Azide yeast dextrose-ethyl violet azide citrate broth method (AYD-EAC) for enterococcal test, reported by Horie in 1960, was reviewed and an improved method, AC (37°C) -AC (45°C), was developed in this work.In the new method, azide citrate medium (AC, polypepton, 20g; yeast extract, 5g; dextrose, 5g; Na-citrate·2H2O, 10g; NaCl, 5g; K2HPO4, 4g; KH2PO4, 1.5g; NaN3, 0.25g; distilled water, 1, 000ml) was employed both in presumptive and confirmatory test, respectively. Although the composition of AC medium resembled closely to that of EAC medium used in original method, ethyl violet was not used in the new medium.AC medium tubes inoculated with serial dilutions of the samples were incubated at 37°C for 48±3 hours. Tubes showing turbidity were transfered to fresh tubes of AC medium, using a wire loop (3mm in diameter). After the tubes were incubated for 48±3 hours at 45°C, tubes showing turbidity were considered positive confirmatory test.Using AYD-AC and AC-AC method, MPN values of enterococci in 27 samples of various contaminated waters, raw oyster and frozen foods were determined. It was shown that although MPN values obtained by both methods were comparable, AC medium gave less false positive tubes than AYD medium in presumptive test. Of 188 tubes recorded as positive in confirmatory test of AC-AC method, 187 were found to contain Gram-positive cocci, in pairs or short chains, which grew in 6.5% NaCl containing glucose broth and showed negative catalase test.It was known that AC-AC method was highly specific for the determination of entero-cocci in samples, such as contaminated waters or foods, which contain heterogeneous bacterial flora.
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HORIE et al. (1971) studied this question.