sampling microorganisms in water gwy-am shin department of environmental and occupational health...
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Sampling microorganisms in water
Gwy-Am ShinDepartment 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
Different type of waters
• Wastewater• Surface water• Ground water• Source water• Drinking water• Recreational water• Sea water• Sediments and sludges
Low numbers of pathogens in water
Incidence and concentration of enteric pathogens in feces (USA)
Pathogen Incidence (%) Concentration(/gram)
Enteric virus 10-40 103-108
Hepatitis A 0.1 108
Rotavirus 10-29 1010-1012
Salmonella 0.5 104-1010
Giardia 3.8
18-54
106
106
Cryptosporidium 0.6-20
27-50
106-107
106-107
Concentration of enteric pathogens in raw sewage (USA)
Organism Concentration (/liter)
Enteric virus 104-105
Salmonella 103-105
Clostridium perfringens 104-107
Cryptosporidium oocysts 102-104
Giardia cysts 102-105
Helminth ova 104-105
Conventional Community (Centralized) Sewage Treatment
Pathogen Reductions Vary from: low (<90%) to Very High (>99.99+%)
Transmission of enteric pathogens
Low number of microbes in natural waters
• Need large volumes
• Need to separate microbes from other materials
Steps in pathogen sampling in water
• Concentration
• Purification/Reconcentration
• Analysis
Sampling enteric viruses in water
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)
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
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 - - - - - -
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Electronegative viruses adsorb to electropositive filter surface
Different types of filters
Field sampling device for viruses
Sampling procedure for viruses
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
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
Immunomagnetic Separation
Y
Y
Y
Y
Bead
Antibody
Microbe
Immonomagnetic separation assay
Application of sCAR with Para-Magnetic Beads for Virus Particle Capture and then RT-PCR
: Virus Particle
: sCAR
Culture + media; :sCAR produced
(RT-) PCR
sCARpurification
: Blocking protein
Amine Terminated Support Magnetic Bead : BioSpheres(Biosource)Pre-coated to provide available amine groups for covalent couplingof proteins or other ligands by glutaraldehyde-mediated coupling method
Covalent coupling to paramagnetic beads
Blocking post-coupling
Sample containing viruses
NA extraction
Sampling protozoan parasites in water
Concentration methods (protozoa)
• Small volume– Flocculation with calcium carbonate– Membrane filtration– Ultrafiltration
• Large volume– Filtration (size exclusion filters)
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
Different types of filters
Sampling procedure for protozoa
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
Reconcentration and Purification (Protozoa)
• Floatation/Sedimentation• IMS (Immunomagnetic separation)
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 layerFlotation: microbe density < medium density
Isopycnic density gradient: microbe density = medium density at one depth
Sampling and analysis for bacteria in water
Membrane filtration technique
• Waters with relatively high bacteria numbers
• Filtration (0.45 µm nitrocellulose)
• Growth on a selective solid medium
Bacteria on membrane filters
E. coli (blue), total coliforms (red-orange) & Salmonella (colorless) colonies
Total coliform
Fecal coliform
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