Practical direct plaque assay for coliphages in 100-ml samples of drinking water

A practical single-agar-layer plaque assay for the direct detection of coliphages in 100-ml samples of water was designed and evaluated. With this assay a 100-ml sample of water, an agar medium containing divalent cations, and the host Escherichia coli C (ATCC 13706) were mixed in a single container, and the mixture was plated on 10 14-cm-diameter petri dishes. It was more sensitive, reliable, and accurate than various other methods and proved rapid, simple, and economic.     

Duplicate split samples for internal quality control in routine water microbiology

In the microbiological examination of water, indicator organisms are sought by membrane filtration of 100-ml sub-samples of water. The same sample cannot be examined twice but the use of split samples, where two 100-ml sub-samples from the same batch are processed, has been explored. Binomial theory was used to determine the 95% confidence intervals for the results obtained. Two sets of 50 results were assessed by the method and the resulting control charts are illustrated.     

Direct quantitation and detection of salmonellae in biological samples without enrichment, using two-step filtration and real-time PCR

A new two-step filtration protocol followed by a real-time PCR assay based on SYBR green I detection was developed to directly quantitate salmonellae in two types of biological samples: i.e., chicken rinse and spent irrigation water. Four prefiltration filters, one type of final filter, and six protocols for recovery of salmonellae from the final filter were evaluated to identify an effective filtration protocol. This method was then combined with a real-time PCR assay based on detection of the invA gene. The best results were obtained by subsequent filtration of 100 ml of chicken rinse or 100 ml of spent irrigation water through filters with pore diameters of >40 mum to remove large particles and of 0.22 microm to recover the Salmonella cells. After this, the Salmonella cells were removed from the filter by vortexing in 1 ml of physiological saline, and this sample was then subjected to real-time quantitative PCR. The whole procedure could be completed within 3 h from sampling to quantitation, and cell numbers as low as 7.5 x 10(2) CFU per 100-ml sample could be quantified. Below this limit, qualitative detection of concentrations as low as 2.2 CFU/100 ml sample was possible on occasion. This study has contributed to the development of a simple, rapid, and reliable method for quantitation of salmonellae in food without the need for sample enrichment or DNA extraction.     

Quantitative detection of Escherichia coli O157 in surface waters by using immunomagnetic electrochemiluminescence.

A protocol for the quantitative detection of Escherichia coli O157 in raw and concentrated surface waters using immunomagnetic electrochemiluminescence (IM-ECL) was developed and optimized. Three antibody sandwich formats were tested: commercial anti-O157:H7 IM beads, IM beads made in-house with a polyclonal anti-O157:H7 immunoglobulin G (IgG), or IM beads made in-house with a monoclonal anti-O157:H7 IgG coupled with a polyclonal anti-O157:H7 IgG to which an electrochemiluminescent label (TAG) was attached. The monoclonal IM bead-polyclonal TAG format was chosen for optimization because it gave lower background levels and linear regression slopes of ca. 1.0, indicative of a constant ECL signal per cell. The dynamic range was ca. 10(1) to 10(5) cells ml(-1) in phosphate-buffered saline and in raw water samples. The monoclonal IM beads selectively captured E. coli O157 cells in the presence of ca. 10(8) cells of a non-O157 strain of E. coli ml(-1). Background ECL signals from concentrated (100-fold) water samples were substantially higher and more variable than raw water samples. The background signal was partially eliminated by the addition of polyvinylpolypyrrolidone. Successive cell capture incubations, termed sequential bead capture (SBC), were optimized for establishing baseline ECL values for individual water samples. The linear dynamic range with SBC was ca. 10(2) to 10(5) E. coli O157 cells ml of concentrated water(-1). To validate the protocol, 10-liter surface water samples were spiked with ca. 5,000 E. coli O157 (Odwalla) cells and concentrated by vortex filtration, and 1- or 3-ml aliquots were analyzed by IM-ECL. Differential ECL signals (SBC) from 1- and 3-ml samples were statistically significant and were generally consistent with standard curves for these cell concentrations. Enrichments were conducted with aliquots of spiked raw water and concentrated water using EC broth and minimal lactose broth (MLB). All tubes with concentrated water became turbid and gave a positive ECL response for E. coli O157 (>10,000 ECL units); MLB gave a somewhat higher detection rate with spiked raw water. The potential sensitivity of the IM-ECL assay is ca. 25 E. coli O157 cells ml of raw water(-1), 25 cells 100 ml of 100-fold concentrated water(-1), or 1 to 2 viable cells liter(-1) with concentration and enrichment. The IM-ECL assay appears suitable for routine analysis and screening of water samples.     

