Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving viable micro-organism
Patent
1997-07-14
1999-01-19
Leary, Louise N.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving viable micro-organism
435 38, 435 14, 435848, 435849, 435968, C12Q 104, C12Q 110, C12Q 154, G01N 3353
Patent
active
058612700
DESCRIPTION:
BRIEF SUMMARY
RELATED APPLICATIONS
This application claims the priority of PCT Application No. PCT/BE95/00102/, filed Nov. 7, 1995, and PCT Application No. PCT/BE94/00083, filed Nov. 7, 1994, which are incorporated herein by reference.
The present invention relates to an enzymatic method for detecting coliform bacteria, in particular total coliform bacteria faecal coliform bacteria or E. coli, in a liquid or liquefied sample, for example drinking or recreational water, comprising the steps of: growth medium containing nutrients to support propagation of the bacteria and an inducer for inducing a marker enzyme in the course of their growth and metabolism; to form microcolonies of these bacteria on the membrane filter and to produce said marker enzyme; and
Coliform bacteria are indicators of the sanitary quality of water and food. Total coliforms (TC) in water originate from soil or organic vegetal material. Faecal (thermotolerant) coliforms (FC) and E. coli in particular inhabit the intestine of humans and animals and are indicators of faecal pollution.
Traditional processes for detecting coliforms and E. coli by membrane filtration are based on lactose fermentation in conjunction with confirmatory tests and require 48 to 96 hours to complete. A procedure is conventionally considered to be rapid if it takes 24 hours or less to perform. However, a 24 hours method is still not rapid enough to be used for the analysis of drinking water in emergency situations, e.g. after breakdowns in the water supply or construction works to the distribution system. In those cases, the detection of at least 1 coliform bacterium per 100 ml of water should be feasible within the ordinary work shift of 8 hours and preferably in maximum 7 hours to demonstrate the potability of the water and, hence to avoid unnecessary warnings to the public about the contrary.
Existing rapid (24 hours) membrane filtration methods for the detection of coliform bacteria, in particular TC and E. coli rely on the demonstration of the activity of 2 specific marker enzymes in the bacterial colonies, i.e. .beta.-galactosidase and .beta.-glucuronidase, respectively, which the bacteria produce as they grow and metabolize. The presence of these enzymes is revealed by the ability of the bacteria present on the membrane filter to cleave chromogenic substrates added to the growth medium such as 5-bromo-4-chloro-3-indolyl-.beta.-D-galactopyranoside (X-gal) for .beta.-galactosidase and 5bromo-4-chloro-3-indolyl-.beta.-D-glucuronide (X-gluc) for .beta.-glucuronidase. The chromogenic substrates themselves are not colored so that the detection of colored colonies on the membrane filter indicates the presence of the enzyme and, hence, of the bacteria. See e.g. Manafi and Kneifel, Zentralbl. Hyg. 189:225-234 (1989), Brenner et al., Appl. Environ. Microbiol. 59:3534-3544 (1993) and Frampton and Restaino, J. Appl. Bacteriol. 74:223-233 (1993).
Similarly, fluorogenic substrates, e.g. 4-methylumbelliferyl-.beta.-D-galactopyranoside (MU-gal) or 4-methylumbelliferyl-.beta.-D-glucuronide (MUG) added to the growth medium can be cleaved by bacterial .beta.-galactosidase and .beta.-glucuronidase, respectively, to yield a fluorescent product, 4-methylumbelliferone (4-MU). The fluorogenic substrates themselves do not fluoresce so that the detection of fluorescent colonies on the membrane filter indicates the presence of the enzyme and, hence, of the bacteria. Currently, the most rapid fluorescent method to detect TC on a membrane filter using MU-gal as a substrate for .beta.-galactosidase takes 16-24 hours to complete (Brenner, cited above). For E.coli the minimal detection time obtained by using MUG as a substrate for .beta.-glucuronidase is 7.5 hours (Sarhan and Foster, J. Appl. Bacterial. 70:394-400 1991)). The Berg et al. U.S. patent and scientific publication disclose a method to detect faecal (thermotolerant) coliforms on a membrane filter using an agar growth medium containing MU-gal as a substrate for .beta.-galactosidase and an incubation temperature of 41.5.degree. C., in a time p
REFERENCES:
Nelis et al; Proceedings of the Water Quality Tech. Conference AWWA; pp. 1663-1673; Nov. 1993.
De Marie et al; J. of Clinical Microbiology; pp. 255-258, Aug. 1984.
Leary Louise N.
Studie- en Samenwerkingsverband Vlaams Water
Universiteit Gent
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