Evaluation of filters for recovery of Campylobacter jejuni from water.

Campylobacter jejuni has been incriminated in several large waterborne outbreaks, but it has rarely been isolated from water itself. Better methodology is needed for the isolation of C. jejuni from water. We evaluated three types of 0.45-micron microporous filters and three different pore sizes of positively charged depth filters for their ability to recover C. jejuni from seeded, sterile tap and surface water. The microporous filters tested were Millipore HA, Gelman GN6, and Zetapor. Three pore sizes of Zeta Plus depth filters (05S, 30S, and 50S) were evaluated by using an adsorption-elution technique. The overall percent recovery in both tap and surface water by microporous filters was: Zetapor, 66%; Millipore HA, 33%; and Gelman GN6, 33%. Adsorption-elution with Zeta Plus 50S allowed 89% recovery of C. jejuni. These data suggest that both the positively charged Zetapor microporous filter and the Zeta Plus 50S depth filter are effective filters for the recovery of C. jejuni from water.     

Comparison of gauze swabs and membrane filters for isolation of Campylobacter spp. from surface water.

The epidemiology of Campylobacter jejuni indicates that waterborne transmission is important; the organism has been isolated from seawater, fresh water, and estuarine sites. Membrane filtration, with and without use of an enrichment broth, has been the most common method for isolating C. jejuni from water. We evaluated two methods for isolating C. jejuni from water: membrane filtration and gauze filtration. The membrane filters evaluated included 0.22- and 0.45-micron-pore Millipore filters (Millipore Corp., Bedford, Mass.), 0.2- and 0.4-micron-pore Nuclepore filters (Nucleopore Corp., Pleasanton, Calif.), and a 0.45-micron-pore Zetapor filters (AMF Cuno, Meridian, Conn.). The gauze filters included both Moore and Spira swabs. Of the membrane filters evaluated, the 0.45-micron-pore Millipore and Zetapor filters were the most sensitive for recovery of C. jejuni from seeded waters. The 0.45-micron-pore Millipore filter placed in Oosterom broth was better for recovery of C. jejuni from seeded stationary surface waters than either the Spira or Moore swab. However, the 0.45-micron-pore Millipore filter placed on a plate or in enrichment broth was equivalent to the Spira gauze swab when used to examine water from Atlanta area streams. C. jejuni organisms were isolated from 9 of 24 surface water samples representing 5 of 12 streams.     

Comparison of gauze swabs and membrane filters for isolation of Campylobacter spp. from surface water

The epidemiology of Campylobacter jejuni indicates that waterborne transmission is important; the organism has been isolated from seawater, fresh water, and estuarine sites. Membrane filtration, with and without use of an enrichment broth, has been the most common method for isolating C. jejuni from water. We evaluated two methods for isolating C. jejuni from water: membrane filtration and gauze filtration. The membrane filters evaluated included 0.22- and 0.45-micron-pore Millipore filters (Millipore Corp., Bedford, Mass.), 0.2- and 0.4-micron-pore Nuclepore filters (Nucleopore Corp., Pleasanton, Calif.), and a 0.45-micron-pore Zetapor filters (AMF Cuno, Meridian, Conn.). The gauze filters included both Moore and Spira swabs. Of the membrane filters evaluated, the 0.45-micron-pore Millipore and Zetapor filters were the most sensitive for recovery of C. jejuni from seeded waters. The 0.45-micron-pore Millipore filter placed in Oosterom broth was better for recovery of C. jejuni from seeded stationary surface waters than either the Spira or Moore swab. However, the 0.45-micron-pore Millipore filter placed on a plate or in enrichment broth was equivalent to the Spira gauze swab when used to examine water from Atlanta area streams. C. jejuni organisms were isolated from 9 of 24 surface water samples representing 5 of 12 streams.     

Concentration of poliovirus from tap water using positively charged microporous filters.

