FOODMICRO 99
 
 
 
 
 

M. MANAFI and H. ROSMANN, Hygiene Institute, University of Vienna, Kinderspitalgasse 15, A-1095, Vienna, Austria. 
Tel. 43/1/40490/79450, Fax 43/1/40490/9794, e-mail: Mohammad.Manafi@univie.ac.at

Because of the potential consequences of waterborne diseases, microbial contamination is still considered to be the most critical risk factor in drinking water quality. The WHO Guidelines for Drinking-water Quality (WHO, 1993) states that it is impractical to monitor drinking water for every possible microbial pathogen. Monitoring the microbiological quality of drinking water relies largely on examination for indicators bacteria such as coliforms, E. coli and enterococci using membrane filtration technique (MF), the multiple-tube fermentation test (MPN), and the presence-absence test (P/A). MPN and P/A tests require a minimum of 2 days, followed by a further 2 days for the confirmatory identification of typical isolates. There is a need for rapid methods particularly in emergencies, to quickly determine the indicator bacteria and pathogens in water. As both, coliforms and E. coli are still important indicators of water pollution, there arises the necessity to create media which is able to detect both bacteria. This would then guarantee a better performance of microbiological quality control. Several attempts have been made to simultaneously detect coliforms and E. coli and novel methods have been introduced, based on the detection of ß-D-galactosidase (ßGAL) and ß-D-glucuronidase (GUD) using fluorogenic and/or chromogenic substrates. 
The enzymatic definition of coliforms, which is not method related, is the possession of ß-galactosidase gene which is responsible for the cleavage of lactose into glucose and galactose by the enzyme ßGAL. ßGAL is an inducible enzyme and enzyme production in bacteria requires induction of the lac-operon. GUD is an enzyme that catalyzes the hydrolysis of ß-D-glucopyranosiduronic acids into their corresponding aglycons and D-glucuronic acid. GUD is a characteristic enzymatic reaction expressed by E. coli strains. 
Readycult Coliforms (RC, Merck) is a new selective and differential medium for determination of the presence or absence of total coliforms (TC) and E. coli in drinking and surface water. This medium, containing 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (X-Gal) and 4-methylumbelliferyl-ß-D-glucuronide (MUG), is presented as a pre-dispensed, sterile powder which can be added directly to the water sample. MUG is hydrolyzed by GUD yielding 4-MU, which shows blue fluorescence when irradiated with long-wave UV light (365 nm). The activity of the enzyme ß-D-galactosidase produces a blue-green coloration in the watersamples, thereby indicating the presence of coliforms. 

A total of 300 drinking, and surface water samples were collected from different sources in Austria. All samples were transported in an icebox and bacteriological analysis was initiated immediately. Two internationally used standards procedures (the DIN procedure and the E.P.A. precedure) and the Readycult system were used to recover the total coliforms and E. coli in each water sample. To confirm presence of E. coli indole production can be demonstrated using indole reagent. Any positive bottles were subcultured by streaking on eosin methylene blue lactose sucrose agar (EMB). The organisms were isolated and identified using API 20E test strips.

The comparison of tested methods for detecting E. coli and coliforms in water samples and number of confirmed positive and false positive samples are presented. Out of 300 samples, a total of 90 (62 E. coli) confirmed vessels were positive by the E.P.A test, 94 (60 E. coli) by the DIN test and 100 (63 E. coli) by the RC system. False-positive results for coliforms and E.coli  were by the E.P.A. test 1(1), by the DIN test 1 (2) and by the RC system 3 (0). The false positive results were Aeromonas hydrophila strains. With the EPA method one E. coli strain and with the DIN method two E. coli strains could not be detected. 

Media containing X-Gal are already used in water monitoring. It was found that coliform strains produced sharp blue colonies on the agar plate because of insolubility of the indigo dye, which does not alter the viability of the colonies. Looking for E. coli and coliforms in watersamples, X-Gal was a faster and more sensitive parameter for total coliforms than gas production from lactose. 

In the present study, only a few non-coliform bacteria such as strains of A. hydrophila gave false-positive reactions with X-Gal, which are considered ubiquitous waterborne organisms and should not be present in drinking water. On the other hand, adding Cefsulodin at 5 to 10µg/ml to media inhibited the growth of Aeromonas and Flavobacterium species. Our results showed that Readycult detected coliforms and E coli in significantly more samples at 24 hours than standard methods. The detection of total coliforms and E. coli with Readycult is reduced to 1 day, compared to 4 days by EPA method and 3 days by DIN method. RC method has proven by experience to be reasonably adequate for practical purposes. The Readycult system was as sensitive as Standard Methods and it simultaneously enumerated E. coli and coliforms in the same analysis. The efficiency and rapidity of the detectable reactions make this medium a very useful tool in routine water microbiology.
 
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