Glass Wool Filters for Concentrating Waterborne Viruses and Agricultural Zoonotic Pathogens

Abstract

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 Cryptosporidium 1 , 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 viruses 2 . 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. Video LinkThe video component of this article can be found at http://www.jove.com/video/3930/ Protocol 1. Preparing the Glass Wool 1. Before and after making each batch of filters, sterilize the work area with 10% bleach solution. 2. Put on gloves and gown. Sterilize a bucket by autoclaving at 121°C and 15 psi for at least 20 minutes. Place the glass wool in the sterile bucket. 3. Saturate the glass wool with reverse osmosis water and let soak for 15 minutes. 4. Drain the reverse osmosis water from the bucket. 5. Saturate the glass wool with 1 M HCl and let soak for 15 minutes. 6. Drain the 1 M HCl from the bucket. 7. Rinse the glass wool with reverse osmosis water. 8. Mix thoroughly. 9. Check the pH using pH paper and repeat the reverse osmosis water rinse until a neutral pH is achieved. 10. Pour off the rinse water. 11. Saturate the glass wool with 1 M NaOH and let soak for 15 minutes. 12. Drain the 1 M NaOH from the bucket. 13. Repeat the reverse osmosis rinse until a neutral pH is achieved. 14. Pour off the rinse water. 15. Instead of a bucket, a glass wool washer can be constructed, which is similar in design to a glass pipette washer (Figure 1). 16. Cover the glass wool completely with sterile Phosphate Buffered Saline (PBS) adjusted to pH 6.8. 17. Use prepared glass wool immediately or store at 4°C...