Sampling microorganisms in water GwyAm Shin Department of

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

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

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

Incidence and concentration of enteric pathogens in feces (USA) Pathogen Enteric virus Hepatitis A Rotavirus Salmonella Giardia Cryptosporidium Incidence (%) 10 -40 0. 1 10 -29 0. 5 3. 8 18 -54 0. 6 -20 27 -50 Concentration(/gram) 103 -108 1010 -1012 104 -1010 106 106 -107

Transmission of enteric pathogens

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

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 – 1 MDS Virozorb – cellulose/fiberglass – not so efficient with seawater or water with p. H >8 • Negatively charged – Millipore HA – cellulose ester/fiberglass – Need p. H 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 (I) Y Bead Y Microbe Y Y Antibody

Immonomagnetic separation (II)

Sampling protozoan parasites 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

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) • Flocculation with calcium carbonate • Membrane filtration • Ultrafiltration • IMS (Immunomagnetic separation) • Floatation/ Buoyant density gradient centrifugation

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) Flotation: microbe density < medium density Isopycnic density gradient: microbe density =

Sampling and analysis for bacteria in water

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

Membrane filtration technique • Waters with relatively high bacteria numbers • Filtration (0. 45 µm nitrocellulose) • Growth on a selective solid medium

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

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