Frequently Asked Questions: <61> Microbial Examination of Nonsterile Products: Microbial Enumeration Tests

  1. Can I use strains other than those that are cited in the USP?
  2. Is there a method that provides verification that there is 100 CFU in the inoculum?
  3. When are you supposed to do the negative control: when testing the suitability of the method, when testing the product, or both?
  4. What is the purpose of the negative control?
  5. Does it have to be done every time the product is tested or during the method validation or is it possible to do it periodically?
  6. Is it necessary to test the growth promotion on all received batches or does it serve just for microbiological validation? Do we have to test the growth promotion of diluted broth?
  7. Do we have to test systematically in parallel a previous and approved batch in order to compare with the new batch?
  8. Why do growth promotion tests have to be performed on sabouraud-dextrose agar (SDA) and casein soya bean digest agar (CSA) for C. albicans and A. niger? Is it because colonies of fungi detected on CSA are counted as part of TAMC?
  9. As bacteria growing on SDA are also counted as part of TYMC, why aren't the growth promotion tests required to be performed on SDA with the bacterial strains?
  10. What does the factor of 2 mean? How is this factor calculated?
  11. What is the sufficient volume of the microbial suspension of not more than 100 CFU? What does 100 CFU refer to?

  1. Can I use strains other than those that are cited in the USP?

    You should use the strains that are cited in General Chapter <61> or equivalent strains from other culture collections. For example, if Pseudomonas aeruginosa ATCC 9027 is indicated, you should use this strain or strains from other culture collections claiming equivalence to ATCC 9027. Other strains such as ATCC 14149 are not appropriate.

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  2. Is there a method that provides verification that there is 100 CFU in the inoculum?

    You may establish a turbidimetric calibration curve or use another suitable method and then you will be able to get an estimate of the concentration of your inoculum. This can be later confirmed by standard plating methods, such as described in USP General Chapter <51>. You can also use ready-to-use certified strains

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  3. When are you supposed to do the negative control: when testing the suitability of the method, when testing the product, or both?

    You are supposed to include the negative control at the same time you test the product.

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  4. What is the purpose of the negative control?

    The purpose of this negative control is to show that there is no contamination during the testing of the product. If a positive result is obtained with a negative control, the test can be regarded as invalid and may be repeated.

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  5. Does it have to be done every time the product is tested or during the method validation or is it possible to do it periodically?

    Negative controls should be included every time the product is tested.

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  6. Is it necessary to test the growth promotion on all received batches or does it serve just for microbiological validation? Do we have to test the growth promotion of diluted broth?

    Growth promotion must be tested for each new batch of medium. Growth promotion must be checked on agar media and nutritive broth but not on diluted broth.

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  7. Do we have to test systematically in parallel a previous and approved batch in order to compare with the new batch?

    You do not have to test a previous batch in parallel. You can do the comparison 'on paper' if growth was clearly described.

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  8. Why do growth promotion tests have to be performed on sabouraud-dextrose agar (SDA) and casein soya bean digest agar (CSA) for C. albicans and A. niger? Is it because colonies of fungi detected on CSA are counted as part of TAMC?

    This is a matter of definition. TAMC by definition includes yeast and molds. Therefore the media have to be checked with these micro-organisms.

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  9. As bacteria growing on SDA are also counted as part of TYMC, why aren't the growth promotion tests required to be performed on SDA with the bacterial strains?

    TYMC is by definition yeasts and molds count so growth promotion with bacteria is not essential. SDA with antibiotics may be used as an alternative when the TYMC is expected to exceed the acceptance criterion due to the bacterial growth.

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  10. What does the factor of 2 mean? How is this factor calculated?

    It means that the result can be twice that of the inoculum. For example with an inoculum of 100 CFU, acceptable counts are: 100/2 = 50 CFU to 100 x 2 = 200 CFU. The factor is introduced to take account of the variability of the method.

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  11. What is the sufficient volume of the microbial suspension of not more than 100 CFU? What does 100 CFU refer to?

    The micro-organisms are to be added to the diluted/suspended product at the end of the preparation (usually a 1 in 10 dilution is prepared) or after the neutralization (in the last fraction of the rinsing fluid in the case of filtration or simultaneously with the preparation in/on the Petri dish in the case of the plate count method) if inhibition of growth by the sample cannot otherwise be avoided. The 100 CFU refers to the inoculum (e.g., what will be on the filter or on the plate).

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