1Wisconsin Water Science Center, United States Geological Survey, 2University of Wisconsin – Madison, 3Agricultural Research Service, United States Department of Agriculture, 4Alaska Science Center, United States Geological Survey
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Millen, H. T., Gonnering, J. C., Berg, R. K., Spencer, S. K., Jokela, W. E., Pearce, J. M., et al. Glass Wool Filters for Concentrating Waterborne Viruses and Agricultural Zoonotic Pathogens. J. Vis. Exp. (61), e3930, doi:10.3791/3930 (2012).
The key first step in evaluating pathogen levels in suspected contaminated water is concentration. Concentration methods tend to be specific for a particular pathogen group, for example US Environmental Protection Agency Method 1623 for Giardia and Cryptosporidium1, which means multiple methods are required if the sampling program is targeting more than one pathogen group. Another drawback of current methods is the equipment can be complicated and expensive, for example the VIRADEL method with the 1MDS cartridge filter for concentrating viruses2. In this article we describe how to construct glass wool filters for concentrating waterborne pathogens. After filter elution, the concentrate is amenable to a second concentration step, such as centrifugation, followed by pathogen detection and enumeration by cultural or molecular methods. The filters have several advantages. Construction is easy and the filters can be built to any size for meeting specific sampling requirements. The filter parts are inexpensive, making it possible to collect a large number of samples without severely impacting a project budget. Large sample volumes (100s to 1,000s L) can be concentrated depending on the rate of clogging from sample turbidity. The filters are highly portable and with minimal equipment, such as a pump and flow meter, they can be implemented in the field for sampling finished drinking water, surface water, groundwater, and agricultural runoff. Lastly, glass wool filtration is effective for concentrating a variety of pathogen types so only one method is necessary. Here we report on filter effectiveness in concentrating waterborne human enterovirus, Salmonella enterica, Cryptosporidium parvum, and avian influenza virus.
1. Preparing the Glass Wool
2. Assembling the Glass Wool Filter
5. Representative Results
|Pathogen||Water Turbidity Level (NTU) a||Amount Seeded/L b,c,d||% Recovery ± 1 SD||Number Independent Trials|
|Enterovirus- Poliovirus Sabin III||0.5||500||81% ± 11||7|
|Enterovirus- Poliovirus Sabin III||0.5||5000||67% ± 12||8|
|Enterovirus- Poliovirus Sabin III||215||500||59% ± 32||7|
|Enterovirus- Poliovirus Sabin III||215||5000||38% ± 22||6|
|Enterovirus- Poliovirus Sabin III||447||500||56% ± 18||8|
|Enterovirus- Poliovirus Sabin III||447||5000||63% ± 37||8|
|Cryptosporidium parvum||0.5||5||38% ± 14||7|
|Cryptosporidium parvum||0.5||50||53% ± 19||8|
|Cryptosporidium parvum||215||5||40% ± 16||7|
|Cryptosporidium parvum||215||50||30% ±6||6|
|Cryptosporidium parvum||447||5||33% ± 13||8|
|Cryptosporidium parvum||447||50||28% ± 11||8|
|Salmonella enterica||0.5||5||29% ± 24||7|
|Salmonella enterica||0.5||500||56% ± 16||8|
|Salmonella enterica||215||5||32% ± 24||7|
|Salmonella enterica||215||500||34% ±11||6|
|Salmonella enterica||447||5||34% ± 18||8|
|Salmonella enterica||447||500||31% ± 24||8|
Table 1. Glass wool concentration with varying water turbidities and pathogen densities.
|Pathogen||Water Sample Location||Amount Seeded/La||% Recovery|
|Avian Influenza H5N2||Sundi Lake, Anchorage Borough||2500||42.9%|
|Avian Influenza H5N2||Minto Flats, Fairbanks North Star Borough||2500||36.7%|
|Avian Influenza H5N2||Portage Valley, Anchorage Borough||2500||7.8%|
|Avian Influenza H5N2||Potter Marsh, Anchorage Borough||2500||41.5%|
|Avian Influenza H5N2||Willow Lake, Yukon-Koyukuk Borough||2500||15.5%|
Table 2. Glass wool concentration of avian influenza virus using water from five sites in Alaska.
Figure 1. Diagram of glass wool washer. This can be used instead of a bucket, saving time from rinsing. The concept is similar to a glass pipette washer.
Figure 2. Glass wool filtration with acid injection by peristaltic pump. Note the "T" connector where the pump tubing introduces acid into the sample line between the water tap and glass wool filter. pH adjustment is necessary only if the water sampled has a pH > 7.5.
Glass wool filters are effective in concentrating pathogens from water with a wide range of turbidity levels and pathogen densities (Table 1). To test this, 20 liters dechlorinated tap water was mixed with dried silt loam soil (0, 1.27, or 2.75 g/L) to achieve the desired level of turbidity and then seeded with pathogens at various densities. The water was passed through a glass wool filter, the concentrated pathogens in the eluate were enumerated, and this value was the numerator in the percent recovery calculation. The quantity of pathogens seeded into the water, that is, the denominator of the percent recovery calculation, was determined by first seeding the pathogens into a negative eluate then enumerating the pathogens. The negative eluate was prepared by passing an unseeded 20 liter sample through a filter and eluting. Quantifying the seeded pathogens in a negative eluate avoids differences in pathogen enumeration that could result from matrix differences created by the glass wool filter. The importance of this step when quantifying pathogens by qPCR is discussed in Lambertini et al.3. A 20 liter negative control sample was concentrated via glass wool filtration to determine there were no native pathogens present that could confound the percent recovery calculation. A 10 μm nominal pore size prefilter was used when the turbidity level was ≥ 215 NTU.
