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欧洲药典2.6.13的问题的回答

2021-11-28 来源:钮旅网


Frequently asked questions on chapter 2.6.13 Microbiological examination of non-sterile products: test for specified micro-organisms B. Harmonised method

Can I use other strains than You must use the micro-those that are cited in the organisms that are cited Ph.Eur.? in this chapter, or

equivalent strains from other culture collections.

3.2 Negative control When are you actually You are supposed to do

supposed to do the negative the negative control at control: when testing the the same time as when suitability of the method, or you are testing the when testing the product, or in product. both situations?

What is the purpose of the The purpose of this negative control? 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.

Does it have to be done every It is preferable to do a time the product is tested or negative control every during the method validation or time that the product is is it possible to do it tested. periodically? PARAGRAPH CONCERNED 3.1 Preparation of the test strain

QUESTIONS

ANSWERS

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3.3 Growth promotion and inhibitory properties

of the media

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 systematically in parallel a previous and approved batch in order to compare with the new batch?

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?

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

Growth promotion must be tested for each new batch of medium (either by the supplier or the analyst).

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 2.6.12 can be used. No strict prescription 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).

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3.3 Growth promotion and inhibitory properties

of the media

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?

3.4 Suitability of the Does it mean that for each test

test method strain, individual suitability tests have to be performed, or is it possible to use a mixed inoculum of all 4 strains?

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 micro-organisms are added individually to the growth medium for each of the specific test methods?

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 sub-culturing 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.

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3.4 Suitability of the Which test micro-organisms

test method should one use? Just the same

micro-organisms as used for testing the growth promoting properties of the respective media, or also the micro-organisms used for testing inhibitory properties of the media?

4.1 Bile-tolerant What are “the Bile-tolerant Gram-negative Gram-negative bacteria”?

bacteria

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. 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 Pseudomonads such as Burkholderia cepacia. Gram negative bacteria that grow in the presence of bile salts and which are non-lactose fermenting but able to utilise glucose, can grow on the agar eg. some Pseudomonas,

Aeromonas and Yersinia species.

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4.2 Escherichia coli In 4-2-2 selection and

subculture, what is the purpose of the elevated temperature 42 - 44°C?

Can one utilise an alternative temperature range, eg. 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 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 Ph. Eur. method, but you can always use alternatives methods as described in the general notices of the Ph. Eur. (chapter 1.2).

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4.1.3-2 Interpretation In chapter 5.1.4, table 5.1.4-1,

acceptance criteria for microbiological quality of non-sterile dosage form the specification for bile-tolerant gram-negative bacteria is given in CFU. In chapter 2.6.13 under 4. Testing of products, it is stated that the probable number of bacteria is determined for bile-tolerant gram-negative bacteria. This is a discrepancy, because CFU is a measure of viable bacterial numbers whereas the determined number of bacteria would enfolded all cells, dead and living. Could you advise on the correct units to be used for the Bile-tolerant test? Should it be most probable of bacteria/mL or CFU/mL or both these units are interchangeable?

4.2.3 Interpretation 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\".

There is a little discrepancy, because \"bacteria\" per g in itself is not correct since these are indeed colonies. However in this context the words can be considered as interchangeable.

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.

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4.5.3 Interpretation

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.

4.6 Clostridia

Your product can be contaminated, maybe not by the species described in the Ph.Eur. but by another micro-organism. Good laboratory practice should make you think that there is a problem and that you should investigate (eg 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 which pharmaceutical For powdered animal products or raw materials is it organs, products with prescribed to search for risk of faecal Clostridia? 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.

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5 Recommended solutions and culture media

Are there alternatives media Methods using that are acceptable? alternative media are considered as

alternative methods, which may be used, provided that their equivalence to the Ph. Eur. methods has been demonstrated.

How can we do the pH You may use a robust measurement for culture electrode. There are media? It is written to measure electrodes for the pH at 25°C. At this measurement in temperature, agar is solid. semisolid samples such Therefore, how can we adjust as meat, cheese and the pH? 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 criteria for pH is met in the final medium.

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5 Recommended solutions and culture media

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?

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.

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