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Bob Friedel <[log in to unmask]>
Reply To:
The Pharmaceutical Microbiology Forum Email List <[log in to unmask]>
Sat, 29 Apr 2017 15:20:44 -0400
text/plain (176 lines)
There are a couple of ways to nullify the effect of antimicrobials:

1) Chemically-neutralize or dilute to sub-inhibitory levels
2) Physically separate (involving membrane filtration)
3) Both 1 & 2

Your method validation exercise should be conducted prior to routine implementation in the QA/QC Microbiology Lab.  The organisms of interest are inoculated directly into the Petri dish containing all of the experimental diluents/media/product (post-manipulation).  Post-incubation recoveries are then compared against one another as follows: 


Control #1 = Agar + diluent
-Tells us whether the organisms can be recovered.

Control #2 = Agar + diluent + antimicrobial neutralizing chemical(s)
-Tells us whether the organisms can be recovered in the presence of the neutralizing chemicals.

Control #3 = Agar + diluent + antimicrobial neutralizing chemical(s) + raw material or product
-Tells us whether the organisms can be recovered in the presence of actual product.

For filtration, inoculating directly into the filter funnel containing the 3rd rinse is appropriate.  Post-incubation recoveries are then compared against one another. 

Membrane filtration:

Control #1 = Agar + diluent + membrane
-Tells us whether the organisms can be recovered.

Control #2 = Agar + diluent + membrane + antimicrobial neutralizing chemical(s)
-Tells us whether the organisms can be recovered in the presence of the neutralizing chemical(s).

Control #3 = Agar + diluent + membrane + antimicrobial neutralizing chemical(s) + raw material or product
-Tells us whether the organisms can be recovered in the presence of actual product.

The overall goal is to ascertain if there are any factors which (if present) impede the organisms from being recovered in the Petri dish or on the membrane filter.

Note: I do not have the latest copy of the ASTM Protocol E1054-08 (2013)  “Standard Practices for Evaluating Inactivators of Antimicrobial Agents Used in Disinfectant, Sanitizer, Antiseptic or Preserved Products.” <> but “back in the day” they advocated the surface-streak methodology when pour-plating actually took place.  This was in reference to plates that showed no growth after incubation.  The problem is that pour-plating and surface-streaking are two different methodologies.  And while having good intentions, the procedure [at that time] was misleading (and in my opinion “flawed”), as it advocated determining neutralization effectiveness post-incubation; not at the time the particular sample was manipulated and pour-plated.  ASTM’s position may have changed but I am unable to confirm this.

Sutton, S.V.W. (1996) "Neutralizer Evaluations as Control Experiments for Antimicrobial Efficacy Tests,” In: Handbook of Disinfectants and Antiseptics, J.M. Ascenzi (ed), Chapter 3, pp. 43-62.

Dey, B.P and F.B. Englué Jr. (1994) “Neutralization of Antimicrobial Chemicals by Recovery Media,” J. Microbial. Methods, Vol. 19, pp. 51-58.

Casetta, P. and F. Negletti (1992) “Microbiological Controls of Antiseptics and Disinfectants by Membrane Filtration: Experimental Evaluation of the Interferences,” Boll. Chim. Farmaceutico, Vol. 131, No. 6, pp. 238-241.

Proud, D.W. and S.V.W. Sutton (1992) “Development of a Universal Diluting Fluid for Membrane Filtration Sterility Testing,” Appl. Environ. Microbial., Vol. 58, No. 3, pp. 1035-1038.

Negretti, F. (1989) “Experimental Observations on the Bacteriological Control of the Antibiotics- I. Antibacterial Activity of Membranes Employed in Bacteriological Assays,” J. Pharm. & Biomed. Analysis, Vol. 7, No. 12, pp. 1861-1865.

Singer, S. (1987) “The Use of Preservative Neutralizers in Diluents and Plating Media,” Cosmet. & Toiletries, Vol. 102, Dec., pp. 55-60.

Naidoo, N.T., Price, C.H., and T.J. McCarthy (1971) “Adsorption of Benzalkonium Chloride onto Different Filter Media During Bacteriological Filtration,” Pharm. Weekblad, Vol. 106, pp. 509-514.

