Comparison of the direct enzyme assay method with the membrane filtration technique in the quantification and monitoring of microbial indicator organisms – seasonal variations in the activities of coliforms and E. coli, temperature and pH

Vc Wutor,Ca Togo,Bi Pletschke

doi:10.4314/wsa.v33i1.48783

Abstract

The aim of this project was to monitor variations and relationships between coliform and E. coli counts, the activities of their marker enzymes GAL and GUD, and temperature and pH over a period of 12 months in river samples obtained from the Eastern Cape, South Africa. Several polluted water samples were collected for direct coliform ƒÀ-D-galactosidase (B-GAL) and Escherichia coli ƒÀ-D-glucuronidase (B-GUD) assays and the membrane filtration technique. While all the samples showed enzyme activities, not all exhibited growth on CM1046 media. Variation in B-GAL activity (40%) was observed between November (highest activity month) and May (lowest activity month). The highest and lowest B-GUD activities were observed in the months of September and May/June, respectively. The sensitivity of the spectrophotometric assay method was indicated by a limit of detection (LOD) of 1 coliform forming unit (CFU)/100 m. and 2 CFU/100 m. for coliforms and E. coli, respectively. There was a significant (P < 0.05) positive correlation between E. coli counts and GUD activity (R2 = 0.8909). A correlation of R2 = 0.9151 was also observed between total coliforms and B-GAL activity, even though the CFUs were not evenly distributed. Direct enzyme assays were also shown to be more sensitive than the membrane filtration (MF) technique.

Highlights

Coliform bacteria have the ability to produce the enzyme β-Dgalactosidase (B-GAL) while Escherichia coli produce β-D-glucuronidase (B-GUD). These two enzymes have been used as selective markers for coliforms and E. coli respectively in the water quality industry for over 100 years
Chlorophenol red β-D-galactopyranoside (CPRG), disodium hydrogen phosphate, dihydrogen sodium phosphate were all obtained from Merck (Darmstadt, Germany)
Microbial counts by membrane filtration (MF), as outlined by Farnleitner et al (2001), were adapted and performed on Oxoid CM 1046

Summary

Introduction

Coliform bacteria have the ability to produce the enzyme β-Dgalactosidase (B-GAL) while Escherichia coli produce β-D-glucuronidase (B-GUD). These two enzymes have been used as selective markers for coliforms and E. coli respectively in the water quality industry for over 100 years. The traditional techniques of detecting and enumerating these micro-organisms require up to 72 h to perform. This required time is too long to provide useful information about a decrease in water quality that may require immediate attention to protect the public (Berg and Fiksdal, 1988). Direct enzyme assays, which are faster and not limited by VBNC cells, have become popular amongst water quality scientists

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