Rapid enumeration of Fecal Coliforms in water by a colorimetric beta-galactosidase assay.

The colorimetric beta-galactosidase assay is based upon the enzymatic hydrolysis of the substrate o-nitrophenyl-beta-D-galactoside (ONPG) by fecal coliforms. This technique provides an estimate of the fecal coliform concentration within 8 to 20 h. A 100-ml portion of test sample was passed through a 0.45 micrometer membrane filter. This filter was then incubated at 37 degrees C for 1 h in EC medium followed by the addition of filter-sterilized ONPG. The incubation was continued at 44.5 degrees C until a half-maximum absorbance (at 420 nm) was reached. The time between the start of incubation and the half-maximum absorbance was proportional to the concentration of fecal coliforms present. Escherichia coli (K-12) was used to measure the kinetics of substrate hydrolysis and the response time of different cell concentrations. High cell densities produced an immediate response, whereas 1 cell/ml will produce a response in less than 20 h. In field studies in which samples were taken from a range of grossly polluted streams to relatively clean lake water, a linear correlation between ONPG hydrolysis times and fecal coliform most-probable-number values was established. A total of 302 isolates randomly selected from positive ONPG-EC media, which were derived from 11 different habitats, were identified as E. coli (96.69 percent), Enterobacter cloacae (2.32 percent), Klebsiella pneumoniae (0.66 percent), and Citrobacter freundii (0.33 percent).     

Release of Sediment-Bound Fecal Coliforms by Dredging

Fecal coliform concentrations increased significantly (F test) in the immediate vicinity of a maintenance dredging operation in the Mississippi River navigation channel. Increased counts were attributed to the disturbance and relocation of bottom sediments by dredging and a concomitant release of sediment-bound fecal coliforms.     

Rapid Method for Detection and Enumeration of Fecal Coliforms in Fresh Chicken

A rapid method for enumerating fecal coliforms in foods was developed employing an agar pour-plate medium. After 7 h of incubation at 41.5 +/- 0.05 C, this medium effectively allows the growth of fecal coliforms only. This rapid method was compared with the Association of Official Analytical Chemists multiple-tube dilution method for Escherichia coli, by using 21 samples of fresh, cut-up chicken and a surface rinsing procedure for sample preparation. Verification of picked colonies was carried out in EC broth using parallel incubation temperatures of 45.5 and 44.5 +/- 0.05 C. Verifications for these temperatures averaged 79 and 98%, respectively. All positively verified isolates were E. coli types I and II, as were the negatives. Geometric means for the verified 7-h plate count were within 12% of the standard means for both EC broth incubation temperatures.     

Rapid colorimetric assay for intestinal peptide hydrolases

A simple two-step method is described for quantitating the release of free l-phenylalanine, l-leucine, l-methionine, or l-isoleucine from di- or polypeptides. The colorimetric assay is based on the ability of l-amino acid oxidase to catalyze the oxidation of free l-amino acid, but not of peptides. The potent metal chelator 1,10-phenanthroline, which is included in the second step of the assay, effectively inhibits peptide hydrolase activity thus permitting the assay to be carried out in two sequential steps in the same test tube with no intervening enzyme-destroying step. Although the assay is indirect and subject to interference by some chemicals, it is not affected by a large number of compounds frequently used in enzyme studies. The method was used to study the subcellular distribution of a number of peptide hydrolase activities in rat intestinal mucosa. For nine substrates, more than 80% of the total recovered activity was present in the cytoplasmic fraction while for two substrates, phenylalanylglycine and phenylalanylglycylglycine, more than 60% of the activity recovered was present in the particulate fraction.     

