1. Sampling microorganisms in water Gwy-Am Shin Department of Environmental and Occupational Health Sciences

2. The challenges Different microbe types Different water types Low numbers of pathogens in natural waters

3. Different waterborne pathogens Viruses Bacteria Protozoa Helminths

4. Different type of waters Wastewater Surface water Ground water Source water Drinking water Recreational water Sea water Sediments and sludges

5. Low numbers of pathogens in water

6. Incidence and concentration of enteric pathogens in feces (USA) PathogenIncidence (%)Concentration(/gram) Enteric virus10-4010 3 -10 8 Hepatitis A0.110 8 Rotavirus10-2910 10 -10 12 Salmonella0.510 4 -10 10 Giardia3.8 18-54 10 6 Cryptosporidium0.6-20 27-50 10 6 -10 7

7. Concentration of enteric pathogens in raw sewage (USA) OrganismConcentration (/liter) Enteric virus10 4 -10 5 Salmonella10 3 -10 5 Clostridium perfringens10 4 -10 7 Cryptosporidium oocysts10 2 -10 4 Giardia cysts 10 2 -10 5 Helminth ova10 4 -10 5

8. Conventional Community (Centralized) Sewage Treatment Pathogen Reductions Vary from: low ( 99.99+%)

10. Transmission of enteric pathogens

11. Low number of microbes in natural waters Need large volumes Need to separate microbes from other materials

12. Steps in pathogen sampling in water Concentration Purification/Reconcentration Analysis

13. Sampling enteric viruses in water

14. Concentration methods (viruses) Small volume –Adsorption to minerals (e.g. aluminum hydroxide, ferric hydroxide) –Hydroextration (dialysis with Polyethylene Glycol (PEG)) –Ultrafiltration (hollow fiber filters) Large volume –Filtration (adsorption filters)

15. Filters for sampling viruses (I) Adsorbent filters – pore size of filters (0.2 -0.45 µm) larger than viruses – viruses retained by adsorption – electrostatic and hydrophobic interactions Positively charged and negatively charged filters

16. Filters for sampling viruses (II) Positively charged –1MDS Virozorb –cellulose/fiberglass –not so efficient with seawater or water with pH >8 Negatively charged –Millipore HA –cellulose ester/fiberglass –Need pH adjustment and addition of cations - - - - - - Virus - - - - - - ++++++++++ Electronegative viruses adsorb to electropositive filter surface

17. Different types of filters

18. Field sampling device for viruses

19. Sampling procedure for viruses

20. Elution from Adsorbent Filters Choice of eluants –Beef extract –Amino acids –w/mild detergents Considerations –Efficiency of elution –Compatibility with downstream assays –Volume –Contact time

21. Reconcentration and Purification (Viruses) Organic Flocculation Adsorption to minerals (e.g. aluminum hydroxide, ferric hydroxide) Hydroextraction (dialysis with Polyethylene Glycol (PEG)) Spin Column Chromatography (antibodies covalently linked to gel particles) IMS (Immunomagnetic separation) Ligand capture

22. Immunomagnetic Separation Y Y Y Y Bead Antibody Microbe

23. Immonomagnetic separation assay

24. Application of sCAR with Para-Magnetic Beads for Virus Particle Capture and then RT-PCR : Virus Particle : sCAR Culture + media; :sCAR produced (RT-) PCR sCAR purification : Blocking protein Amine Terminated Support Magnetic Bead : BioSpheres(Biosource) Pre-coated to provide available amine groups for covalent coupling of proteins or other ligands by glutaraldehyde-mediated coupling method Covalent coupling to paramagnetic beads Blocking post-coupling Sample containing viruses NA extraction

25. Sampling protozoan parasites in water

26. Concentration methods (protozoa) Small volume –Flocculation with calcium carbonate –Membrane filtration –Ultrafiltration Large volume –Filtration (size exclusion filters)

27. Filters for sampling protozoa in water Size exclusion filters –1-several µm pore size –Protozoa retained by their sizes Various formats –Cartridge, capsule, and disk filters

28. Different types of filters

29. Sampling procedure for protozoa

30. Elution from size exclusion filters Choice of eluants –PBS with Tween 80 and SDS (sodium dodecyl sulfate) –Tris buffer with laureth-12, EDTA, and antiform A

31. Reconcentration and Purification (Protozoa) Floatation/Sedimentation IMS (Immunomagnetic separation)

32. Flotation/sedimentation Flotation centrifugation –Layer or suspend samples or microbes in medium of density greater than microbe density; centrifuge; microbes float to surface; recover them from top layer Isopycnic or buoyant density gradient centrifugation –Layer or suspend samples or microbes in a medium with varying density with depth but having a density = to the microbe at one depth. –Microbes migrate to the depth having their density (isopycnic) –Recover them from this specific layer Flotation: microbe density < medium density Isopycnic density gradient: microbe density = medium density at one depth

33. Sampling and analysis for bacteria in water

34. Membrane filtration technique Waters with relatively high bacteria numbers Filtration (0.45 µm nitrocellulose) Growth on a selective solid medium

36. Bacteria on membrane filters E. coli (blue), total coliforms (red-orange) & Salmonella (colorless) colonies Total coliform Fecal coliform

37. Conclusions Sampling methods are lagging behind detection methods Difficulties with a single platform for any one media because of wide range of organisms and environmental conditions Speed isn’t everything Negative results don’t necessarily mean target not there There is a need to focus on the reliability and sensitivity of concentration methods