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Subject:
From:
Scott Sutton <[log in to unmask]>
Reply To:
The Pharmaceutical Microbiology Forum Email List <[log in to unmask]>
Date:
Tue, 30 May 2006 11:44:05 -0400
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Jeff,

No, that discussion is if the specification is 10 CFU/ml or gram, then the
maximum allowable observed value is 20.  In other words, a two-fold
variability for the specifications is allowed on the measured value.  The
two topics are apples and oranges - this section has nothing to do with
acceptable numbers of CFU on a plate.

You are correct though - this section was a compromise between USP (which
currently has no such allowance) and EP (which currently has a five-fold
allowance).

As to the specific phrase you cite last, I agree that the USP Microbial
Limits Chapter is not correct in this regard - the authors (predates my
involvement from 1993) confused the Limit of Detection for the plate count
method (1 CFU) with the Limit of Quantification (25 CFU).  

I share the blame for failing to correct this, but to be honest it has only
really become an issue as we are validating alternate methods and realizing
that the arbitrary values found in some guidance documents are unworkable.
This has forced a re-thinking of the situation.   Add to that the absurd EM
specifications (1 CFU is OK, 3 CFU is a major investigation) and you begin
to wonder if it might be time to apply some scientific rigor to the
regulatory arena in microbiology. 

Scott

=========
Scott Sutton, Ph.D.
Pharma Consultant, Microbiology
Vectech Pharmaceutical Consultants
585-594-8273
[log in to unmask]
This Message is privileged, confidential and protected by law from
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-----Original Message-----
From: The Pharmaceutical Microbiology Forum Email List
[mailto:[log in to unmask]] On Behalf Of Jeff S One
Sent: Tuesday, May 30, 2006 11:11 AM
To: [log in to unmask]
Subject: Re: [PMFLIST] Bioburden

Scott,

How does the new harmonized <61> statement affect your conclusion?  It 
seems they are suggesting 20 vs. 25 as the limiting reportable plate 
count.

When an acceptance criterion for microbiological quality is prescribed, it 
is interpreted as follows: 
.       101 cfu: maximum acceptable count = 20; 
.       102 cfu: maximum acceptable count = 200; 
.       103 cfu: maximum acceptable count = 2000; 
and so forth.
This harmonize chapter seems to say something different from the current 
<61> which states,  If no microbial colonies are recovered from the dishes 
representing the initial 1:10 dilution of the specimen, express the 
results as "less than 10 microorganisms per g or per mL of specimen."



Scott Sutton <[log in to unmask]> 
Sent by: The Pharmaceutical Microbiology Forum Email List 
<[log in to unmask]>
05/30/2006 06:42 AM
Please respond to
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Subject
Re: [PMFLIST] Bioburden






Linda,

The question, as I see it, isn't so much whether or not it sounds
reasonable.  "Reasonableness" is frequently based on our desires and
preconceptions.  The question is rather is it scientifically supportable.

Let's look at the question fresh - we can agree that in normal situations
the valid number of bacterial colonies to count on a plate is 25-250 (my
apologies, I was a little sloppy with this range on my previous Email). We
can agree that if there are more than 250 colonies on a plate, we report 
the
data out as TNTC (Too Numerous To Count).  So we agree on a countable 
range
and we agree on how to report excess at the high end.

How do we report excess at the low end of the countable range?  Do we 
ignore
it?  I suggest we change "accepted practice" and opt for good science in
this regard.  As we introduce more and more alternate, rapid 
microbiological
methods, we are going to be more often faced with the questions of 
accuracy
and precision in our test results. 

ISO 17025 "General Requirements for the Competence of Testing and
Calibration Laboratories" is an interesting read in this regard.  In 
section
5.4.5.2 of "Validation of Methods" you can find the statement "The
techniques used for the determination of the performance of a method 
should
be one of, or a combination of, the following: . . . assessment of the
uncertainty of the results based on scientific understanding of the
theoretical principles of the method and practical experience."  This
statement is only one of several dealing with validation (accuracy,
precision, range, etc - see USP <1223> or Pharm Eur 5.1.6) and uncertainty
of measurements to be found in that ISO standard. 

A question for testing labs certified under ISO 17025 - How do you meet 
the
requirements for understanding and reporting uncertainty in the data?

Back to the question at hand.   How do we report out data from a plate 
that
has too few colonies to be in the countable range?  Common practice is to
ignore the situation and report it out as if it were an accurate count.  I
have even seen situations were a lab will take duplicate plates with 
counts
of 0 and 1 and report out 0.5 CFU at a 1/10 dilution = 5 CFU/mL.  This
practice is not scientifically supportable.

Merely because it would be convenient to report out CFU down to 1 
CFU/plate
is not a justification for doing so.  These numbers are not even remotely
accurate and we are only digging deeper holes for ourselves by 
participating
in the charade - holes that will come to present major obstacles when we
attempt to validate an alternate method and are FORCED to apply rigorous
measures under USP or Pharm Eur guidance.

Yes, in answer to your question, the only defensible act in this situation
(in my opinion) is to acknowledge that the plate count method has a Limit 
of
Quantification, and the LOQ is 25 CFU/plate.  Pursuing this argument, if 
you
have counts that fall below the countable range of 25-250 CFU/plate on the
1/10 dilution you would report the data out as <250 CFU/mL.  Even if the
duplicate plate counts are 24 and 23 - they fall below the LOQ of the 
plate
count method.

Scott

=========
Scott Sutton, Ph.D.
Pharma Consultant, Microbiology
Vectech Pharmaceutical Consultants
585-594-8273
[log in to unmask]
This Message is privileged, confidential and protected by law from
disclosure. If you received this message in error, then forward it to
[log in to unmask] and delete it from your system.

-----Original Message-----
From: The Pharmaceutical Microbiology Forum Email List
[mailto:[log in to unmask]] On Behalf Of Linda K. Skowronsky
Sent: Friday, May 26, 2006 5:57 PM
To: [log in to unmask]
Subject: Re: [PMFLIST] Bioburden

In reply to Scott's response....I'm confused. Based on what you said, then 

if we have no growth on 2 plates from a 1:10 dilution and we can only 
report cfu in the countable range of 20-200, does this mean the minimum 
detection limit would be <200? Does this sound reasonable??? Or perhaps I 
misunderstood...

Best Regards,
Linda

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------------------
The PMFList (http://microbiol.org/PMFList_info.htm) is operated from
The Microbiology Network (http://microbiol.org) and supported by
our sponsors (http://microbiol.org/sponsor.htm) as a service to
the scientific community.

Please take a second to visit our sponsors' web sites and say thank you for their support of this service.

Accugenix - http://www.accugenix.com

American Type Culture Collection - http://www.atcc.org

ATS Laboratories - http://www.ats-labs.com

Biolog - http://www.biolog.com

Biomerieux - http://industry.biomerieux-usa.com

Dupont Qualicon - http://www.qualicon.com

EMD Chemicals - http://www.emdchemicals.com

EMSL Analytical, Inc. - http://www.emsl.com

Genomic Profiling Systems, Inc. - http://www.genprosys.com

MIDI, Inc. - http://www.midi-inc.com

Millipore - http://www.millipore.com

Pall - http://www.pall.com

NovaTek International - http://www.ntint.com

Raven Biological Labs - http://www.ravenlabs.com

Veltek Associates, Inc - http://www.sterile.com

=================================
The nature of this service is to provide a medium for communication.  The specific statements and endorsements of individuals participating in the discussions are not necessarily those of The Microbiology Network, Inc., the PMF, or the sponsors of the list.

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