FAQs: <62> Microbial Enumeration of Nonsterile Products: Tests for Specified Microorganisms

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

You should use the strains that are cited in this chapter, 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.

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

You are supposed to do the negative control at the same time as when you are testing the product.

3. What is the purpose of the negative control?

The purpose of the 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.

4. 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 that the product is tested.

5. Is it necessary to test the growth promotion on all received batches or does it serve just for microbiological validation?

Growth promotion must be checked for each new batch of medium.

6. 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

7. What are the specifications when we compare a fresh batch with a previous batch for growth promotion properties? Do we need to take a factor of 2 into account?

The factor of 2, as described in USP <61> can be used. No strict requirement was deliberately given in this chapter because the test is qualitative, not quantitative. You can define the comparability criterion yourself. For example, colony size at the shortest incubation time prescribed.

8. You should not incubate more than the « incubation time prescribed ». How exactly are the given times (e.g. 18 – 24 h) to be followed?

For growth promoting properties of media you must incubate not more than 18 h (worst case conditions).

For inhibitory properties of media you must incubate not less than 24 h (worst case conditions). For indicative properties of the media you could incubate within the specified range (here, from 18 h to 24 h).

9. In the growth promotion test of Rappaport Vassiliadis Salmonella enrichment broth there is no visible growth after the incubation time, but after subculturing on selective agar there is typical growth. Is this the case only in our laboratory?

This can be observed, since for Rappaport Vassiliadis Salmonella enrichment broth, we inoculate low numbers of Salmonella sp (usually the inoculum is around 20 CFU per 10 mL Rappaport Vassiliadis Salmonella enrichment broth).

Even if the enrichment broth seems clear, you must confirm recovery of Salmonella by subculturing the Rappaport Vassiliadis Salmonella enrichment broth to solid agar.

10. Does it mean that for each test strain, individual suitability tests have to be performed, or is it possible to use a mixed inoculum of all 4 strains?

The method states that the strains are inoculated individually. No mixed inoculum is permitted.

11. Test strains must be inoculated individually using a number of micro-organisms equivalent to not more than 100 CFU, could you clarify if this means that only the specific micro-organism under detection in the test method is inoculated into the growth medium or if each of the 4 microorganisms are added individually to the growth medium for each of the specific test methods?

It is only the specified micro-organism under detection which is inoculated.

12. Which test micro-organisms should one use? Just the same micro-organisms as used for testing the growth promoting properties of the respective media, or also the microorganisms used for testing inhibitory properties of the media?

You do not have to use an inhibitory strain in order to test the suitability of the method. For example if you test the suitability of the method for E. coli, you should use only E. coli as test micro-organism for growth promotion.

13. Must all products be tested?

Not always. For products differing only in amount of active ingredient a bracketing approach may be applied.

14. What is meant by "at the time of mixing"? Bile-tolerant gram-negative bacteria: At the time of sample preparation, or at the time of addition to the resuscitation broth, or at the time of inoculation of the Mossel Broth? E.coli: At the time of sample preparation, or at the time of addition to pre- broth, or at the time of inoculation of the MacConkey broth etc.?

In both cases, the micro-organisms should be added at the time of mixing with the preincubation or resuscitation broth. If Bile-tolerant gram-negative bacteria are taken as an example it refers to the sub-section "Sample Preparation and Pre-Incubation". The micro-organisms are added to the casein soy bean digest broth (SCDB) immediately before or after the product to be examined is added. The micro-organisms are therefore present during the whole resuscitation period of 2 – 5 hours.

15. What does "100 cfu in the inoculated test preparation" mean? How should this be done?

This is to simulate the situation that 100 cfu’s are present in the sample to be examined, usually 1 g of the product but 10 g for Salmonella (cf. section Salmonella), resp.

Example: Suppose 10 g of product is diluted in 100 ml of CSDB. Then less than 100 cfu’s are added to this suspension.

16. Should the test organism (<100 cfu) be added to the 1:10 diluted test solution (with 10g Product) and then the inoculated test solution be inoculated into the selective medium? If the inactivators (see Table 2, USP<61>) cannot inactivate the antimicrobial activity of the product, can we proceed as in USP<61> to add the test organisms to a higher dilution of the product (<100cfu in 1g product) or at a later time in the test?

The test organisms should be added at the time of mixing with the medium for pre-incubation. Inactivation of antimicrobial activity should be attempted as far as possible. Addition at a later time of the test is not a reasonable measure. If inactivation cannot be achieved, the test can be abandoned for the product because it is assumed that the specified micro-organism will not be able to survive in the product.

