Monday, July 30, 2007
P84

Determining gramicidin potency using Enterococcus hirae grown in an improved Antibiotic Assay Medium #3 formulation

Ned T. Barden, Biological Sciences, Massachusetts College of Pharmacy and Health Sciences, 179 Longwood Avenue, Boston, MA 02115

Official microbiological assay protocols for determining antibiotic potencies are published in the U.S. Pharmacopoeia (USP) that dictates both the specific bacterial test species and its growth medium for each antibiotic.  For gramicidin potency the specified medium, Antibiotic Assay Medium #3 (AAM), especially formulations from certain manufacturers, often have not supported adequate growth of the fastidious test species, Enterococcus hirae ATCC 10541 (Eh).  Recently, experimental enhancements in the AAM formulation have been shown to provide vastly improved growth of Eh.  In this current study various gramicidin assay trials were performed to determine the suitability of using the improved AAM in the official assay protocol.  In the trials the improved medium consistently provided Eh inocula 2-3X the cell concentration after the protocol’s 3-4 hr incubation period compared to the original AAM formulation.  The improved inocula gave very good turbidimetric 4-dose response curves (good linear portion with slightly sigmoid portions at high and low concentrations) using the protocol’s specified gramicidin range for establishing the standard curve.  Multiple trials using the improved medium were consistently accurate in determining the gramicidin potency of coded, blind “unknowns” prepared in the lab.  Based on the successful demonstration of the suitability of the improved medium to perform well in this assay for gramicidin the USP organization will be notified regarding the AAM modifications.

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