positive control for antagonism assay

[Mike Dew]

This will sound like a very basic question, anyway...

In an experiment to detect bacteriocin-producing bacteria against L. 
monocytogenes, I am trying to make a positive control with nisin in an 
(adapted) deferred antagonism assay, but I have a problem. Briefly the 
procedure is:

-adding nisin (0,3 mg/mL, from Sigma 2.5% nisin) in a sterilised culture 
supernatant (no proteolytic enzymes added);
-multiple pH adjustments from 4.8 to 7.1 in tubes to fit the best pH;
-2 uL spots on a TSA plate;
-thin overlay with BHI agar seeded with Listeria
-incubation at 30 deg C 24 h.

Result: no inhibition zones at all ! Listeria feels pretty good in the 
overlay. But I have inhibition zones around the lactic acid bacteria 
colonies cultured by the same method... What is wrong ?

Thank you for help ...

M. Dew
Quebec, Canada
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