17. Why shall I use the shortest incubation period?

You have to show that the worst conditions work. Moreover you are working with healthy cells and these should give the required response in the shortest time.

18. What does "The specified micro-organisms must be detected with the indication reactions as described under 'Testing of Products'" mean?

Characteristic colonies are observed on the selective agar, and no such colonies are observed with a non-inoculated product, examined simultaneously as a negative blank.

19. What do I have to show to be able to proceed as stated: "If for a given product the antimicrobial activity with respect to a micro-organism for which testing is prescribed cannot be neutralized, then it is to be assumed that the inhibited micro-organism will not be present in the product."

Once you demonstrate that you have tried all possible approaches, then you can refer to the clause cited in your question.

20. What are "the Bile-tolerant Gram-negative bacteria"?

There is no strict definition of this group of micro-organisms. They are defined operationally as those micro-organisms that show growth in the stated conditions on Violet Red Bile Glucose Agar medium. They include, Gram negative bacteria that grow in the presence of bile salts, non-lactose fermenting but able to utilize glucose, e.g., some Bile Tolerant Gram Negative Bacteria includes members of the family Enterobacteriaceae, Pseudomonads and Aeromonas.

21. In the sub-section Selection and Subculture under Escherichia coli, what is the purpose of the elevated temperature 42 – 44°C?

Can one utilize an alternative temperature range, e.g. 35 – 37°C provided that one demonstrates the recovery of E. coli during qualification?

The purpose of the elevated temperature, 42 – 44°C is to allow for selective conditions for fecal E. coli. The selected temperature is usually a compromise between sensitivity and specificity as not all strains of E. coli will grow, or grow and produce gas, at these higher incubation temperatures.

An alternative temperature range would depart from the USP method, but you can always use alternatives methods as described in the General Notices of the USP and USP<1223>.

22. With accumulation of the minimal incubation (3 x 18h) for pre-culture, enrichment culture, and sub-culture, it is unavoidable to work at night-hours to perform the validation. How can we avoid these awkward working hours for our personnel?

You have to confirm that the test works for the minimum time for routine testing. In fact, should a company find during suitability testing, that the minimum incubation time is not sufficient for a given product but a longer incubation time is needed, prolongation would be a necessary variation of the test.

23. If 10 g of sample is added to the initial broth for a test such as E. coli (4.2), but an amount is immediately subcultured into a second broth that is only representative of 1g of actual product and this broth is incubated. At the end of testing, can this test be classified, for a negative result, as "none detected per 10 g" or as "none detected per g".

The quantity that is pre-incubated is 1 g therefore the outcome of the test is "absence in 1 g". For Salmonella, you will note that an "absence in 10 g" test has been implemented.

24. It is observed that on selective media of S. aureus, yellow colonies of gram-positive cocci in chains are seen, but the yellow colonies are without clear zones in the test sample. Whereas positive culture shows yellow colonies of gram-positive cocci in clusters surrounded by yellow zones.

Your product can be contaminated, maybe not by the species described in the USP but by another micro-organism. Good laboratory practice should make you think that there is a problem and that you should investigate (e.g. identify the species and find out where it comes from). Probably the product cannot be released, but it is up to the QC laboratory manager to decide.

25. For which pharmaceutical products or raw materials is it prescribed to search for Clostridia?

For powdered animal organs, products with risk of fecal contamination by Clostridia and possible proliferation, natural raw materials, possible telluric contamination. However it has not been introduced in any monograph yet. The test is particularly relevant where a preparation is exposed to anaerobic or low-oxygen conditions during use.

26. Are there alternatives media that are acceptable?

Methods using alternative media are considered as alternative methods, which may be used when consistent with General Notices 6.30.

27. How can we do the pH measurement for culture media? It is written to measure the pH at 25°C. At this temperature, agar is solid. Therefore, how can we adjust the pH?

You may use a robust electrode. There are electrodes for measurement in semisolid samples such as meat, cheese and fruit. These electrodes are certainly suitable for measurements in solid agar. Adjustment of pH must be made during preparation of the medium for ensuring that the criterion for pH is met in the final medium.

28. If we have growth problems of S. aureus and inhibitory problems of E. coli with mannitol salt agar medium that is recommended in the harmonized method, what is the cause?

The composition of mannitol salt agar has been optimized to recover S. aureus and inhibit E. coli (pH, nutritive qualities…). You should verify that the temperature of incubation is correct. Moreover there could be a problem of stability of the medium and you should therefore verify that the medium has been stored in adequate conditions. Lastly, you could try to use different media suppliers, which may give better results.