Microporous filters that are more electropositive than the negatively charged filters currently used for virus concentrations from water by filter adsorption-elution methods were evaluated for poliovirus recovery from tap water. Zeta Plus filters composed of diatomaceous earth-cellulose-"charge-modified" resin mixtures and having a net positive charge of up to pH 5 to 6 efficiently adsorbed poliovirus from tap water at ambient pH levels 7.0 to 7.5 without added multivalent cation salts. The adsorbed virus were eluted with glycine-NaOH, pH 9.5 to 11.5. Electropositive asbestos-cellulose filters efficiently adsorbed poliovirus from tap water without added multivalent cation salts between pH 3.5 and 9.0, and the absorbed viruses could be eluted with 3% beef extract, pH 9, but not with pH 9.5 to 11.5 glycine-NaOH. Under water quality conditions in which poliovirus recoveries from large volumes of water were less than 5% with conventional negatively charged filters and standard methods, recoveries with Zeta Plus filters averaged 64 and 22.5% for one- and two-stage concentration procedures, respectively. Electropositive filters appear to offer distinct advantages over conventional negatively charged filters for concentrating enteric viruses from water, and their behavior tends to confirm the importance of electrostatic forces in virus recovery from water by microporous filter adsorption-elution methods.     

Concentration of poliovirus from tap water using positively charged microporous filters.

Microporous filters that are more electropositive than the negatively charged filters currently used for virus concentrations from water by filter adsorption-elution methods were evaluated for poliovirus recovery from tap water. Zeta Plus filters composed of diatomaceous earth-cellulose-"charge-modified" resin mixtures and having a net positive charge of up to pH 5 to 6 efficiently adsorbed poliovirus from tap water at ambient pH levels 7.0 to 7.5 without added multivalent cation salts. The adsorbed virus were eluted with glycine-NaOH, pH 9.5 to 11.5. Electropositive asbestos-cellulose filters efficiently adsorbed poliovirus from tap water without added multivalent cation salts between pH 3.5 and 9.0, and the absorbed viruses could be eluted with 3% beef extract, pH 9, but not with pH 9.5 to 11.5 glycine-NaOH. Under water quality conditions in which poliovirus recoveries from large volumes of water were less than 5% with conventional negatively charged filters and standard methods, recoveries with Zeta Plus filters averaged 64 and 22.5% for one- and two-stage concentration procedures, respectively. Electropositive filters appear to offer distinct advantages over conventional negatively charged filters for concentrating enteric viruses from water, and their behavior tends to confirm the importance of electrostatic forces in virus recovery from water by microporous filter adsorption-elution methods.     

Effects of humic materials on virus recovery from water.

Humic and fulvic acids were tested for their ability to interfere with virus recovery by microporous filters. Two electropositively charged types of filter (Seitz S and Zeta Plus 60S) were used to concentrate poliovirus in the presence of humic materials. Humic acid inhibited virus adsorption, but even at the highest humic acid concentrations tested (200 mg/liter), 30 to 40% of the virus was recovered by the filters. Fulvic acid, tested with Zeta Plus filters, did not affect virus recovery. For comparison, two electronegatively charged filter types were tested (Cox and Balston). These two types of filter were more sensitive to interference at lower concentrations of humic acid than the more positively charged filters. With Balston filters, at humic acid concentrations above 10 mg/liter, most of the virus was recovered in the filtrate. Fulvic acid, tested with Balston filters, did not interfere with virus recovery. With the electropositively charged filters, the humic materials adsorbed efficiently, even at high input concentrations. Interference with virus adsorption occurred at humic acid concentrations which were below the level of saturation of the filters. In addition, in high-volume experiments, humic acid led to premature blockage of the filters. The efficiency of virus recovery by a second concentration step, organic flocculation of the filter eluate, was tested. For all the filter types tested, this procedure was not affected by the presence of humic or fulvic acid in the input water.     

