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Bioburden Test Problems

Bob Friedel rfriedel at perrittlab.com
Thu May 25 08:07:36 EST 2000

Antibiotics are generally inactivated enzymatically or by other chemical
means.  Rinsing with WFI is not going to eliminate the antimicrobial
activity.  DO NOT USE cellulose membranes as they have a history of binding
antibiotic materials (See below).  Then there is the potential problem of
the antibiotic being released from the membrane upon placement into the
broth enrichment.  The following references will give you the information
you need:

Russell, A.D., and Rogers, D.T. (1984) "Laboratory Uses of
Antibiotic-Inactivating Enzymes", J. Antimicrob. Chemother., Vol. 14, p.567.

Corry, J.E.L., Van Doorne, H., Mossel, D.A.A. (1977) "Recovery And Revival
Of Microbial Cells, Especially Those From Environments Containing
Antibiotics", In: Antibiotics in Agriculture, M. Woodbine (ed), Chapter 12,

Negretti, F. (1989) "Experimental Observations on the Bacteriological
Controls of the Antibiotics--I. Antibacterial Activity of Membranes Employed
in Bacteriological Assays", J. Pharma. & Biomed. Anal.,  Vol. 7, No. 12, p.

Options include:

a) increasing the concentration of the chemical inactivator

b) using an alternate membrane filter media (e.g., polyvinylidene difluoride
[PVDF] or polyethersulfone [PES]), as cellulose- based membranes tend to
bind antimicrobial materials.  If the organism(s) do not grow on the
antibiotic-filtered membrane, there is still activity within the membrane
(assuming that the membrane material itself was checked for antimicrobial
activity in the absence of the antibiotic).

c) increase the volume of the rinse

At some point in the process, you may need to state that the material under
consideration has enough intrinsic antimicrobial activity to warrant the
exclusion of the Microbial Limit Tests.  If you've conducted several
validation trials to date (as you have indicated) without much success, this
data will provide supporting justification for such a decision.

Also check the following website:

You can e-mail Professor A.D. Russell.  He will be able to make additional
suggestions if you can't get the references:  russelld2 at cf.ac.uk

Bob Friedel
Quality Assurance Manager
Perritt Laboratories
Hightstown, NJ USA
rfriedel at perrittlab.com

R.Randolph <microbiology at usa.net> wrote in message
news:v24qis4i11svp2tof4tk25r4t9vomi1rns at 4ax.com...
> You might consider adding SPS to your "flushings". R.Randolph
> ==============================
> On Fri, 31 Mar 2000 23:09:04 +1000, "Paul A. Yeatman" <pauly at eisa.net>
> wrote:
> >I am trying to validate a number of antibiotics using a 'bioburden test'.
> >Due to the antimicrobial nature of the products being tested, I am having
> >trouble getting our test organisms to grow.
> >
> >The products are vancomycin and gentamycin.  Out test organisms are
> >subtilis and Pseudomonas aeruginosa (at approx 100cfu/ml).
> >
> >Ideally we would filter 50 mls of the product using either a 2 micron
> >cellulose membrane, or a durapore membrane.  So far I have tried flushing
> >with sterile water WFI, followed by a regime of letting the membrane sit
> >immersed in Tryptone Soya broth  TSB for differeing amounts of time, in
> >affort to kick start the microbial growth.
> >
> >As it stand, I am up to about a 6000ml WFI flush, followed by 3x 20mins
> >TEB (for a total of 2000ml TEB), which is getting rather excessive.
> >
> >Does anyone know of a medium our media department can make that will
> >deactivate the antimicrobial effects of these antibiotics, while allowing
> >our t6est organisms to grow?
> >
> >I have tried a method similar to what we have successfully used for a
> >cytotoxic antimicrobial - 5500ml WFI followed by 3x 20min of TEB (1500ml
> >total) with a cellulose membrane, filtering only 5ml of product, but to
> >effect (returns were around 25% of the controls for gentamycin, and worse
> >for vancomycin).
> >
> >Any insight would be appecitated.
> >

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