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 - - - - - -

+

+

+

+

+

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