defined substrate methodology

Rapid, Specific, Defined Substrate Technology Colilert System for the Simultaneous Detection of Total Coliforms and Escherichia coli from Water

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• S. C. Edberg 4 ,
• M. J. Allen 5 &
• D. B. Smith 6  

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Potable water is subject to microbial contamination from a variety of sources. Microbes may enter a water supply as a consequence of cross-connections, back flow, disturbances during repairs, and back-siphonage. In addition, bacteria, including the total coliform group, may establish residence in distribution systems and become long-term inhabitants of its biofilm population (Ludwig et al. 1985; Edberg et al. 1986). The current means of analysis of water does not provide sufficient information to allow a water utility to ascertain the exact nature of its microbial problem. There is no membrane filter (MF) or multiple-tube fermentation (MTF) method that analyzes for a fecal-specific bacterial indicator. In addition, present methods are labor-intensive and costly and preclude the sampling of water supplies on an on-going basis. Monitoring is also infrequent. It is often days after an event that water companies and public health authorities have enough information to make decisions.

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Department of Laboratory Medicine, Yale University School of Medicine, New Haven, Connecticut, USA

S. C. Edberg

A.W.W.A Research Foundation, Denver, Colorado, USA

M. J. Allen

South Central Connecticut Regional Water Authority, New Haven, Connecticut, USA

D. B. Smith

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Department of Virology, University of Helsinki, Haartmaninkatu 3, SF-00290, Helsinki, Finland

Antti Vaheri

North American Laboratory Group, 1 Lake Street, 06052, New Britain, CT, USA

Richard C. Tilton

105 Bay Colt Road, 30201, Alpharetta, GA, USA

Albert Balows

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Edberg, S.C., Allen, M.J., Smith, D.B. (1991). Rapid, Specific, Defined Substrate Technology Colilert System for the Simultaneous Detection of Total Coliforms and Escherichia coli from Water. In: Vaheri, A., Tilton, R.C., Balows, A. (eds) Rapid Methods and Automation in Microbiology and Immunology. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-76603-9_53

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National field evaluation of a defined substrate method for the simultaneous enumeration of total coliforms and Escherichia coli from drinking water: comparison with the standard multiple tube fermentation method

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• 1 Department of Laboratory Medicine, Yale University School of Medicine, New Haven, Connecticut 06510.
• PMID: 3046490
• PMCID: PMC202702
• DOI: 10.1128/aem.54.6.1595-1601.1988

A defined substrate method was developed to simultaneously enumerate total coliforms and Escherichia coli from drinking waters without the need for confirmatory or completed tests. It is a new method based on technology that uses a hydrolyzable substrate as a specific indicator-nutrient for the target microbes. No equipment other than a 35 degrees C incubator and long-wavelength (366-nm) light is necessary. To perform the test, one only has to add water to the powdered ingredients in a tube or flask. If total coliforms are present in the water sample, the solution will change from its normal colorless state (no target microbes present) to yellow. The specific presence of E. coli will cause the same tube to fluoresce under a longwave (366-nm) UV lamp. The test, called Autoanalysis Colilert (AC), was compared with Standard Methods for the Examination of Water and Wastewater 10-tube multiple tube fermentation (MTF) in a national evaluation. Five utilities, representing six U.S. Environmental Protection Agency regions, participated. All water samples came from distribution systems. Split samples from a wide variety of water sources were analyzed for the MPN-versus-MPN comparison. A total of 1,086 tubes were positive by MTF, and 1,279 were positive by AC. There was no statistical difference between MTF and AC. Species identifications from positive tubes confirmed the sensitivity of the AC. A national evaluation of the AC test showed that it: (i) was as sensitive as Standard Methods MTF, (ii) specifically enumerated 1 total coliform per 100 ml, in a maximum of 24 h, (iii) simultaneously enumerated 1 E. coli per 100 ml in the same analysis, (iv) was not subject to false-positive or false-negative results by heterotrophic bacteria, (v) did not require confirmatory tests, (vi) grew injured coliforms, (vii) was easy to inoculate, and (viii) was very easy to interpret.

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• Bacteriological Techniques
• Enterobacteriaceae / growth & development*
• Escherichia coli / growth & development*
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Defined Substrate Technology Method for Rapid and Specific Simultaneous Enumerationof Total Coliforms and Escherichia coli from Water: Collaborative Study

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Stephen C Edberg, Martin J Allen, Darrell B Smith, Defined Substrate Technology Method for Rapid and Specific Simultaneous Enumerationof Total Coliforms and Escherichia coli from Water: Collaborative Study, Journal of Association of Official Analytical Chemists , Volume 74, Issue 3, 1 May 1991, Pages 526–529, https://doi.org/10.1093/jaoac/74.3.526

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The defined substrate technology (DST) method Is a reagent system designed to enumerate specific target microbes(s) from a mixture of bacteria. The system simultaneously enumerates total coliforms and Escherichia coll directly from a water sample. The reagent contains o-nitrophenyl-β-D-galactopyranoside (ONPG), which is hydrolyzed by total conforms to produce a yellow chromogen, and 4-methylumbelllferyl-β-D-glucuronide (MUG), which is hydrolyzed and fluoresces when E. coll organisms grow. Noncoliform bacteria are suppressed and cannot metabolize the indicator nutrients. Nine laboratories participated in a field evaluation of the method, which covered a wide range of surface and subsurface water sources and water-processing modalities, including the examination of natural samples. The DST system was compared to multiple-tube fermentation (MTF) (quantitative) and presence-absence (P-A) (qualitative) Standard Methods formats. Comparison of water samples from natural sources by using the most probable number (MPN) procedure showed that the DST test was equivalent to the currently used MTF test. Results from the DST and the qualitative P-A procedure showed that these tests agreed with each other in 94% of the water samples analyzed. Specificity of the DST method was established by subculturing a species consistent with a total conform or E. coll from each positive tube. Eight laboratories participated in a collaborative study of the method. Each laboratory received 3 concentrations of E. coll (organisms/100 mL): 10 (low); 60 (medium); and 120 (high). The DST test was inoculated from a split sample of each bacterial density In parallel with Standard Methods brilliant green lactose broth. Statistical analyses for repeatability and reproducibility showed the DST system to be equivalent to currently used standard methods. The DST method has been adopted official first action by AOAC for detection and enumeration of total coliforms and E. coll in water.

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