Determination of clenbuterol in bovine urine using gas chromatography–mass spectrometry following clean-up on an ion-exchange resin

A gas chromatographic–mass spectrometric method was developed for the determination of residues of clenbuterol in bovine urine. The method involves a simple cation-exchange clean-up and concentration of clenbuterol in the acidified urine, followed by ethyl acetate extraction. The analyte is determined as the di-trimethylsilyl derivative and quantitated against an internal standard of penbutolol. Using a 5-ml sample of urine, a detection limit of 0.07 ng/ml can be achieved with recoveries close to 100% for fortification levels of 0.2 and 1 ng/ml. By increasing the sample volume to 50 ml, a detection limit below 0.01 ng/ml was achievable with recovery averaging 70%. The coefficient of variation of the assay ranged from 15% at 0.01 ng/ml (50-ml sample) to 6% at 1 ng/ml (5-ml sample). It was demonstrated that the method can detect the presence of clenbuterol in bovine urine at sub-ppb (ng/ml) levels using low resolution GC–MS with electron impact (EI) ionization.     

Bioanalysis of m-iodobenzylguanidine in plasma by high-performance liquid chromatography after derivatization with benzoin

A sensitive chromatographic assay has been developed for m-iodobenzylguanidine (MIBG) in human plasma based on the derivatization with benzoin. MIBG is first isolated from plasma using solid-phase extraction on a cyanopropyl-modified silica phase. After evaporation of the eluate, a fluorescent derivative is formed using benzoin. The derivative is analysed by reversed-phase liquid chromatography using a mixture 60% (v/v) acetonitrile, 30% (v/v) water and 10% (v/v) of the 0.5 M Tris buffer (pH 8.0) as the eluent and fluorescence detection at 320 nm for excitation and 435 nm for emission, respectively. In the evaluated concentration range (2–200 ng/ml) precisions 10% and accuracies in between 90 and 100% have been found, with 2 ng/ml being the lower limit of quantification using a 0.5-ml plasma sample volume. The assay can also be used without the internal standard benzylguanidine. The assay was successfully used to obtain a pharmacokinetic curve of MIBG.     

Direct Quantitation and Detection of Salmonellae in Biological Samples without Enrichment, Using Two-Step Filtration and Real-Time PCR

A new two-step filtration protocol followed by a real-time PCR assay based on SYBR green I detection was developed to directly quantitate salmonellae in two types of biological samples: i.e., chicken rinse and spent irrigation water. Four prefiltration filters, one type of final filter, and six protocols for recovery of salmonellae from the final filter were evaluated to identify an effective filtration protocol. This method was then combined with a real-time PCR assay based on detection of the invA gene. The best results were obtained by subsequent filtration of 100 ml of chicken rinse or 100 ml of spent irrigation water through filters with pore diameters of >40 μm to remove large particles and of 0.22 μm to recover the Salmonella cells. After this, the Salmonella cells were removed from the filter by vortexing in 1 ml of physiological saline, and this sample was then subjected to real-time quantitative PCR. The whole procedure could be completed within 3 h from sampling to quantitation, and cell numbers as low as 7.5 × 10² CFU per 100-ml sample could be quantified. Below this limit, qualitative detection of concentrations as low as 2.2 CFU/100 ml sample was possible on occasion. This study has contributed to the development of a simple, rapid, and reliable method for quantitation of salmonellae in food without the need for sample enrichment or DNA extraction.     

Field assessments of control agents for lesser mealworm (Coleoptera: Tenebrionidae) using litter sampling

Spinosad, diatomaceous earth, and cyfluthrin were assessed on two broiler farms at Gleneagle and Gatton in southeastern Queensland, Australia in 2004-2005 and 2007-2009, respectively to determine their effectiveness in controlling lesser mealworm, Alphitobius diaperinus (Panzer) (Coleoptera: Tenebrionidae). Insecticide treatments were applied mostly to earth or 'hard' cement floors of broiler houses before the placement of new bedding. Efficacy of each agent was assessed by regular sampling of litter and counting of immature stages and adult beetles, and comparing insect counts in treatments to counts in untreated houses. Generally, the lowest numbers of lesser mealworm were recorded in the house with hard floors, these numbers equalling the most effective spinosad applications. The most effective treatment was a strategic application of spinosad under feed supply lines on a hard floor. In compacted earth floor houses, mean numbers of lesser mealworms for two under-feed-line spinosad treatments (i.e., 2-m-wide application at 0.18 g of active insecticide (g [AI]) in 100-ml water/m2, and 1-m-wide application at 0.11 g ([AI] in 33-ml water/m2), and an entire floor spinosad treatment (0.07 g [AI] in 86-ml water/m2) were significantly lower (i.e., better control) than those numbers for cyfluthrin, and no treatment (controls). The 1-m-wide under-feed-line treatment was the most cost-effective dose, providing similar control to the other two most effective spinosad treatments, but using less than half the active component per broiler house. No efficacy was demonstrated when spinosad was applied to the surface of bedding in relatively large volumes of water. All applications of diatomaceous earth, applied with and without spinosad, and cyfluthrin at the label rate of 0.02 g (AI)/100-ml water/m2 showed no effect, with insect counts not significantly different to untreated controls. Overall, the results of this field assessment indicate that cyfluthrin (the Australian industry standard) and diatomaceous earth were ineffective on these two farms and that spinosad can be a viable alternative for broiler house use.     

