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USP 51 and Microbial Recovery Incubation Time

incubation usp recovery antimicrobial

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#1 GaoYuQing

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Posted 10 February 2012 - 08:46 AM

Well, I'm looking to base a test method on USP 51 Antimicrobial Effectiveness Testing and I'm having trouble understanding what the USP is intending. Anyone done this before? I'll copy the relevant section:

PREPARATION OF INOCULUM



Preparatory to the test, inoculate the surface of a suitable volume of solid agar medium from a recently revived stock culture of each of the specified microorganisms. The culture conditions for the inoculum culture are described in Table 2 in which the suitable media are Soybean–Casein Digest or Sabouraud Dextrose Agar Medium .
To harvest the bacterial and C. albicans cultures, use sterile saline TS, washing the surface growth, collecting it in a suitable vessel, and adding sufficient sterile saline TS to obtain a microbial count of about 1 × 108 colony-forming units (cfu) per mL. To harvest the cells of A. niger, use sterile saline TS containing 0.05% of polysorbate 80, and add sufficient sterile saline TS to obtain a count of about 1 × 108 cfu per mL.
Alternatively, the stock culture organisms may be grown in a suitable liquid medium (i.e., Soybean–Casein Digest Broth or Sabouraud Dextrose Broth) and the cells harvested by centrifugation, then washed and resuspended in sterile saline TS to obtain a microbial count of about 1 × 108 cfu per mL. [Note—The estimate of inoculum concentration may be performed by turbidimetric measurements for the challenge microorganisms. Refrigerate the suspension if it is not used within 2 hours. ]
Determine the number of cfu per mL in each suspension, using the conditions of media and microbial recovery incubation times listed in Table 2 to confirm the initial cfu per mL estimate. This value serves to calibrate the size of inoculum used in the test. The bacterial and yeast suspensions are to be used within 24 hours of harvest, but the fungal preparation may be stored under refrigeration for up to 7 days.

The table 2 they reference has a column for incubation temp, inoculum incubation time and microbial recovery time. This is what's throwing me off.
Way I see it, you get a starting population of 10^8 CFU's based on turbidometric measurements (and if anyone knows of a good chart to show this it'd be appreciated) and then you...what? Where do the incubation and recovery times come into play? They say to use the suspensions within 24 hours. How do you use these times to determine the population? I'd just take a sample and do a membrane filtration test on it to get the population, run concurrently with the actual inoculated samples.
Anyone run this before or understand what they are talking about with these "inoculum incubation time" and "microbial recovery times?" I suspect they are making it more complicated than it is.
First time post, hope to scan the archives soon to glean some good info Posted Image

#2 pito

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Posted 10 February 2012 - 10:11 AM

I dont really understand what your problem is.
I think you are misreading something or maybe just misunderstanding something?

The way how I read/understand it, is that the "inoculum incubation time" and "microbial recovery times" refer to "how long it takes to get a 10*8 cfu inoculation". (altough microbial recovery time might refer to something else too, but I dont know this test)

You just need to grow the bacteria till 10*8 cfu and then you can start working with them.. and how don you know they are 10*8, well by doing a counting or turbidity test..or..
I think you are just misled by the text , not so much the proces itself.

If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.


#3 GaoYuQing

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Posted 10 February 2012 - 10:26 AM

It certainly is possible that I'm misunderstanding or misreading something. Would not be the first time and would be a neat solution to my quandry. However, the table 2 gives, for instance, 18-24 hours for inoc inc time and 3-5 days for microbial recovery inc time for the same organism. they're talking about two completely different things there. Also it appears as if they are telling you to use those conditions to determine the population, after you've already determined the 10^8 population density, which seems a non sequitor. It'd be easier to just grow it to 10^8, but all the rest of the stuff they are talking about muddy the waters.

#4 pito

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Posted 11 February 2012 - 01:47 AM

Eum, maybe they mean it takes 18-24 hours to incubate your sample (for example flesh or fish or whatever you test) and 3-5 days for the microbial recovery time.
As a positive test, to see if they grow at all.
(I mean: if you would like to test an antibiotic and the product itself does not allow growth... then there is no point in testing that antibiotic at all on that product).

I dont know the test , so thats what I think about it and while reading this (http://www.antimicro...llenge_test.htm) it does seem to be something like that (check Summary of the USP <51> Antimicrobial Effectiveness Test: and then this: A recovery analysis is performed to verify that microorganisms present in a sample can be adequately recovered and enumerated using the chosen dilution and plating scheme.)

But again, I dont know this test.
Maybe you should mail the seller of the test? THey should be able to give you all the information.
Sometimes its just about "words" (english not being your native langauge?) rather then the test itself.

If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.


#5 GaoYuQing

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Posted 13 February 2012 - 05:14 AM

But again, I dont know this test.
Maybe you should mail the seller of the test? THey should be able to give you all the information.
Sometimes its just about "words" (english not being your native langauge?) rather then the test itself.


Heh, the writer of the test is the USP. Not that that means they have any better understanding of simple English ;) Will check that link you sent.

#6 pito

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Posted 13 February 2012 - 05:51 AM


But again, I dont know this test.
Maybe you should mail the seller of the test? THey should be able to give you all the information.
Sometimes its just about "words" (english not being your native langauge?) rather then the test itself.


Heh, the writer of the test is the USP. Not that that means they have any better understanding of simple English Posted Image Will check that link you sent.


I dont mean they dont speak good english, I mean that you can ask/mail them for more information.
Mail the seller for more info, normally they have a customerservice where you can ask questions.

If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.


#7 Phil Geis

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Posted 17 February 2012 - 07:54 PM

USP 51 is Antimicribial Effectiveness Testing. You don;t know the microbial cocnentration precisely - you can only estuimate that it has reached the right density turbidometrically. You need to plate so you have an accurate estimate of the inoculum as performance in the test is judged vs. that count.





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