USP Microbial Limits Prep Test

Enigl enigl at aol.com
Mon Nov 20 10:15:26 EST 1995

In article <48nntc$l3i$1 at mhafn.production.compuserve.com>, ATG
<76235.2473 at CompuServe.COM> writes:

>>Industrial microbiologists: what do you do when Prep Testing for 
>a pharmaceutical product does not validate your ability to 
>recover the test species, despite addition of sorbate, lecithin, 
>attempts at membrane filtration, etc.? >>

I wrote an SOP covering the process of Microbial Limit Tests validation.  
I used a flow chart approach.   Each step has a pass/fail (growth/
no-growth) question.  The USP preparatory test is followed with some
additions found in a newsletter, (I think it was)  _Microbiological
Update_ published by  Microbiological Applications, Inc. Edited by Dr.
Murray S. Cooper. 

PROBLEM 1:  The problem of neutralization of antimicrobials is compounded
because the USP requires a 10 grams minimum of test article (product). 
So, you can not simply dilute the product by adding less than 10 grams to
a diluent. . . Adding 0.001 grams of product to 90 mL of broth will not
comply with the 10 gram minimum.

In my procedure:  
To start, 10 grams is added to the inoculated conventional USP methods
media (separately) for the six tests:  total plate count (TPC), total
combined yeast and mold count (YMC), _Staph. aureus_ (SA)  _Ps.
aeruginosa_ (PA), _E. coli_ (EC) and _Salmonella_ sp.(SAL).  BTW we only
run the tests required by the UPS.  Not all six tests are required for all
our products. 

If growth is not found in a test, that part of the microbial limit test is
invalid and requires following down the flow chart to improve recovery of
the inoculum.  Since even TPC and YMC are validated by _ANY_ growth even
substantial killing by the product still validates the test.  

PROBLEM 2:  I believe validating the TPC/YMC by _any_ growth is an
oversight in the UPS.  I would prefer running a D-value to determine the
speed of kill (if any) and if a D-value is shown not to be infinitely
long,  the slope of the linear regression is negative, (i.e. not a
positive slope, growth-generation time), neutralization, dilution ,
filtration etc. should *still* take place.   

PROBLEM 3:  The inoculation suggested in the UPS is too high IMO.  I
calculated 10^5 CFU/mL.   I suggest testing at low high inoculum levels
e.g. 10-100 CFU, too.

First, the flow chart tries various neutralization methods (Modified
Letheen, D/E Neutralizing broth, HC Agar (YMC) , Microbial Content Test
Agar) _without_ dilution.  My reasoning is to reduce the amount of culture
media to made.  

Second, if neutralization by itself fails, the flow chart calls for
increasing dilution using the maximum neutralizing medium so far found
(Letheen, D/E Neutralizing broth, HC Agar, TSALT (microbial Content Test
Agar). If this fails, membrane filtration is attempted.  

(I have left out some steps, but you get the idea).

>> If the product is so 
>inhibitory, does it make sense to run microbial analysis on it in 
>the first place. <<

You have a good point. And, this is where the suggestions from
_Microbiological Update_ come in.

Dr. Cooper's newsletter (I hope this is where I got the suggestion, if not
sorry...) said if all else fails, try to define the antimicrobial 
spectrum.  Is the product killing mostly Gram negatives or positives? 
Enterics or pseudomonads?  yeast, mold?   Also, has there been a
processing change or vendor change that increases or decreases the
antimicrobial effect.  Use non-USP test strains to get a better picture.

The USP itself says: If all else fails the product is "Inherently
antimicrobial"  and the Microbial Limit Test(s) that fail need not be
performed except to occasionally check and make sure the product is still
killing what you think it is killing.

>>Or more importantly, can any one predict the 
>Agency's interpretation of this oxymoron (microbial limits on an 
>antimicrobial product/ingredient)?

We don't ever try to predict the FDA.  We try to produce a valid
scientific study to show th

More information about the Microbio mailing list

Send comments to us at biosci-help [At] net.bio.net