antibiotic assay - (Feb/09/2012 )

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.

I am not so familiar with this assay, but I have done a lot of antifungal assays. From my experience, it is not all that uncommon for drugs to lose their activity. This could come from freeze-thaw, light exposure, etc. I would try a broth microdilution assay with the compounds to ensure that they are still active. Spectrophotometry is not even needed for this as just visual inspection of the plates will give you your answer. Also, just check that the range of drug concentrations is sufficient to observe decreased growth. The last thing, you could try taking the readings earlier. Maybe there is outgrowth after the antibiotics have had their effect and this could explain the same turbidity for all of the samples.