[maycrobiology]

Anyone who is familiar with Gramicidin (antibiotic) assay? I have been performing this test for quite a while, but I could not come up with a valid result. I am using USP as my reference for the procedure (Spectrophotometry). The problem is, the turbidity of my assay do not have a trend even if I increase the concentration of the antibiotic used for the Standard. I am working with a pure culture of Enterococcus hirae, and all the materials used was sterilized, so I am confident that my problem with turbidity is not caused by contamination. Any insight will be highly appreciated. Thanks in advance.

[CharonY]

Have you tried counting live-titers (i.e. plate assays?). Also have you established a good calibration curve between absorbance and actual cell count?

[maycrobiology]

CharonY, on 10 February 2012 - 05:01 PM, said:

Have you tried counting live-titers (i.e. plate assays?). Also have you established a good calibration curve between absorbance and actual cell count?

We have tried cylinder plate assay in other antibiotic assays that we do (like neomycin,bacitracin,polymyxin etc.). But according to our reference (USP 33), Gramicidin assay should be performed using spectrophotometry by reading the transmittance at 580 nm.

[CharonY]

Depends a little bit on how established the methods in your lab are. If results are unexpected I would unleash the whole range of controls to identify the problem.