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USP 61
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Quick understanding of the test
USP 61 - Microbial Enumeration Test
- 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
Results
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
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
USP 61 Microbial Enumeration Test Method Explained
USP 61 outlines three commonly accepted methods for estimating microbial counts in non-sterile products. The method used depends on the sample type, physical and chemical composition of the product, and expected microbial load.
1. Membrane Filtration Method
- The product is diluted using sterile buffered diluent or neutralizing solution (as necessary).
- A suitable quantity of the sample is filtered through the sterile membrane filter under aseptic conditions.
- The membrane with sterile diluent is rinsed to remove product residues.
- The filter membrane is placed on Soybean–Casein Digest Agar for Total Aerobic Microbial Count (TAMC) and on Sabouraud Dextrose Agar for Total Combined Yeast and Mold Count (TYMC).
- The plates are incubated under controlled test conditions (Soybean– Casein Digest Agar plates: 30–35°C for 3–5 days and Sabouraud Dextrose Agar plates: 20–25°C for 5–7 days).
- After incubation, colonies on each plate are counted and expressed as CFU per volume or weight of the product.
2. Pour-Plate Method
- Serial dilutions of the product are performed using sterile diluent.
- 1 mL of each dilution is transferred into separate sterile Petri dishes. 15–20 mL of molten agar is added and swirled to ensure proper mixing. The plates are allowed to solidify.
- The plates are incubated under controlled test conditions (same as membrane filtration method).
- Plates with 30–300 colonies are selected and the microbial count is calculated and expressed as CFU/g or mL.
3. Most Probable Number (MPN) Method
- Serial dilutions of the product are prepared using sterile diluent.
- From each level of dilution, 3 aliquots of 1ml are used to inoculate three tubes of Soybean–Casein Digest Broth media.
- All test tubes are incubated at 30–35°C for not more than 3 days.
- The number of positive tubes in each dilution set is recorded.
- The most probable number of microorganisms per gram or mL of sample is estimated using an MPN table.
Suitable Method for USP 61 Microbial Enumeration Test
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
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
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.
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.
At MIS, we offer precise and compliant USP 61 testing services. 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
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.
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.
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.
A controlled environment, working with sterile equipment, exercising good laboratory practices, and maintaining strict aseptic conditions can help reduce the possibility of contamination.
Sample ingredients that inhibit microbial growth, such as preservatives or poor sample preparation techniques, can give false positives for a microorganism’s presence.
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.
The recent USP <61> update, approved by the Pharmacopeial Discussion Group (PDG), refines the membrane filtration method suitability criteria. It now requires that the count of each test organism must not differ by more than a factor of 2 from the control value.
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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