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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

Sampling microorganisms in water

Gwy-Am Shin

Department of Environmental and Occupational Health Sciences

the challenges
The challenges
  • Different microbe types
  • Different water types
  • Low numbers of pathogens in natural waters
different waterborne pathogens
Different waterborne pathogens
  • Viruses
  • Bacteria
  • Protozoa
  • Helminths
different type of waters
Different type of waters
  • Wastewater
  • Surface water
  • Ground water
  • Source water
  • Drinking water
  • Recreational water
  • Sea water
  • Sediments and sludges
source
Source
  • Environment
    • Mycobacterium avium complex (MAC)
    • Legionella pneumophila
  • Infected hosts
    • Humans
    • Animals
conventional community centralized sewage treatment
Conventional Community (Centralized) Sewage Treatment

Pathogen Reductions Vary from: low (<90%) to Very High (>99.99+%)

low number of microbes in natural waters
Low number of microbes in natural waters
  • Need large volumes
  • Need to separate microbes from other materials
steps in pathogen sampling in water
Steps in pathogen sampling in water
  • Concentration
  • Purification/Reconcentration
  • Analysis
filters for sampling viruses i
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

Filters for sampling viruses (II)

- - -

- -

- Virus -

- -

- - -

+

+

+

+

+

Electronegative viruses adsorb to electropositive filter surface

elution from adsorbent filters
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
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 i
Immunomagnetic Separation (I)

Y

Antibody

Bead

Y

Y

Y

Microbe

filters for sampling protozoa in water
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
elution from size exclusion filters
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
Reconcentration and Purification (Protozoa)
  • Flocculation with calcium carbonate
  • Membrane filtration
  • Ultrafiltration
  • IMS (Immunomagnetic separation)
  • Floatation/ Buoyant density gradient centrifugation
flotation sedimentation
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

indicator bacteria
Indicator bacteria
  • Total coliforms
  • Fecal coliforms
  • E. coli
  • Enterococcus
membrane filtration technique
Membrane filtration technique
  • Waters with relatively high bacteria numbers
  • Filtration (0.45 µm nitrocellulose)
  • Growth on a selective solid medium
bacteria on membrane filters
Bacteria on membrane filters

Total coliform

E. coli (blue), total coliforms (red-orange) & Salmonella (colorless) colonies

Fecal coliform

conclusions
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