Sampling microorganisms in water

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Sampling microorganisms in water

• Gwy-Am Shin
• Department of Environmental and Occupational
  Health Sciences

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The challenges

• Different microbe types
• Different water types
• Low numbers of pathogens in natural waters

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Different waterborne pathogens

• Viruses
• Bacteria
• Protozoa
• Helminths

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Different type of waters

• Wastewater
• Surface water
• Ground water
• Source water
• Drinking water
• Recreational water
• Sea water
• Sediments and sludges

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Low numbers of pathogens in water
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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
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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
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Conventional Community (Centralized) Sewage
Treatment
Pathogen Reductions Vary from low (lt90) to Very
High (gt99.99)
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Transmission of enteric pathogens
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Low number of microbes in natural waters

• Need large volumes
• Need to separate microbes from other materials

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Steps in pathogen sampling in water

• Concentration
• Purification/Reconcentration
• Analysis

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Sampling enteric viruses in water
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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)

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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

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Filters for sampling viruses (II)

• Positively charged
• 1MDS Virozorb
• cellulose/fiberglass
• not so efficient with seawater or water with pH
  gt8
• Negatively charged
• Millipore HA
• cellulose ester/fiberglass
• Need pH adjustment and addition of cations

- - - - - - Virus
- - - - - -

Electronegative viruses adsorb to electropositive
filter surface
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Different types of filters
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Field sampling device for viruses
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Sampling procedure for viruses
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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

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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

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Immunomagnetic Separation
Y
Antibody
Bead
Y
Y
Y
Microbe
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Immonomagnetic separation assay
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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-mediat
ed coupling method
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Sampling protozoan parasites in water
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Concentration methods (protozoa)

• Small volume
• Flocculation with calcium carbonate
• Membrane filtration
• Ultrafiltration
• Large volume
• Filtration (size exclusion filters)

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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

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Different types of filters
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Sampling procedure for protozoa
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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

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Reconcentration and Purification (Protozoa)

• Floatation/Sedimentation
• IMS (Immunomagnetic separation)

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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 lt medium density
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Sampling and analysis for bacteria in water
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Membrane filtration technique

• Waters with relatively high bacteria numbers
• Filtration (0.45 µm nitrocellulose)
• Growth on a selective solid medium

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Bacteria on membrane filters
Total coliform
E. coli (blue), total coliforms (red-orange)
Salmonella (colorless) colonies
Fecal coliform
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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 isnt everything
• Negative results dont necessarily mean target
  not there
• There is a need to focus on the reliability and
  sensitivity of concentration methods