meets microbiological safety standards.
Household products
- Testing household cleaning products, detergents, and disinfectants for microbial contamination.
- Ensuring that these products are safe for use in homes and do not harbor harmful microorganisms.
Packaging materials
- Assessing the microbiological quality of packaging materials used for pharmaceuticals, cosmetics, and food products.
- Ensuring that packaging materials do not introduce microbial contamination to the products they contain.
Research and development
- Conducting microbiological testing during the development of new pharmaceutical and cosmetic products.
- Evaluating the effectiveness of preservatives and antimicrobial agents in product formulations.
Microbe Investigations Switzerland has incorporated fully equipped bio-suites and analytical facilities for microbial enumeration tests. We also perform USP 60 and USP 62 tests to assess the growth of specific microorganisms in non-sterile products. Our experts understand the client’s needs and expectations first and take complete responsibility to provide reliable and accurate results.
Contact our experts now to discuss your Microbial enumeration test requirements and other related antimicrobial tests.
Frequently Asked Questions
DR. Martinoz Scholtz
MIS Labs
1. How are samples prepared for the microbial limits test?
Samples are prepared by aseptic dilution in a sterile diluent to provide low-concentration levels for the plating. This is necessary to avoid errors in counting microbial colonies.
2. What happens if a product fails the microbial limits test?
In case a product fails the test, an investigation is done to identify the source of contamination. The product can either be reformulated or rejected depending on the results obtained.
3. How does the microbial enumeration test ensure product safety for consumers?
The test ensures that the microbial levels are within the acceptable limits in non-sterile products thus reducing the possible adverse health effects like infections and allergic reactions.
4. How can you minimize contamination during the microbial enumeration test process?
A controlled environment, working with sterile equipment, exercising good laboratory practices, and maintaining strict aseptic conditions can help reduce the possibility of contamination.
5. What are the potential interferences that can affect the test results?
Sample ingredients that inhibit microbial growth, such as preservatives or poor sample preparation techniques, can give false positives for a microorganism’s presence.
6. What is the difference between USP 61 and USP 62?
USP 61 is for the determination of the presence of total viable aerobic bacteria and fungi. USP 62 deals with the detection of specific microbiological agents that may be pathogenic.
7. What is the USP 61 update?
A recent update of the standard was recently approved by the Pharmacopeial Discussion Group (PDG). The update included changes in the procedure used to verify the suitability of the membrane filtration method.
8. What is the most current version of the USP?
The most current version of the USP is USP–NF 2024, Issue 1 and is available on the official USP website.
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