Development of a rapid positive/absent test for coliforms using sensitive bioluminescence assay

Abstract

I. MASUDA‐NISHIMURA, S. FUKUDA, A. SANO, K. KASAI and H. TATSUMI.2000.We have developed a sensitive bioluminescence assay for β‐galactosidase using a luminescent substrate, d‐luciferin‐O‐β‐galactopyranoside (LuGal). The detection limit for β‐galactosidase was 3 × 10−20 mol per assay, which was approximately 50‐fold more sensitive than the test using a fluorescent substrate. This assay was applied to a positive/absent (P/A) test for coliforms. Observations made after 7 h of culture followed by a 10‐min enzyme assay using LuGal were comparable to those made after a 22–24‐h culture by the current method. Therefore, the LuGal method allows a rapid P/A test for coliforms.

Bioluminescent bacteria: lux genes as environmental biosensors

“…Advances in molecular biology have made possible the cloning and expression of the luxAB genes in bacteria that are normally non-luminescent, transforming them into bioluminescent bacteria. The resulting transformations provide enormous applications (9,25) in industry (53), medicine (11,35), microbial ecology (6,29), and environmental fields (50).…”

A simple bioluminescence procedure for early warning detection of coliform bacteria in drinking water

“…The enzyme b-D-galactosidase has been targeted in a range of colorimetric and fluorometric assays focusing on coliform bacteria in water and food (Apte et al 1995;Van Poucke and Nelis 1995;Geisler et al 2000;George et al 2001;Tryland et al 2001;Van Poucke and Nelis 2000). However, enzyme assays based on luminescence have the potential to be more sensitive than detection methods based on chromogenic of fluorogenic substrates (Bronstein et al 1989;Van Poucke and Nelis 1995;Masuda-Nishimura et al 2000). Masuda-Nishimura et al (2000) used a custom made luminescent substrate and a special enrichment broth in a bioluminescence assay for detection of coliforms in laboratory samples.…”

“…However, enzyme assays based on luminescence have the potential to be more sensitive than detection methods based on chromogenic of fluorogenic substrates (Bronstein et al 1989;Van Poucke and Nelis 1995;Masuda-Nishimura et al 2000). Masuda-Nishimura et al (2000) used a custom made luminescent substrate and a special enrichment broth in a bioluminescence assay for detection of coliforms in laboratory samples. The study showed that bioluminescence results were equivalent to observations made using conventional methods (Masuda-Nishimura et al 2000).…”

“…Masuda-Nishimura et al (2000) used a custom made luminescent substrate and a special enrichment broth in a bioluminescence assay for detection of coliforms in laboratory samples. The study showed that bioluminescence results were equivalent to observations made using conventional methods (Masuda-Nishimura et al 2000). In the present study, the proposed bioluminescence procedure presented is based on a commercial bioluminescence assay (Beta-Glo), and can be used with commercial enrichment media such as Fluorocult or Colilert.…”

Rapid Enumeration of Microbial Cells in Solubilized Soymilk Using an Automatic Cell Counting System with LED Illumination

“…Therefore, a simple, rapid, and accurate detection method is desirable. Several non-culture methods have been used for the rapid detection of bacteria in food (Fujikawa and Morozuki, 2003;Gracias and Mckillip, 2004;Kawasaki et al, 2004;Kawasaki et al, 2007;Masuda-Nishimura et al, 2000;Miyamoto et al, 1998). PCR is a useful technique because of its high sensitivity.…”