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 second of two Parts discussing additional FAQ from USP <62>.
Q. Why shall I use the shortest incubation period?
A. You have to show that the worst conditions work. Moreover you are working with healthy cells, these should give the required response in the shortest time.
Q. What does “The specified micro-organisms must be detected with the indication reactions as described under “Testing of Products” mean?
A. 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.
Q. 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 neutralised, then it is to be assumed that the inhibited microorganism will not be present in the product.”
A. Once you demonstrate that you have tried all possible approaches, then you can refer to the escape clause.
Q. What are “the Bile-tolerant Gram-negative bacteria”?
A. There is no strict definition of this group of microorganisms. 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 utilise glucose, e.g., some Bile Tolerant Gram Negative Bacteria includes members of the family Enterobacteriaceae, Pseudomonads and Aeromonas.
Q. In the sub-section Selection and Subculture under Escherichia coli , what is the purpose of the elevated temperature 42 – 44°C?
Can one utilise an alternative temperature range, e.g. 35 – 37°C provided that one demonstrates the recovery of E. coli during qualification?
A. The purpose of the elevated temperature, 42 – 44°C is to allow for selective conditions for faecal 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>.
Q. 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?
A. 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.
Q. 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”.
A. 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.
Q. 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.
A. 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.
Q. For which pharmaceutical products or raw materials is it prescribed to search for Clostridia?
A. For powdered animal organs, products with risk of faecal 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.
Q. Are there alternatives media that are acceptable?
A. Methods using alternative media are considered as alternative methods, which may be used when consistent with General Notices 6.30.
Q. 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?
A. 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.
Q. If we have growth problems of S. aureus and inhibitory problems of E. coli with mannitol salt agar medium that is recommended in the harmonised method, what is the cause?
A. The composition of mannitol salt agar has been optimised 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.