[Microbiology] Re: Reporting of enumeration results

John Gentile via microbio%40net.bio.net (by yjgent from nospamcox.net)
Fri Nov 7 11:38:45 EST 2008


On 2008-11-06 18:58:51 -0500, "N10" <limbic_lesion from hotmail.com> said:

> 
> "Loh Han Liat" <hanliat from tp.edu.sg> wrote in message
> news:mailman.643.1225999747.29717.microbio from net.bio.net...
> Hi All,
> 
> 
> 
> I've got questions regarding the reporting of enumeration results based
> on plating. The same issue occurs with several testing methods from
> FDA-BAM as well as AOAC, and I really need some good advice.
> 
> 
> 
> I'll use the example of enumerating for S. aureus, according to FDA-BAM.
> 
> 
> 
> Accordingly, I'll divide 1 ml of inoculum over 3 Baird-Parker agar
> plates. After incubation, I'll select plates that have 20-200 colonies,
> add up the S. aureus colonies on the 3 plates and report as count per g
> X the dilution factor. Of course prior to this, I would have determined
> the fraction of "true" S. aureus among the presumptive colonies and all
> that.
> 
> 
> 
> Now, the problem is I never get anywhere close to 20 colonies on any of
> my plates, for example, as in the table below.
> 
> 
> 
> Dilution
> 
> Plate 1 (0.4 ml)
> 
> Plate 2 (0.3 ml)
> 
> Plate 3 (0.3 ml)
> 
> 10-1
> 
> 5 colonies
> 
> 4 colonies
> 
> 5 colonies
> 
> 
> 
> Assume that I've already confirmed all 14 colonies are S. aureus, how do
> I report my results?
> 
> 
> 
> There's a clause somewhere in the manual that says if the count from the
> lowest dilution is < 20, I could still use the plates. Does this mean I
> could report the results as 140 cfu/g? If not, how should I report the
> results?
> 
> 
> 
> Also how should I report the results if there're no colonies on any of
> the plates?
> 
> 
> 
> Any advice, opinions or comments from anyone with any experience in this
> area would be greatly appreciated.
> 
> 
> 
> Thank you very much!
> 
> 
> 
> Regards
> 
> HL
> 
> 
> Hi Hl
> 
> 
> If you are following  your protocol exactly then you are no doubt returning
> the correct result. The problem with  microbiological assays of this type is
> that at low levels of isolation there is usually a high degree of "
> Uncertainty"  associated with the result of obtained   At  high  levels of
> uncertainty you really should report your result  with a caveat expresing
> the range for  upper an lower limits within which  results might reside.
> 
> YOu need to carry out a validation exercise to calculate the degree of
> uncertainty associated with this assay in your lab and possibley with
> respect to  concensus results in ring trials.   Uncertainty is a statiscally
> quanity related to  confidence limits ( therefore functions of varience and
> SD)  and which takes into account repeatability, reporducability , precision
> and bias. There are no set rules for the calculation of uncertainty  but
> some stable guide lines do exist based on sound statiscal criteria.
> 
> Thsi link might help as will many other ; google "Microbiological
> uncertainty of meadsurement
> 
> www.ifcc.org/ejifcc/vol13no4/130401006.htm
> 
> Until then report your results :- Any isolation of  confirm SA is
> significant ..usually.
> 
> Hope this helps

Your calculation seems to be correct, and if so, direct plating of your 
suspension should give sufficient countable colonies in the 20 - 200 
cfu range. It would confirm your dilution results.

-- 
John Gentile MS, M(ASCP)
Laboratory Information Mgr.
VA Medical Center
Providence, RI 
yjgent from cox.net



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