USP Microbial Limits Prep Test

Enigl enigl at aol.com
Thu Nov 23 09:13:24 EST 1995

Thank you for your questions:

<< Question 1: How long does it take for you to validate each material

Your question has two answers: technician time and time including
incubation.  I only run the prep test once a month in one batch.  

1.  Starting on a Friday I rehydrate Culti-loops with the ATCC strains,
streaking for isolation.  

2.  On Monday I pick the isolated colonies to the cultivation media. 

3.  On Tuesday I inoculate the test broths(TSB and Lactose)/TPC/YMC media
and add the product.  

4.  Each test incubates and on Wednesday the broths are subcultured.  

5.  On Thursday the PA and  SAL are subcultured and EC (MacConkey) is read
and subcultured, Vitek I.D. etc. 

6.  On Friday the SAL media is read and subcultured to blood agar for
Vitek I.D.  Also on Friday, I read the TPC plates and PA is subcultured
from Cetrimide to PAAP and PAAF and incubated over the weekend.   

7.  On Monday, the PAAP and PAAF are subcultured to blood agar for Vitek

8. On Tuesday, the YMC is read. Viteks are finalized. Report is ready for
analysis and possible re-running due to inhibition.

So, in technician time it takes very little more than the regular test(s)
that is positive. Only the inoculation step is added.  Including
incubation time, it takes only two days extra (Culti-Loop on the first
Friday and subculture on the first Monday prior to inoculation).  Total
time is 8 working days.  If re-running is required, it takes 8 more days.

<<Question 2: Why not start with filtration? If you go by the easiest
route to 
<<validate, then all materials are tested the same, simplifying

The USP forbids _starting_ with the filtration method and puts severe
restrictions for using it e.g. the product must be substantially dissolve
and filterable.  Only 3 of our 60 products meet this requirement.  The
filter method must be "validated" itself.  This is assumed to mean using
the same criteria as the sterility test including bacteriostasis testing,
filter degradation/retention testing.  This work adds up.  The FDA expects
us to show that the filter is not harmed by the products so holes would be
created and let bacteria go through the filter.  If this happens, we would
get a (potentially very dangerous) false negative (e.g. Salmonella).  I
once had an alcohol based product dissolve the CN filter.  WOOPS!  I
switched to Nylon 66, but could not get retention of  _Pseudomonas
diminuta_ or even some _Ps. aeruginosa_ strains.  They passed straight
through the filter.  

Filtration can work and I developed a method using a modified DE
Neutralizing broth rinse I saw in _Applied and Environmental

<<Question 3: What kind of materials are you routinely testing? 

We are a pharmaceutical manufacturer.  Some of our (60) products are: 
Amoxapine, Clorazepate, Fenoprofen, Hydrocodone, Imdomethacin, Lorazepam,
Loxapine, Maprotiline, Triamterene, Verapamil, Albuterol sulfate and
Cyclobenzaprine.  Many of them are highly antimicrobial. All are
non-sterile. Most are solid (dry or low water activity aw < 0.80) dosage
forms,  transdermal patch and preserved liquids.

<<Question 4: What do you do with antibiotic raw materials which are to be

I would suggest, filtration is the current best way.  See specific
restrictions in the USP and validate as described above.  If you have
another way please let me know.  I'm always looking for a better, easier

Retention of the antibiotic by the filter is a problem.  I've seen where
the center of the filter is cutout  and cultured separately from the
edges.   The antimicrobial effects are lessened on bacteria by culturing
the part of the filter with less antibiotic retention.   The part of the
filter with more antibiotic retention is cultured for fungus because
fungus may not be affected at all by antibacterial antibiotics.


Dynamically Optimistic!
Davin C. Enigl
Send Internet e-mail to: enigl at aol.com
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