Detection of Pseudomonas aeruginosa from clinical and environmental samples by amplification of the exotoxin A gene using PCR.

PCR was used to detect Pseudomonas aeruginosa from water samples by amplifying a 396-bp region of the exotoxin A (ETA) structural gene sequence. The identify of the amplified 396-bp fragment was confirmed by digesting it with PvuI restriction endonuclease, which produced the predicted 246- and 150-bp fragments. Specific primers amplified ETA-positive P. aeruginosa DNA, whereas other species of Pseudomonas and GC-rich bacteria did not yield any 396-bp fragment. The specificity and sensitivity of the assay were 100 and 96%, respectively, which confirms the assay's reliability for diagnostic and epidemiological studies. The assay can detect as few as 5 to 10 cells in a 10-ml water sample or 0.1 pg of P. aeruginosa DNA per reaction mixture (5 microliters) by ethidium bromide staining of an agarose gel. Ten-times-lower concentrations were detected by hybridization with a digoxigenin-labeled oligonucleotide probe internal to the PCR product. With this PCR method, ETA-positive P. aeruginosa was detected in animal cage water samples at a level of 40 cells per ml. This method is rapid and less cumbersome than other diagnostic methods for the identification of P. aeruginosa strains. The method described can be used to detect a low level of P. aeruginosa from environmental and clinical samples without the use of selective media or additional biochemical tests.     

Latex and vinyl nonsterile examination gloves: status report on laboratory evaluation of defects by physical and biological methods.

We have reported previously (H. R. Kotilainen, J. P. Brinker, J. L. Avato, and N. M. Gantz, Arch. Intern. Med. 149:2749-2753, 1989) that the quality of nonsterile examination gloves available for clinical use may be extremely variable. In view of the concern over human immunodeficiency virus and hepatitis B virus transmission to health care workers, the continuing variability of gloves available for use, and the need for a simple and safe test, we have evaluated 2,500 vinyl (five brands) and 2,000 latex (four brands) gloves by the 300-ml and the newly proposed 1,000-ml water tests and for permeability to herpes simplex virus type 1 and poliovirus type 1, respectively. While all 300-ml watertight gloves were unlikely to leak herpes simplex virus type 1 (1.3% vinyl; 0.5% latex), poliovirus was recovered much more frequently (8.9% vinyl, 6.1% latex). In all gloves that passed the 1,000-ml test, herpes simplex virus type 1 was not recovered. Poliovirus was recovered infrequently (1.4% vinyl, 1.5% latex). Preliminary analyses suggest that the 1,000-ml water test has significantly increased sensitivity over the 300-ml water test in the detection of small holes in both vinyl and latex gloves that may allow the passage of viral particles. Gloves that pass a 1,000-ml water challenge are unlikely to allow the passage of a small virus such as poliovirus. Given that human immunodeficiency virus, hepatitis B virus and herpes simplex virus type 1 are larger particles than poliovirus, gloves that pass the 1,000-ml water test theoretically could provide better protection.     

Comparison of bacterial indicators and sampling programs for drinking water systems

A total of 48 French villages were selected for a survey of water quality from February 1983 to June 1984 as part of an epidemiological study conducted in the rural area of the Rh?ne-Alpes region. Water samples were collected and analyzed on a weekly basis in each village. Bacteriological analysis of each water sample included enumeration of standard plate count bacteria, total and thermotolerant coliforms, and fecal streptococci. The water quality regulations are examined as to the analysis frequency, the volume of samples, and the relationship between the various bacterial indicators. Analyzing 300-ml, instead of 100-ml, samples tends to generate better information on single water samples. However, if many samples are analyzed over time from the same community, the value of using large volumes of water samples is diminished. The comparisons between bacterial indicators showed that the information obtained from the various indicators was very similar. However, fecal streptococci had a better predictive value of a negative test than coliforms with respect to the French standards.     

