Ensuring the microbiological safety of products in the development process is of prime importance for pharmaceuticals and cosmetics. It is important to ensure that these products meet the required standards of microbiological safety to protect consumers against infections or other adverse reactions. USP test for specified microorganisms describes standard test methods for the detection of the presence of specific microorganisms in non-sterile products. The following sections of the article will discuss the key elements of USP 62 such as the test organisms and the test procedure to be followed for testing non-sterile products.
USP 62 standard
USP 62 sets the standard guidelines for assessing the presence or absence of harmful microorganisms in non-sterile products. The USP test for specified microorganisms ensures that every tested product meets the established microbiological specifications before reaching consumers.
The standard primarily deals with the detection of pathogenic microorganisms that, when present in sufficient numbers could be a health hazard. This standard assists manufacturers in conducting necessary tests that ensure that the required microbiological quality standards are met. Products that can be tested using USP 62 guidelines are pharmaceuticals, cosmetics, dietary supplements and personal care products.
These tests described in USP 62 are used to test either for the absence or limited occurrence of microorganisms such as Escherichia coli, Salmonella, Staphylococcus aureus and others. Other methods for microbiological testing can be used by the manufacturer provided that the methods used are equivalent to the USP test for specified microorganisms.
Sample preparation
Sample preparation is an important step in USP 62 testing. Product samples must be prepared with great caution and interfering antimicrobial agents must be removed or neutralized from the product. This precautionary step is highly essential in those products that may contain inherent antimicrobial activity which may interfere with the outcome of microbial enumeration or testing.
The methods for sample preparation are outlined in USP 61 (Microbiological Examination of Non-Sterile Products: Microbial Enumeration Tests). When surface-active substances are used in sample preparation, it is necessary to test their nontoxic effect on microorganisms. Furthermore, compatibility with inactivators used in the method must be confirmed to avoid errors in the test results.
Suitability of the test methods
For every product that is to be tested, the suitability of the test methods should be verified to ensure that it can detect microorganisms in the presence of the product. This includes assessing the possible interference of the products with the test either due to its chemical composition or physical properties. Suitability testing must be conducted when there is any change in the product formulation or testing performance that could affect the outcome.
The test is conducted using standard suspensions of test strains, which should not be more than five passages distant from the original master seed lot. The suitability of the test method assures consistent, reliable and accurate detection of the specified microorganisms.
Negative controls
A negative control is performed to ensure that any microbial growth observed is due to the presence of that particular microorganism in the sample and not due to external contamination. Thus conducting a negative control test helps check the test conditions before an actual USP test for specified microorganisms is carried out. To perform a negative control test, a diluent is used instead of the test sample. Any microbial growth observed in the negative control is indicative of faulty test procedures and must be investigated to determine the problem before other tests can be done.
Testing procedures for specific microorganisms
USP 62 specifically tests the presence of microorganisms which are hazardous to the health of consumers. These microorganisms have been identified as pathogenic and can cause infections or other adverse health effects when present in non-sterile products. The following are the key microorganisms specified under USP test for specified microorganisms:
Bile-tolerant gram-negative bacteria
Bile-tolerant Gram-negative bacteria is tested by using a 1 in 10 dilution of the product in Soybean-Casein Digest Broth. The product is incubated to restore the bacteria without promoting its multiplication. It is then subcultured on Violet Red Bile Glucose Agar to detect the presence of bile-tolerant bacteria.
Salmonella
The sample is incubated in soybean-casein digest broth followed by its subculturing in Rappaport Vassiliadis Salmonella enrichment broth. These are further plated on xylose lysine deoxycholate agar and incubated. The probable presence of Salmonella is indicated by red colonies with black centers. Identification tests need to be carried out to confirm these results.
Escherichia coli
The testing for Escherichia coli includes preparation of a sample, incubation in soybean-casein digest broth and then in MacConkey Broth at high temperatures for the isolation of E. coli. The growth of colonies on MacConkey Agar, followed by identification tests indicates the presence of E.coli. The product meets USP 62 standards if no colonies are observed or when confirmatory tests are negative.
Staphylococcus aureus
Product samples are prepared and incubated in Soybean-Casein Digest Broth. It is then subcultured on Mannitol Salt Agar. The growth of yellow colonies with yellow zones indicates the presence of S. aureus and additional identification tests must be carried out for confirmation.
Clostridia
In the case of testing for Clostridia, the sample is divided into two portions; one of which undergoes heat treatment in order to detect spore-forming bacteria. The samples are then incubated in Reinforced Medium for Clostridia and subcultured on Columbia Agar. The presence of Clostridia is determined by the presence of anaerobic rods that may or may not have endospores, after which identification tests must be conducted as confirmation.
Pseudomonas aeruginosa
Samples are incubated in soybean-casein digest broth to test for Pseudomonas aeruginosa. These samples are further subcultured on cetrimide agar. The presence of P. aeruginosa is indicated by the growth of green colonies. However, the colonies must be verified through identification tests.
Candida albicans
Candida albicans is tested by inoculating the sample in Sabouraud Dextrose Broth, with subsequent subculturing in Sabouraud Dextrose Agar. The growth of white colonies would indicate the probable presence of C. albicans which should be confirmed through identification tests.
Interpretation of results
The result of the test should be interpreted as per the USP test for specified microorganisms. The presence of a particular microorganism in the sample implies that the product does not meet the specification. The probable number of microorganisms present is determined by the smallest quantity of product giving a positive result. The products should maintain the microbial examination limit for the safety and acceptability of the customer. The product could be accepted and released to the market for safe sale if the product shows negative test results on specified microorganisms.
Importance of USP 62 testing
In essence, the intent of the USP test for specified microorganisms is to confirm that non-sterile products are not contaminated with harmful microbes. Testing protects the end-users against infections and illnesses that may result from the use of contaminated products. USP 62 has rigid guidelines that ensure high and efficacious microbiological quality for pharmaceutical products. This is important for manufacturers to achieve regulatory compliance.
At Microbe Investigations Switzerland, we offer comprehensive USP 62 testing services to detect and evaluate specified microorganisms in your products. Our expert team ensures your products meet stringent regulatory standards for microbial safety.
For more details on our USP 62 testing services or to consult an expert, please contact us today.