Effects of humic materials on virus recovery from water

Humic and fulvic acids were tested for their ability to interfere with virus recovery by microporous filters. Two electropositively charged types of filter (Seitz S and Zeta Plus 60S) were used to concentrate poliovirus in the presence of humic materials. Humic acid inhibited virus adsorption, but even at the highest humic acid concentrations tested (200 mg/liter), 30 to 40% of the virus was recovered by the filters. Fulvic acid, tested with Zeta Plus filters, did not affect virus recovery. For comparison, two electronegatively charged filter types were tested (Cox and Balston). These two types of filter were more sensitive to interference at lower concentrations of humic acid than the more positively charged filters. With Balston filters, at humic acid concentrations above 10 mg/liter, most of the virus was recovered in the filtrate. Fulvic acid, tested with Balston filters, did not interfere with virus recovery. With the electropositively charged filters, the humic materials adsorbed efficiently, even at high input concentrations. Interference with virus adsorption occurred at humic acid concentrations which were below the level of saturation of the filters. In addition, in high-volume experiments, humic acid led to premature blockage of the filters. The efficiency of virus recovery by a second concentration step, organic flocculation of the filter eluate, was tested. For all the filter types tested, this procedure was not affected by the presence of humic or fulvic acid in the input water.     

Concentration of simian rotavirus SA-11 from tap water by membrane filtration and organic flocculation

Simian rotavirus SA-11 was concentrated from tap water by adsorption to and elution from microporous filters, followed by organic flocculation. Two types of filters were compared for their ability to concentrate the virus. Both Zeta Plus 60S and Cox AA type M-780 filters were efficient for virus adsorption, but the efficiency of virus elution was higher with Zeta Plus than with Cox filters. Optimum conditions for virus recovery from Zeta Plus filters included an input water pH of 6.5 to 7.5 and the use of 3% beef extract (pH 9.0) for elution. Under these conditions, an average of 62 to 100% of the virus was recovered in the concentrate. Organic flocculation was used as a second-step concentration method, with average recoveries of 47 to 69%. When the two methods were used to concentrate small numbers (7 to 75 PFU/liter) of input rotavirus, an average of 75 +/- 40% recovery was achieved. With large volumes of input water, however, recovery was reduced to 16 +/- 7%.     

Concentration of coliphages from large volumes of water and wastewater.

Membrane filter adsorption-elution technology has been extensively used for the concentration and detection of animal viruses from large volumes of water. This study describes the development of positively charged microporous filters (Zeta Plus) for the concentration of coliphages from large volumes of water and wastewater. Four different coliphages were studied: MS-2, phi X174, T2, and T4. Positively charged microporous filters were found to efficiently adsorb these coliphages from tap water, sewage, and lake water at neutral pH. Adsorbed viruses were eluted with a 1:1 mixture of 8% beef extract and 1 M sodium chloride at pH 9. Using this method, coliphages could be concentrated from 17-liter volumes of tap water with recoveries ranging from 34 to 100%. Coliphages occurring naturally in raw and secondarily treated sewage were recovered with average efficiencies of 56.5 and 55.0%, respectively. This method should be useful in isolation of rare phages, the ecology of phages in natural waters, and the evaluation of water quality.     

Concentration of Simian Rotavirus SA-11 from Tap Water by Membrane Filtratiòn and Organic Flocculation

Simian rotavirus SA-11 was concentrated from tap water by adsorption to and elution from microporous filters, followed by organic flocculation. Two types of filters were compared for their ability to concentrate the virus. Both Zeta Plus 60S and Cox AA type M-780 filters were efficient for virus adsorption, but the efficiency of virus elution was higher with Zeta Plus than with Cox filters. Optimum conditions for virus recovery from Zeta Plus filters included an input water pH of 6.5 to 7.5 and the use of 3% beef extract (pH 9.0) for elution. Under these conditions, an average of 62 to 100% of the virus was recovered in the concentrate. Organic flocculation was used as a second-step concentration method, with average recoveries of 47 to 69%. When the two methods were used to concentrate small numbers (7 to 75 PFU/liter) of input rotavirus, an average of 75 ± 40% recovery was achieved. With large volumes of input water, however, recovery was reduced to 16 ± 7%.     

