agar and 4-methylumbelliferyl-galactopyranoside

The essential trace element selenium (Se) is involved in the form of selenocysteine at the active site of several prokaryotic and eukaryotic proteins called selenoproteins. These proteins have recently attracted attention particularly in relation to their application to human health and new characteristics of the genetic code. We have recently described a selenium bioassay based on a recombinant DNA construct in which the expression of the lac' Z gene in Escherichia coli is proportionally and specifically driven by UGA-directed selenocysteine incorporation. Here we have further developed this bioassay for more rapid and sensitive detection and measurement of selenium that permits screening of the selenium status on agar plates. Again, the inclusion of selenium into the lac'Z-fusion product is reflected by the level of beta-galactosidase activity, which in turn is reflected by the intensity of fluorescence on agar plates. This fluorescing agent is a 4-methylumbelliferyl moiety which is released through the cleavage by the enzyme of 4-methylumbelliferyl-beta-D-galactoside. The intensity of the fluorescence is easily detected by uv irradiation and photographed by polaroid or video cameras.

Topics: Agar; beta-Galactosidase; Biological Assay; Cloning, Molecular; Escherichia coli; Fluorescent Dyes; Galactosides; Hymecromone; Selenium; Sensitivity and Specificity

Drinking water regulations under the Final Coliform Rule require that total coliform-positive drinking water samples be examined for the presence of Escherichia coli or fecal coliforms. The current U.S. Environmental Protection Agency-approved membrane filter (MF) method for E. coli requires two media, an MF transfer, and a total incubation time of 28 h. A newly developed MF method, the MI agar method, containing indoxyl-beta-D-glucuronide and 4-methylumbelliferyl-beta-D-galactopyranoside for the simultaneous detection of E. coli and total coliforms, respectively, by means of their specific enzyme reactions, was compared with the approved method by the use of wastewater-spiked tap water samples. Overall, weighted analysis of variance (significance level, 0.05) showed that the new medium recoveries of total coliforms and E. coli were significantly higher than those of mEndo agar and nutrient agar plus MUG (4-methylumbelliferyl-beta-D-glucuronide), respectively, and the background counts were significantly lower than those of mEndo agar (< 5%). Generally, the tap water source, overall chlorine level, wastewater source, granular activated carbon treatment of the tap water, and method of grouping data by E. coli count for statistical analysis did not affect the performance of the new medium.

Topics: Agar; Analysis of Variance; Chlorine; Colony Count, Microbial; Enterobacteriaceae; Escherichia coli; Galactosides; Glucuronates; Hymecromone; Indoles; Micropore Filters; United States; United States Environmental Protection Agency; Waste Disposal, Fluid; Water Microbiology; Water Supply

A total of 78 strains, representing 21 Vibrio species, were examined by using taurocholate-tellurite-gelatin agar (TTGA) medium and modified TTGA medium containing 4-methylumbelliferyl-beta-D-galactoside (150 micrograms/ml). Modified TTGA medium allowed for simple and direct detection of beta-D-galactosidase (beta-gal) activity. This feature, in conjunction with other differential characteristics of TTGA medium, gave improved differentiation of the vibrios tested. The modified TTGA medium allowed for easy differentiation of Vibrio cholerae (beta-gal+) from Vibrio parahaemolyticus (beta-gal-). The 4-methylumbelliferyl-beta-D-galactoside substrate is inexpensive and very stable. Incorporation into the agar did not affect the performance of TTGA as a differential medium. The assay for beta-gal activity with this substrate was specific and sensitive.

Topics: Agar; beta-Galactosidase; Galactosides; Gelatin; Humans; Hymecromone; Species Specificity; Taurocholic Acid; Tellurium; Vibrio