Poliovirus was quantified by real-time qPCR using the secondary concentration and nucleic acid extraction procedures and primers and probe described in Lambertini et al.3. Cryptosporidium parvum was quantified in the final concentrated sample volume (FCSV) created by the secondary concentration procedure for poliovirus. Oocysts were visualized by immunofluorescence (MeriFluor Cryptosporidium & Giardia Detection Kit, Meridian Life Science, Inc., Cincinnati, OH). Salmonella enterica was quantified in the FCSV by plating onto XLD agar (Remel, Lenexa, KS) and counting colony-forming-units.
Glass wool filters are effective in concentrating avian influenza viruses (Table 2). Low pathogenicity avian influenza virus (H5N2) was seeded into water from several locations in Alaska and the percent recovery calculated as described above. Secondary concentration and nucleic acid procedures were performed as for poliovirus; the virus was quantified by qPCR using the primers and probe described in Spackman et al.4
Glass wool filters have been used by several research teams3,5,6 to concentrate human enteric viruses from a variety of water sources such as finished drinking water7, groundwater8,9, surface water10, sea water11, wastewater12, and agricultural runoff13. Here we report the filters are also effective in concentrating avian influenza virus as well as the bacterial and protozoan pathogens Salmonella enterica (serovar Typhimurium) and Cryptosporidium parvum, respectively. Deboosere et al. also recently reported glass wool concentration of avian influenza virus14.
The filters are advantageous in that they are inexpensive, highly portable, usable in a wide range of water matrices, and effective for concentrating many types of waterborne pathogens. They can be constructed to any size, depending on research needs. After disinfection, filter housings are reusable.
Glass wool filters, however, do have limitations. As with any virus concentration method that relies on electropositively charged media for virus adsorption (e.g., 1MDS filter, CUNO Inc., Meriden, CT), filter effectiveness depends on ambient water pH. In our laboratory we have selected pH 7.5 as the cut-off, above which the water pH is adjusted downward by continuously pumping 0.25 M HCl into the filter input line during sampling. Higher pH waters can be sampled without pH adjustment, but at the cost of filter effectiveness3. Another limitation is shelf-life. We have shown for filters stored at 4°C for six weeks that pathogen concentration effectiveness does not decline (unpublished data). Nonetheless, longer storage times have not been tested so, conservatively, to ensure data quality, we do not use filters older than 30 days. Usually, filters are made as needed. Another potential roadblock in some countries is obtaining the glass wool from the French source specified in earlier papers. Recently, we demonstrated standard unfaced fiberglass insulation is equally effective, and this is readily available from hardware or home improvement stores (see Materials list online).
For all experiments, it is important to run two sets of controls, an equipment blank to ensure glass wool filters were not contaminated during construction and a recovery control to ensure the filters work as designed. These controls are necessary for any waterborne pathogen concentration method.
Using a glass wool filter can be as simple as attaching it to a faucet and turning on the tap or as complicated as sampling a sediment-laden river in a remote location, requiring pumps, pH adjustment, and a prefilter to prevent clogging. For our research group, the largest benefit of using glass wool filters is the capability to collect and analyze thousands of water samples for human and livestock pathogens, yielding data on pathogen abundance and distribution in the environment that would not have been as feasible with more costly, complicated methods13,15.
No conflicts of interests declared.
We thank William T. Eckert for narrating the video. Development of the glass wool protocol was part of the Wisconsin Water And Health Trial for Enteric Risks (WAHTER Study), funded by US EPA STAR Grant R831630. Alaska samples were collected by A. Reeves, A. Ramey, and B. Meixell with financial support from USGS. Any use of trade, product, or firm names is for descriptive purposes only and does not imply endorsement by the U.S. Government.
|Hydrochloric acid||Fisher Scientific||A144-500|
|Sodium hydroxide||Fisher Scientific||BP359-212|
|Phosphate Buffered Saline Sodium chloride Potassium phosphate-dibasic Potassium phosphate-monobasic||Fisher Scientific||BP358-212 BP363-500 BP362-500|
|Sodium hypochlorite i.e., household bleach||Clorox|
|Sodium thiosulfate, anhydrous||Fisher Scientific||S 475-212|
|Beef extract, desiccated||BD Biosciences||211520|
|Oiled sodocalcic glass wool Or R-11 unfaced fiberglass insulation||Johns Manville||Bourre 725 QN|
|Polypropylene mesh||Industrial Netting||xN4510|
|2"x4" Sch 80 PVC threaded pipe nipple||Grainger||6MW35|
|2" Sch 40 PVC cap||Grainger||5WDW3|
|Male adapter nylon fitting (1/2"x1/2")||US Plastic Corp.||62178|
|Sample bottles for eluate- 1 liter||Fisher Scientific||03-313-4F|
|60 mL syringe||Fisher Scientific||NC9661991|
|pH strips||Whatman, GE Healthcare||2614 991|
|Prefilter, Polypropylene, 10 inch cartridge, 10 Ám||McMaster-Carr||4411K75|