Efficiency of several micro-fiber glass filters for recovery of poliovirus from tape water.

Micro-fiber glass filters from Gelman, Filterite, Johns-Manville, and Whatman were compared with Millipore membrane filters on the basis of their virus adsorbancy, flow rate, clogging resistance, and virus concentration efficiency by using tap water at 2 nephelometric turbidity units. As virus adsorbants the Johns-Manville D39, Filterite 0.25-micron, Filterite 0.45-micron, and Millipore 0.45-micron filters were the most efficient, retaining more than 99% of the added virus in water at pH 3.5 and 0.0005 M aluminum chloride. The Johns-Manville D79 and D49 filters retained 92 and 96% of the virus, respectively, whereas the Whatman GF-D, Whatman GF-F, Gelman A-E, and Millipore AP-20 filters retained only 28, 78, 56, and 34% of the virus, respectively. The best flow rate and clogging resistance were obtained with the Johns-Manville D79 filter or with this filter acting as a prefilter to the Johns-Manville D49, Johns-Manville D39, or Filterite 0.45-micron filter. Finally, poliovirus experimentally seeded in 20 liters of tape water was recovered from Johns-Manville D79-Johns-Manville D39 or Johns-Manville D79-Filterite 0.45 micron 142-mm filter combinations was a efficiencies of 86 and 85%, respectively.     

Comparison of Gelman and Millipore Membrane Filters for Enumerating Fecal Coliform Bacteria

Tests of two leading brands of membrane filters used for enumerating fecal coliform bacteria showed that Gelman GN-6 filters recovered statistically more colonies of bacteria than did Millipore HAWG 047SO filters from pure cultures incubated at either 35 C (the optimal growth temperature) or 44.5 C (the standard temperature for the fecal coliform test). Standard membrane filter procedures with M-FC broth base were used to enumerate the organisms. Densities of colonies incubated on Gelman filters at 44.5 C averaged 2.3 times greater than those on Millipore filters. Plate counts of the bacteria at both temperatures indicated that incubation at 44.5 C did not inhibit propagation of fecal coliform bacteria. For the pour plates, M-FC broth base plus 1.5% agar was used. This modified medium compared favorably to plate count agar for enumerating Escherichia coli. At 35 and 44.5 C, colony counts on Gelman filters agreed closely with plate counts prepared concurrently, but Millipore counts were consistently lower than plate counts, especially at 44.5 C. Comparative analyses of river water for fecal coliform bacteria by the membrane filter technique gave results comparable to those for the pure cultures.     

Influence of Coliform Source on Evaluation of Membrane Filters

Four brands of membrane filters were examined for total and fecal coliform recovery performance by two experimental approaches. Using diluted EC broth cultures of water samples, Johns-Manville filters were superior to Sartorius filters for fecal coliform but equivalent for total coliform recovery. Using river water samples, Johns-Manville filters were superior to Sartorius filters for total coliform but equivalent for fecal coliform recovery. No differences were observed between Johns-Manville and Millipore or Millipore and Sartorius filters for total or fecal coliform recoveries using either approach, nor was any difference observed between Millipore and Gelman filters for fecal coliform recovery from river water samples. These results indicate that the source of the coliform bacteria has an important influence on the conclusions of membrane filter evaluation studies.     

Comparison of the New Millipore HC with Conventional Membrane Filters for the Enumeration of Fecal Coliform Bacteria

Fecal coliform recoveries were determined for six types of membrane filters using 65 nonchlorinated water samples. Results showed that the membranes could be ranked in order of decreasing recovery as follows: Millipore HC > Gelman > Johns-Manville ~ Sartorius > Millipore HA > Schleicher & Schuell.     

Comparison of cellular recovery rates and morphologic detail obtained using membrane filter and cytocentrifuge techniques.