Comparison of bacterial indicators and sampling programs for drinking water systems.

A total of 48 French villages were selected for a survey of water quality from February 1983 to June 1984 as part of an epidemiological study conducted in the rural area of the Rhône-Alpes region. Water samples were collected and analyzed on a weekly basis in each village. Bacteriological analysis of each water sample included enumeration of standard plate count bacteria, total and thermotolerant coliforms, and fecal streptococci. The water quality regulations are examined as to the analysis frequency, the volume of samples, and the relationship between the various bacterial indicators. Analyzing 300-ml, instead of 100-ml, samples tends to generate better information on single water samples. However, if many samples are analyzed over time from the same community, the value of using large volumes of water samples is diminished. The comparisons between bacterial indicators showed that the information obtained from the various indicators was very similar. However, fecal streptococci had a better predictive value of a negative test than coliforms with respect to the French standards.     

Latex and vinyl nonsterile examination gloves: status report on laboratory evaluation of defects by physical and biological methods

We have reported previously (H. R. Kotilainen, J. P. Brinker, J. L. Avato, and N. M. Gantz, Arch. Intern. Med. 149:2749-2753, 1989) that the quality of nonsterile examination gloves available for clinical use may be extremely variable. In view of the concern over human immunodeficiency virus and hepatitis B virus transmission to health care workers, the continuing variability of gloves available for use, and the need for a simple and safe test, we have evaluated 2,500 vinyl (five brands) and 2,000 latex (four brands) gloves by the 300-ml and the newly proposed 1,000-ml water tests and for permeability to herpes simplex virus type 1 and poliovirus type 1, respectively. While all 300-ml watertight gloves were unlikely to leak herpes simplex virus type 1 (1.3% vinyl; 0.5% latex), poliovirus was recovered much more frequently (8.9% vinyl, 6.1% latex). In all gloves that passed the 1,000-ml test, herpes simplex virus type 1 was not recovered. Poliovirus was recovered infrequently (1.4% vinyl, 1.5% latex). Preliminary analyses suggest that the 1,000-ml water test has significantly increased sensitivity over the 300-ml water test in the detection of small holes in both vinyl and latex gloves that may allow the passage of viral particles. Gloves that pass a 1,000-ml water challenge are unlikely to allow the passage of a small virus such as poliovirus. Given that human immunodeficiency virus, hepatitis B virus and herpes simplex virus type 1 are larger particles than poliovirus, gloves that pass the 1,000-ml water test theoretically could provide better protection.     

Bronchoalveolar lavage in interstitial lung disease: effect of volume of fluid infused

To evaluate the effect of varying infusate volume on the results of bronchoalveolar lavage (BAL) in patients with interstitial lung disease, 55 patients underwent 58 BAL during which both a 100- and 250-ml lavage was performed in the same lobe of the lung. Although the percent of the fluid that was returned and the total numbers of cells were greater in the 250- vs. the 100-ml lavage, there were no significant differences in cell differentials or numbers of cells per milliliter between the 100- and 250-ml BAL. We conclude that infusate volume does not affect cell differentials or numbers of cells per milliliter of bronchoalveolar lavage fluid in patients with interstitial lung disease.     

Detection of noroviruses in tap water in Japan by means of a new method for concentrating enteric viruses in large volumes of freshwater

A virus concentration method using a cation-coated filter was developed for large-volume freshwater applications. Poliovirus type 1 (LSc 2ab Sabin strain) inoculated into 40 ml of MilliQ (ultrapure) water was adsorbed effectively to a negatively charged filter (Millipore HA, 0.45- micro m pore size) coated with aluminum ions, 99% (range, 81 to 114%) of which were recovered by elution with 1.0 mM NaOH (pH 10.8) following an acid rinse with 0.5 mM H(2)SO(4) (pH 3.0). More than 80% poliovirus recovery yields were obtained from 500-ml, 1,000-ml, and 10-liter MilliQ water samples and from tap water samples. This method, followed by TaqMan PCR detection, was applied to determine the presence of noroviruses in tap water in Tokyo, Japan. In a 14-month survey, 4 (4.1%) and 7 (7.1%) of 98 tap water samples (100 to 532 liters) contained a detectable amount of noroviruses of genotype 1 and genotype 2, respectively. This method was proved to be useful for surveying the occurrence of enteric viruses, including noroviruses, in large volumes of freshwater.     