Occurrence of Coliforms, Fecal Coliforms, and Streptococci on Vegetation and Insects

This study considers the sanitary significance of coliforms, fecal coliforms, and streptococci isolated from 152 species of plants and 40 samples of insects. These specimens were collected from various ecological environments and grouped into several categories. Results indicate that typical coliforms of the warm-blooded animal gut contribute a relatively small percentage of the organisms associated with vegetation (14.1%) and insects, (14.9%). A total of 1,203 coliform strains from vegetation and 1,084 coliform strains from insects were classified as to IMViC type and fecal coliform. No type was predominant in either the vegetation or insect groupings. The biochemical results for 646 streptococci from vegetation and 226 cultures from insects were reported. The predominant group, Streptococcus fecalis, as defined by Sherman criteria, constituted a majority of all strains from vegetation and insects. The “Completed Coliform Test” is recommended for the examination of plant and insect specimens to eliminate the many anaerobic and aerobic sporeforming bacteria that frequently produce false positive reactions by the “Confirmed Test” procedure. These findings support the current interpretation of the significance of the fecal coliform test for stream investigations or for surface water quality evaluations.     

Rapid continuous colorimetric enzyme assay for penicillin G acylase

A rapid, continuous, colorimetric enzyme assay for penicillin G acylase has been developed. The assay measures the formation of the acidic products of penicillin G hydrolysis by following the decrease in pH using Phenol Red as an indicator. The activity measured is directly proportional to the amount of enzyme added to the assay, having a linear relationship with an R ² value of 0.9994.     

Unusual Organism Which Gives a Positive Elevated Temperature Test for Fecal Coliforms

Organisms apparently not of fecal origin isolated from a sulfur hot spring gave a positive elevated temperature test for fecal coliforms.     

Automated kinetic assay of beta-galactosidase activity.

An automated kinetic assay for beta-galactosidase activity in Escherichia coli was developed to permit the measurement of many independent samples simultaneously. Bacteria are grown, lysed from without (by adsorption of a high multiplicity of bacteriophage T4) and assayed in microtiter plates with 96 wells. Absorbance data are collected and analyzed by computer. The growth and lysis procedure, apparatus and software used in this assay can be used for other spectrophotometric enzyme assays.     

Rapid detection and enumeration of Legionella pneumophila in hot water systems by solid-phase cytometry

A new method for the rapid and sensitive detection of Legionella pneumophila in hot water systems has been developed. The method is based on an IF assay combined with detection by solid-phase cytometry. This method allowed the enumeration of L. pneumophila serogroup 1 and L. pneumophila serogroups 2 to 6, 8 to 10, and 12 to 15 in tap water samples within 3 to 4 h. The sensitivity of the method was between 10 and 100 bacteria per liter and was principally limited by the filtration capacity of membranes. The specificity of the antibody was evaluated against 15 non-Legionella strains, and no cross-reactivity was observed. When the method was applied to natural waters, direct counts of L. pneumophila were compared with the number of CFU obtained by the standard culture method. Direct counts were always higher than culturable counts, and the ratio between the two methods ranged from 1.4 to 325. Solid-phase cytometry offers a fast and sensitive alternative to the culture method for L. pneumophila screening in hot water systems.     

Detection of opines by colorimetric assay

A colorimetric procedure for confirming the presence of arginine-derived opines (nopaline and octopine) in plant tissue extracts is described. Those materials are widely used as markers of plant cell transformation and tumorigenesis mediated by the tumor-inducing plasmids of Agrobacterium tumefaciens. Nopaline and octopine are generally detected, following resolution by paper electrophoresis, by observation of the uv-fluorescent products formed upon reaction with phenanthrenequinone. We found that a further heat treatment step, compatible with paper electrophoresis, results in rapid production of a red-purple pigment. Our colorimetric assay is sensitive to 1.25-micrograms quantities of opine and eliminates problems of background fluorescence encountered with crude plant extract in the usual assay.     

Rapid enumeration of Escherichia coli in oysters by a quantitative PCR-ELISA

Direct enumeration of Escherichia coli from oysters was achieved using a polymerase chain reaction (PCR) amplification of the lamB gene coupled with an enzyme-linked immunosorbent assay (ELISA). Amplified PCR products generated using a digoxigenin-labelled primer were heat denatured before being quantified by an ELISA. A biotinylated probe immobilized onto streptavidin-coated microplates was used to capture the digoxigenin-labelled fragments that were detected with a peroxidase antidigoxigenin conjugate. Subsequent enzymic conversion of substrate gave distinct absorbance differences when assaying oyster samples containing E. coli in the range 10-10(5) cfu g-1.     

