Evaluation of indoxyl- β- d-glucuronide and hydrophobic grid membrane filters for electronic enumeration of Escherichia coli

Abstract

Glucuronidase (GUDase) positive organisms convert indoxyl- β - d -glucuronide (IBDG) to indigo. A library of 848 strains of Enterobacteriaceae which included 120 (pathogenic and non-pathogenic) Escherichia coli strains in arrays on three hydrophobic grid membrane filters was replicated and tested for detectability of indigo by an electronic counter using the permutation of conditions: IBDG 100, 200, 400, 600 and 800 μg ml −1 in lauryl sulfate tryptose agar (LSTA), tryptone bile lactose (1%) agar (TBLA) or buffered (pH 7) tryptone bile lactose agar (BTBLA); temperatures of 35.0 and 44.5 °C; and incubation times of 18 and 24 h. In pure cultures with IBDG at 400–800 μg/ml in TBLA or BTBLA the electronic counter detected the GUDase-positive E. coli strains after 18 h incubation at 35 or 44.5 °C, while only 80% were detected on LSTA. GUDase activity was detected in about 120 other strains ( Salmonella, Shigella, Hafnia spp.). A strongly and a weakly GUDase-positive E. coli culture were tested in competitive growth with all the other strains under the same permutation of conditions. Under the best conditions (BTBLA with 600–800 μg ml −1 IBDG at 44.5 °C) only 1% of GUDase-negative non- E. coli strains prevented detection of the strongly GUDase-positive E. coli , whereas about 30% prevented detection of the weakly GUDase-positive E. coli .