MODIFIED FLUOROMETRIC ASSAY FOR ESTIMATING AMPICILLOATE CONCENTRATIONS AND ITS USE FOR DETECTING BETA-LACTAMASE AND PENICILLIN ACYLASE ACTIVITY IN BACTERIA

Sodium ampicilloate concentrations were estimated fluorimetrically by heating solutions with ascorbic acid, EDTA and a modified Lowry A reag ent which was prepared by including copper sulfate and potassium sodiu m tartrate in 0.5 mol dm(-3) acetate buffer at pH 4. A concentration r ange of 0.5-50 mu mol dm(-3) was used for the estimations, The reactio n was used to estimate beta-lactamase activity on ampicillin but the s ubstrate also showed some fluorescence and a calculation was required to determine the amount of ampicilloate formed when both substances we re present in the one reaction mixture, The beta-lactamase was inhibit ed by treatment with trichloroacetic acid so the procedure could be us ed to assay the enzyme activity after a fixed time, 6-Aminopenicillani c acid did not fluoresce on treatment with the modified reagent and or ganisms which contained penicillin acylase lowered the amount of ampic illin which could be converted to ampicilloate. When penicillin acylas e and beta-lactamase co-existed in the one organism, the respective ac tivities were determined by use of the copper-ascorbate-EDTA fluoresce nce assay for ampicilloate coupled with a fluorescamine assay for 6-am inopenicillanic acid determinations, On prolonged incubation, some org anisms containing penicillin acylases lowered the amount of ampicilloa te which formed a fluorescent product, This effect was attributed to d eacylation of ampicilloate by the penicillin acylases.