Rapid method for enumeration of viable Legionella pneumophila and other Legionella spp. in water

A sensitive and specific method has been developed to enumerate viable L. pneumophila and other Legionella spp. in water by epifluorescence microscopy in a short period of time (a few hours). This method allows the quantification of L. pneumophila or other Legionella spp. as well as the discrimination between viable and nonviable Legionella. It simultaneously combines the specific detection of Legionella cells using antibodies and a bacterial viability marker (ChemChrome V6), the enumeration being achieved by epifluorescence microscopy. The performance of this immunological double-staining (IDS) method was investigated in 38 natural filterable water samples from different aquatic sources, and the viable Legionella counts were compared with those obtained by the standard culture method. The recovery rate of the IDS method is similar to, or higher than, that of the conventional culture method. Under our experimental conditions, the limit of detection of the IDS method was <176 Legionella cells per liter. The examination of several samples in duplicates for the presence of L. pneumophila and other Legionella spp. indicated that the IDS method exhibits an excellent intralaboratory reproducibility, better than that of the standard culture method. This immunological approach allows rapid measurements in emergency situations, such as monitoring the efficacy of disinfection shock treatments. Although its field of application is as yet limited to filterable waters, the double-staining method may be an interesting alternative (not equivalent) to the conventional standard culture methods for enumerating viable Legionella when rapid detection is required.     

Phosphate determination by enzymatic colorimetric assay

A direct colorimetric assay for inorganic phosphate in serum is described. The system is based on utilization of the enzymes, purine-nucleoside phosphorylase and xanthine oxidase, to generate superoxide ions. The superoxide is measured in the presence of an electron mediator compound with 3-(4',5'-dimethyl-2-thiazolyl)-2,4-diphenyl-2H-tetrazolium bromide as the chromogen. The high absorbance of this chromogen between 550 and 660 nm affords useful results with a sample/reagent volume ratio as low as 1:100. A single working reagent is used, and the reaction is complete in 15 min at room temperature. The standard curve is linear for inorganic phosphate concentrations as high as 4.9 mmol/liter. Analytical recovery of phosphate in human sera averages 100%. Within-run precision study gives CV less than or equal to 1.0%. The results of this method compare closely (r greater than 0.99) with those obtained by the semidine method (recommended standard). The method lends itself to automation.     

Sunlight Inactivation of Enterococci and Fecal Coliforms in Sewage Effluent Diluted in Seawater

Inactivation (loss of culturability) by sunlight of enterococci and fecal coliforms within sewage effluent diluted in seawater was investigated in field experiments. In most experiments, 500-ml flasks of pure silica were used to confine activated sludge effluent diluted to 2% (vol/vol) in seawater. Inactivation of bacteria in these flasks (diameter, 0.1 m) was faster than in either open chambers (depth, 0.25 m) or patches of dyed effluent (depth of order, 1 m), probably because of the longer light paths in the latter two types of experiment, which caused greater attenuation of sunlight. Inactivation of 90% of enterococci generally required 2.3 times the insolation required for 90% inactivation of fecal coliforms, because of both the presence of larger initial shoulders on survival curves and a lower final inactivation rate. Two parameters are required to model inactivation of enterococci, a shoulder constant as well as a rate coefficient. The depth dependence of inactivation rate for both fecal indicators matched the attenuation profile of UV-A radiation at about 360 nm. Inactivation by UV-B radiation (290 to 320 nm), which penetrates much less into seawater, is of minor importance compared with the UV-A and visible radiation in sunlight, contrary to expectations in consideration of published action spectra for bacterial inactivation.     

Rapid staining and enumeration of small numbers of total bacteria in water by solid-phase laser cytometry

The nucleic acid stain SYBR Green I was evaluated for use with solid-phase laser cytometry to obtain total bacterial cell counts from several water sources with small bacterial numbers. Results were obtained within 30 min and exceeded or equaled counts on R2A agar plates incubated for 14 days at room temperature.     