Two methods commonly used for collecting cells from a large volume of fluid-membrane filters (Millipore, Gelman, and Nuclepore) and cytocentrifugation-were compared for percentage of cell recovery and degree of cell preservation. Twenty samples of body cavity fluid were centrifuged, and the buffy coat of each was resuspended in a balanced electrolyte solution. The cellularity of each suspension was determined using both Coulter Counter and hemocytometer. Exact aliquots of each sample were collected on Millipore, Gelman, and Nuclepore filters and on slides by cytocentrifugation (Shandon). The resultant material was fixed in alcohol (95% ethanol), stained by the Papanicolaou method, mounted, and then evaluated with respect to the number of cells present and the diagnostically significant morphologic detail of the cells. Cell recovery was estimated by counting cells in known areas of each preparation and then ascertaining the total area. The Millipore filter technique consistently recovered the highest percentage of cells and preserved the best morphologic detail.     

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.     

Simple method for concentration of bacteria from large volumes of tap water.

Membrane adsorption-elution techniques have made it possible to concentrate and detect small numbers of viruses in large volumes of water and wastewater, but no such methods are available for quantitative recovery of bacteria. A number of waterborne disease outbreaks of "unknown etiology" in the United States are suspected to have been caused by pathogens present in numbers too small to be detected by currently available methodology. The present study reports on the use of positively charged depth filters for the concentration and detection of bacteria in large volumes of tap water. In this method, dechlorinated tap water was passed, under positive pressure, through positively charged filter media (Zetaplus, 05S). More than 90% of seeded bacteria adsorbed to these filters at ambient pH levels. Adsorbed bacteria were eluted by passing a small volume of Trypticase soy broth in the direction opposite of the influent flow. By this method, Escherichia coli and Salmonella serovar B organisms in 20 liters of tap water were concentrated in a final volume of 50 ml, with an average recovery efficiency of greater than or equal to 30%.     

Comparison of Autoclave and Ethylene Oxide-Sterilized Membrane Filters Used in Water Quality Studies

Autoclave and ethylene oxide-sterilized membrane filters manufactured by Gelman, Millipore, and Sartorius were field tested for their recovery of total coliforms, fecal coliforms, fecal streptococci, and heterotrophs. The data were analyzed by using split-plot analysis of variance and significance tests. Membranes were also tested for pH and toxicity using Escherichia coli. The mean data summaries indicated that Gelman membrane filters generally produced the highest counts during the field studies. Statistical analyses of the March data showed that there were significant differences between membrane filters at 1% level; however, statistical analyses of June data revealed no significant differences except in total coliform recoveries. Toxicity tests at 35 C indicated that Gelman and Millipore autoclaved membrane filters were able to recover 92% of the test organisms. Toxicity tests performed at 44.5 C revealed that no membranes were able to recover more than 40% of the test organisms. Since differences were found in the ability of the three brands of membrane filters to recover bacteria from natural and controlled sources, membrane filters from different manufacturers cannot be readily interchanged. There is a need for a standardized procedure for testing bacterial recovery by membrane filters.     

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.     

Detection and distribution of rotavirus in raw sewage and creeks in São Paulo, Brazil.

Rotaviruses were concentrated from 8-liter samples of raw domestic sewage and sewage-polluted creek water by adsorption to and elution from positively charged microporous filters (Zeta Plus 60S), followed by ultracentrifugation of the filter eluates. Indirect immunofluorescence and direct immunoperoxidase methods allowed detection and enumeration of rotavirus in 6 (20.6%) of 29 sewage samples and in 19 (34.5%) of 55 creek water samples. Levels of rotaviruses ranged from < 3 to 63 focus-forming units (FFU)/liter, and the geometric means were 2.2 FFU/liter in sewage, 2.9 FFU/liter at creek Tremembé, and 2.6 FFU/liter at creek Pirajussara. Wastewater samples examined during autumn and winter months showed a higher rate positivity for rotavirus than those collected in spring and summer, corresponding to the seasonal variation of rotaviral diarrhea in the city of São Paulo.