Detection of Noroviruses in Tap Water in Japan by Means of a New Method for Concentrating Enteric Viruses in Large Volumes of Freshwater

A virus concentration method using a cation-coated filter was developed for large-volume freshwater applications. Poliovirus type 1 (LSc 2ab Sabin strain) inoculated into 40 ml of MilliQ (ultrapure) water was adsorbed effectively to a negatively charged filter (Millipore HA, 0.45-μm pore size) coated with aluminum ions, 99% (range, 81 to 114%) of which were recovered by elution with 1.0 mM NaOH (pH 10.8) following an acid rinse with 0.5 mM H₂SO₄ (pH 3.0). More than 80% poliovirus recovery yields were obtained from 500-ml, 1,000-ml, and 10-liter MilliQ water samples and from tap water samples. This method, followed by TaqMan PCR detection, was applied to determine the presence of noroviruses in tap water in Tokyo, Japan. In a 14-month survey, 4 (4.1%) and 7 (7.1%) of 98 tap water samples (100 to 532 liters) contained a detectable amount of noroviruses of genotype 1 and genotype 2, respectively. This method was proved to be useful for surveying the occurrence of enteric viruses, including noroviruses, in large volumes of freshwater.     

Direct detection of Salmonella spp. in estuaries by using a DNA probe

A method for direct detection of Salmonella spp. in water was developed by using a commercially available DNA probe. Particulate DNA was extracted from 500- to 1,500-ml water samples collected from New York Harbor and Chesapeake Bay and used as a substrate for a salmonella-specific DNA probe in dot blot assays. The method detected salmonellae in water samples from 12 of 16 sites, including 6 sites where salmonellae could not be cultured. The specificity of the probe was evaluated, and cross-hybridization, although negligible, was used to set detection limits for the assay. Salmonella DNA bound the probe quantitatively, and from these results Salmonella DNA in the total particulate DNA in environmental samples could be estimated. The data obtained in this study indicate that Salmonella spp. often are not detected in water samples by culture methods, even when they are present in significant numbers.     

Direct detection of Salmonella spp. in estuaries by using a DNA probe.

A method for direct detection of Salmonella spp. in water was developed by using a commercially available DNA probe. Particulate DNA was extracted from 500- to 1,500-ml water samples collected from New York Harbor and Chesapeake Bay and used as a substrate for a salmonella-specific DNA probe in dot blot assays. The method detected salmonellae in water samples from 12 of 16 sites, including 6 sites where salmonellae could not be cultured. The specificity of the probe was evaluated, and cross-hybridization, although negligible, was used to set detection limits for the assay. Salmonella DNA bound the probe quantitatively, and from these results Salmonella DNA in the total particulate DNA in environmental samples could be estimated. The data obtained in this study indicate that Salmonella spp. often are not detected in water samples by culture methods, even when they are present in significant numbers.     

Development of an immunomagnetic bead-immunoliposome fluorescence assay for rapid detection of Escherichia coli O157:H7 in aqueous samples and comparison of the assay with a standard microbiological method

The objective of this study was to develop and optimize a protocol for the rapid detection of Escherichia coli O157:H7 in aqueous samples by a combined immunomagnetic bead-immunoliposome (IMB/IL) fluorescence assay. The protocol consisted of the filtration or centrifugation of 30- to 100-ml samples followed by incubation of the filter membranes or pellet with anti-E. coli O157:H7 immunomagnetic beads in growth medium specific for E. coli O157:H7. The resulting E. coli O157:H7-immunomagnetic bead complexes were isolated by magnetic separation, washed, and incubated with sulforhodamine B-containing immunoliposomes specific for E. coli O157:H7; the final immunomagnetic bead-E. coli O157:H7-immunoliposome complexes were again isolated by magnetic separation, washed, and lysed with a n-octyl-beta-d-glucopyranoside to release sulforhodamine B. The final protocol took less than 8 h to complete and had a detection limit of less than 1 CFU of E. coli O157:H7 per ml in various aqueous matrices, including apple juice and cider. To validate the protocol at an independent facility, 100-ml samples of groundwater with and without E. coli O157:H7 (15 CFU) were analyzed by a public health laboratory using the optimized protocol and a standard microbiological method. While the IMB/IL fluorescence assay was able to identify E. coli O157:H7-containing samples with 100% accuracy, the standard microbiological method was unable to distinguish E. coli O157:H7-spiked samples from negative controls without further extensive workup. These results demonstrate the feasibility of using immunomagnetic beads in combination with sulforhodamine B-encapsulating immunoliposomes for the rapid detection of E. coli O157:H7 in aqueous samples.