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.
You are supposed to do the negative control at the same time as when you are testing the product.
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.
Negative controls should be included every time that the product is tested.
Growth promotion must be checked for each new batch of medium.
You do not have to test a previous batch in parallel. You can do the comparison 'on paper' if growth was clearly described
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.
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).
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.
The method states that the strains are inoculated individually. No mixed inoculum is permitted.
It is only the specified micro-organism under detection which is inoculated.
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.
Not always. For products differing only in amount of active ingredient a bracketing approach may be applied.
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.
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.
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.
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.
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.
Once you demonstrate that you have tried all possible approaches, then you can refer to the clause cited in your question.
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.
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>.
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.
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.
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.
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.
Methods using alternative media are considered as alternative methods, which may be used when consistent with General Notices 6.30.
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.
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.