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Sampling microorganisms in water

Sampling microorganisms in water. Gwy-Am Shin Department of Environmental and Occupational Health Sciences. The challenges. Different microbe types Different water types Low numbers of pathogens in natural waters. Different waterborne pathogens. Viruses Bacteria Protozoa Helminths.

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Sampling microorganisms in water

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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. Source • Environment • Mycobacterium avium complex (MAC) • Legionella pneumophila • Infected hosts • Humans • Animals

  7. Incidence and concentration of enteric pathogens in feces (USA)

  8. Transmission of enteric pathogens

  9. Conventional Community (Centralized) Sewage Treatment Pathogen Reductions Vary from: low (<90%) to Very High (>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. 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 (I) Y Antibody Bead Y Y Y Microbe

  22. Immonomagnetic separation (II)

  23. Sampling protozoan parasites in water

  24. 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

  25. Different types of filters

  26. Sampling procedure for protozoa

  27. 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

  28. Reconcentration and Purification (Protozoa) • Flocculation with calcium carbonate • Membrane filtration • Ultrafiltration • IMS (Immunomagnetic separation) • Floatation/ Buoyant density gradient centrifugation

  29. 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

  30. Sampling and analysis for bacteria in water

  31. Indicator bacteria • Total coliforms • Fecal coliforms • E. coli • Enterococcus

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

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

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

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