Van Ooteghem, M. and H. Heriots (1969) “The Adsorption of Preservatives on Membrane Filters,” Pharm. Acta, Helv., Vol. 44, pp. 610-619.

Kayser, A. and G. Vad de Ploeg (1965) “Growth Inhibition of Staphylococci by Sodium Thiosulfate,” J. Appl. Bacterial., Vol. 28, No. 2, pp. 286-293.

Bob Friedel

> On Apr 28, 2017, at 6:38 PM, Aaron Fielder <[log in to unmask]> wrote:
> Hi Tony,
>    I must agree with Julie. Surely the intent of the validation is to show that the actual test is capable of identifying and recovering sub-lethally damaged cells from actual raw materials (or finished goods for that matter)? In that case, we have to challenge the inoculae against the product as best we are able before trying to prove that the test method will recover them. That pre-exposure time will necessarily be arbitrary as, in real life, the native bugs will have been exposed for an extended period, from date of manufacture of the material, but an arbitrary lab decided time frame is a better challenge than simply showing that insoluble residues remain to stress the organisms after numerous rinsing steps or partially neutralized by agar. If we apply the USP method as written, and there are no residues, then the challenge organisms actually never meet the product at any stage of the validation, which doesn't seem particularly useful?
> best regards, Aaron
> On 24/04/2017 10:20 PM, Tony Cundell wrote:
>> I must disagree with Julie. The issue is not the survival in the product
>> matrix or diluted product but whether the product residues prevent the
>> recovery of the inoculum in the culture. This is apparent on reading the
>> USP chapters.
>> For example, an aliquot of the dilution and a method suitability
>> microorganisms at <100 CFU is added to the sterile Petri dish, mixed, and
>> the molten agar is added, mixed again to fully disperse the two within the
>> agar. The plate is incubated and the recovery compared to a plate
>> inoculated without the aliquot. The acceptance criterion is between 50 and
>> 200%. Typically it is around 100%.
>> Tony
>> On Sun, Apr 23, 2017 at 11:06 AM, Julie B <[log in to unmask]> wrote:
>>> Saiyem,
>>> it will depend on antimicrobial activity of your product. When you to
>>> execute method suitability via pour plate method you should add
>>> microorganisms into your product matrix (i.e. product and dilutent
>>> combination), and then plate aliquotes out. It should be no different with
>>> filtration method. No, if you experiencing inhibition, then yes you can add
>>> organisms into last rinse as it was recommended previously. However, I
>>> would not jump directly to the adding organisms to the last rinse without
>>> proving that adding organisms into product matrix does not work.
>>> Hope that helps
>>> Julie Barlasov, MBA
>>> [log in to unmask]
>>> Julie Barlasov, MBA
>>> [log in to unmask]
>>> (908) 342-3582
>>> On Fri, Apr 21, 2017 at 12:51 PM, moataz el gaaly <
>>> [log in to unmask]>
>>> wrote:
>>>> During the last third wash
>>>>  Hope it helps
>>>> Sent from my HTC
>>>> ----- Reply message -----
>>>> From: "Muhammod Saiyem" <[log in to unmask]>
>>>> To: "[log in to unmask]" <[log in to unmask]
>>>> Subject: [PMFLIST] MET method validation: recovery of microorganisms
>>>> Date: Fri, Apr 21, 2017 16:59
>>>> Dear All
>>>> During method validation of microbial enumeration test of raw materials
>>> by
>>>> filtration, at which stage should I add test organisms?
>>>> Is it to the prepared sample solution, then filter & wash the filter
>>> paper
>>>> 3 times with suitable diluent (3*100ml)?
>>>> Or at first filter the sample solution, then wash the filter paper 2
>>> times
>>>> and add test organisms into the filtration cup during last time (3rd)
>>>> washing?
>>>> Please let me know your ideas.
>>>> Regards
>>>> Saiyem
>>>> Square Pharmaceuticals Ltd.
>>>> Bangladesh

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