绿僵菌孢子活性的MTT比色法快速检测技术研究

通过比较孢子浓度、MTT终浓度、反应温度、反应时间、MTTf提取时间以及pH值等因素对孢子活性检测的影响,优化了MTT比色法杀蝗绿僵菌孢子活性检测条件,建立起稳定、灵敏、可靠的生化检测体系,适用于绿僵菌等真菌农药原药与制剂中活孢率的快速测定,为真菌农药研制提供了新的质量分析方法。     

Reliable Drosophila body fat quantification by a coupled colorimetric assay

Factors and mechanisms controlling lipometabolism homeostasis share a remarkable evolutionary conservation between humans and Drosophila flies. Accordingly, the Drosophila model has been successfully used to understand the pathophysiology of human metabolic diseases such as obesity. Body fat stores in species as different as humans and flies consist of neutral lipids, mainly triacylglycerols. Changes in body fat storage are a diagnostic phenotype of lipometabolism imbalances of genetic or environmental origin. Various methods have been developed to quantify Drosophila body fat storage. The most widely used method adopts a commercial coupled colorimetric assay designed for human serum triacylglycerol quantification, which is based on glycerol content determination after enzymatic conversion of glycerides into glycerol. The coupled colorimetric assay is compatible with large-scale genetic screen approaches and has been successfully applied to characterize central regulators of Drosophila lipometabolism. Recently, the applicability of the coupled colorimetric assay for Drosophila storage fat quantification has been questioned in principle. Here we compare the performance of the coupled colorimetric assay on Drosophila samples with thin layer chromatography, the "gold standard" in storage lipid analysis. Our data show that the presented variant of the coupled colorimetric assay reliably discriminates between lean and fat flies and allows robust, quick and cost-effective quantification of Drosophila body fat stores.     

Rapid enumeration of microorganisms in foods by the direct epifluorescent filter technique.

Filtration of "stomachered" food suspensions through nylon filters (pore size, 5 microns) removed most of the food debris without affecting the recovery of microorganisms. Two to ten milliliters of these prefiltered suspensions could be filtered in the direct epifluorescent filter technique (DEFT). The technique takes less than 30 min to complete and has a lower sensitivity of less than 60,000 microorganisms per g for all products examined. Vegetative bacterial cells, spores, fungal hyphae, and yeasts could be distinguished with the technique. For fresh meat and fish, the DEFT count of prefiltered suspensions agreed well with the plate count of unfiltered suspensions over the range of 10(4) to 10(10)/g (correlation coefficient of 0.91). For frozen meat and fish and frozen vegetables, the two counting methods had correlation coefficients of 0.87 and 0.66, respectively. The poor correlation for frozen vegetables was due to the inclusion in the DEFT count of nonviable bacteria killed by the blanching process used to inactivate enzymes. Good agreement was obtained between the prefiltered DEFT count and unfiltered plate count for cooked meats, cream doughnut, and whole peppers. Possible reasons for the poor agreement between the DEFT count and plate count for certain products are discussed.     

Incidence of Infectious Drug Resistance Among Fecal Coliforms Isolated from Raw Sewage

Raw sewage was examined for the incidence of antibiotic-resistant coliforms present among both total and fecal coliforms. In both groups, it was found that approximately 3% of the coliform bacteria were resistant to two or more antibiotics. Of these organisms, 48% were capable of transferring all or part of their antibiotic resistance to an antibiotic-sensitive, F⁻, derivative of Escherichia coli K-12. Among the R factors identified, those conferring resistance to streptomycin-tetracycline, ampicillin-streptomycin-tetracycline, and ampicillin or ampicillin-streptomycin accounted for 23, 20, and 15%, respectively, of the total R factors detected. The data indicate a significant level of infectious drug resistance among the fecal coliforms of the urban population. The data indicate further that because of the high incidence of coliform bacteria found to be doubly resistant to streptomycin and tetracyline, the inclusion of these antibiotics in selective media used for routine total or fecal coliform counts may serve to identify domestic sources of pollution.