We add 2 ml of our test sample to 100ml of our growth medium (CYLG I've the
recipe if you want it)
and overcome the inhibitory nature of samples by dilution.
We then aseptically distribute the medium into eight 10 ml aliquots.
The medium is then inoculated with 2 consecutive dilutions of
B. subtilis, Cl. sporogenes, St. aureus and C. albicans (i.e. 2 aliquots per
organism)
to give a inoculum size of up to 100 cfu.
Control blood agars are inoculated with the dilutions of the test orgs to
assess the inoculum size.
--
David Lawton
"Paul A. Yeatman" <pauly at eisa.net> wrote in message
news:8c242u$6b2$1 at news.eisa.net.au...
> 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
Bacillus
> 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 an
> affort to kick start the microbial growth.
>> As it stand, I am up to about a 6000ml WFI flush, followed by 3x 20mins of
> 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 no
> effect (returns were around 25% of the controls for gentamycin, and worse
> for vancomycin).
>> Any insight would be appecitated.
>>
