1. Sampling microorganisms in water
Gwy-Am Shin
Department of Environmental and Occupational Health Sciences

2. The challenges Different microbe types Different water types
Low numbers of pathogens in natural waters

3. Different waterborne pathogens
Viruses
Bacteria
Protozoa
Helminths

4. Different type of waters
Wastewater
Surface water
Ground water
Source water
Drinking water
Recreational water
Sea water
Sediments and sludges

5. Low numbers of pathogens in water

6. Source Environment Infected hosts Mycobacterium avium complex (MAC)
Legionella pneumophila
Infected hosts
Humans
Animals

7. Incidence and concentration of enteric pathogens in feces (USA)
Concentration(/gram)
Enteric virus
10-40
Hepatitis A
0.1
108
Rotavirus
10-29
Salmonella
0.5
Giardia
3.8
18-54
106
Cryptosporidium
0.6-20
27-50

8. Transmission of enteric pathogens

9. Conventional Community (Centralized) Sewage Treatment
Pathogen Reductions Vary from: low (<90%) to Very High (>99.99+%)

11. Transmission of enteric pathogens

12. Low number of microbes in natural waters
Need large volumes
Need to separate microbes from other materials

13. Steps in pathogen sampling in water
Concentration
Purification/Reconcentration
Analysis

14. Sampling enteric viruses in water

15. Filters for sampling viruses (I)
Adsorbent filters
pore size of filters ( µm) larger than viruses
viruses retained by adsorption
electrostatic and hydrophobic interactions
Positively charged and negatively charged filters

16. 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

17. Different types of filters

18. Field sampling device for viruses

19. Sampling procedure for viruses

20. 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

21. 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

22. Immunomagnetic Separation (I)
Antibody Y Y
Bead Y Y
Microbe

23. Immonomagnetic separation (II)

24. Sampling protozoan parasites in water

25. 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

26. Different types of filters

27. Sampling procedure for protozoa

28. 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

29. Reconcentration and Purification (Protozoa)
Flocculation with calcium carbonate
Membrane filtration
Ultrafiltration
IMS (Immunomagnetic separation)
Floatation/ Buoyant density gradient centrifugation

30. 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

31. Sampling and analysis for bacteria in water

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

33. Membrane filtration technique
Waters with relatively high bacteria numbers
Filtration (0.45 µm nitrocellulose)
Growth on a selective solid medium

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

36. 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