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Microbial Sampling in Water: Challenges and Methods

This study explores the challenges of sampling microbes in various water types, including wastewater and drinking water. Different pathogens like viruses, bacteria, protozoa, and helminths are discussed, along with methods for concentration, purifications, and analysis. The text covers the importance of sampling low numbers of pathogens in natural waters and the steps involved in pathogen sampling. Specific techniques for sampling viruses, protozoa, and bacteria are detailed, along with purification methods like flotation/sedimentation and immunomagnetic separation. The text concludes by highlighting the need for improved sampling methods to match advanced detection technologies.

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Microbial Sampling in Water: Challenges and Methods

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  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)

  7. Concentration of enteric pathogens in raw sewage (USA)

  8. Conventional Community (Centralized) Sewage Treatment Pathogen Reductions Vary from: low (<90%) to Very High (>99.99+%)

  9. Transmission of enteric pathogens

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

  11. Steps in pathogen sampling in water • Concentration • Purification/Reconcentration • Analysis

  12. Sampling enteric viruses in water

  13. 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)

  14. 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

  15. 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 Filters for sampling viruses (II) - - - - - - Virus - - - - - - + + + + + Electronegative viruses adsorb to electropositive filter surface

  16. Different types of filters

  17. Field sampling device for viruses

  18. Sampling procedure for viruses

  19. 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

  20. 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

  21. Immunomagnetic Separation Y Antibody Bead Y Y Y Microbe

  22. Immonomagnetic separation assay

  23. Application of sCAR with Para-Magnetic Beads for Virus Particle Capture and then RT-PCR sCAR purification Covalent coupling to paramagnetic beads Culture + media; :sCAR produced Blocking post-coupling (RT-) PCR : sCAR NA extraction Sample containing viruses : Virus Particle : 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

  24. Sampling protozoan parasites in water

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

  26. 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

  27. Different types of filters

  28. Sampling procedure for protozoa

  29. 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

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

  31. 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 Isopycnic density gradient: microbe density = medium density at one depth Flotation: microbe density < medium density

  32. Sampling and analysis for bacteria in water

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

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

  35. 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

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