COMMENT
The USP has recently released a Summary of Frequently Asked Questions (FAQ) for both USP <61> and <62>. The FAQ for USP <61> was previously posted in another Blog. The FAQ listed below is the first of two Parts discussing additional FAQ from USP <62>. Additional FAQ will be posted later in the week.
Microbial Examination of Nonsterile Products: Tests for Specified Microorganisms USP <62>
Q. What is the purpose of the negative control?
A. 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.
Q. 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?
A. Negative controls should be included every time that the product is tested.
Q. Is it necessary to test the growth promotion on all received batches or does it serve just for microbiological validation?
A. Growth promotion must be checked for each new batch of medium.
Q. Do we have to test systematically in parallel a previous and approved batch in order to compare with the new batch?
A. You do not have to test a previous batch in parallel. You can do the comparison ‘on paper’ if growth was clearly described
Q. What are the specifications when we compare a freshly batch with a previous batch for growth promotion properties? Do we need to take a factor of 2 into account?
A. 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.
Q. You should not incubate more than the « incubation time prescribed ». How exactly are the given times (e.g. 18 – 24 h) to be followed?
A. 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).
Q. 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?
Do we have to test systematically in parallel a previous and approved batch in order to compare with the new batch?
A. 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.
Q. 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?
A. The method states that the strains are inoculated individually. No mixed inoculum is permitted.
Q. 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?
A. It is only the specified micro-organism under detection which is inoculated.
Q. 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?
A. 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.
Q. Must all products be tested?
A. Not always. For products differing only in amount of active ingredient a bracketing approach may be applied.
Q. 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.?
A. 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.
Q. What does “100 cfu in the inoculated test preparation” mean? How should this be done?
A. 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.
Q. 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 antimicrobal activity of the product, can 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?
A. 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 microorganism will not be able to survive in the product.
During MLT validation E.coli getting Growth in MacConkey broth with in 4-6 hrs, if we extended incubation for shortest time (24Hrs) Growth not established on MCA at shortest incubation at 18 Hrs. We know E.coli has fast generation time bacteria (20 Min) then why could wait for 24 Hrs.
In Routine MLT Testing Enrichment 18-24 Hrs., in this which time line we have to follow whether shortest or Maximum, If our validation met shortest time 18 Hrs recovery for all specified organisms mean can we follow 18 hrs for Further subculture.
If you are obtaining a complete recovery for all specified microorganisms, and don’t have other “Objectionable organisms” that might not completely meet the 18 hour window, you should be in compliance.
My concern would be that all “bugs” present are observable. Remember, these media are indicative tests, not final tests.
As your concern Maximum Incubation time is more possible to recover all “Objectionable organisms” I agree, So all Test for “specified microorganism” has to follow maximum incubation, Could you tell me the reason USP why defining 18-24 Hrs, 18-72 Hrs 24-48 Hrs.,
Experience on the part of the time its takes for standard microbiological cultures to be observed as individual cultures.