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

2. Sampling

3. The challenges Different microbe types Different media types Low numbers of pathogens in the environment

4. Infectious diseases 1415 human pathogens (2001) –217 viruses and prions –538 bacteria and rickettsiae –307 fungi –66 protozoans –287 helminths

5. Transmission of infectious disease Person-to-person –Direct: person-to-person or animal-to-person –Indirect : droplet, fomites (toys), other vehicles Environment –Airborne –Waterborne –Foodborne –Vectorborne

6. Low numbers of pathogens in the environment

7. Transmission of enteric pathogens

8. Low number of microbes in the environment Need large volumes Need to separate microbes from other materials

9. Steps in pathogen sampling in the environment Concentration Purification/Reconcentration Analysis

10. Sampling microbes in water Filtration is typically used for concentration Several formats utilized: –Membrane, pleated capsule, cartridge, hollow fiber Several types of media –cellulose ester, fiberglass, nylon, polycarbonate, diatomaceous earth, polypropylene, cotton, polysulfone, polyacrylonitrile, polyether sulfone

11. Filters to Recover and Concentrate Microbes from Liquids

12. Sampling microbes in air Filters –Not recommended due to low sampling efficiency Impingers –AGI sampler –Biosampler (SKC) sampler Impactors –Anderson single and multistage sampler –Slit sampler –Rotary arm sampler Centrifugal samplers –Cyclone sampler –Centrifugal sampler

13. Impingers

14. Impactors (I)

15. Impactors (II)

16. Centrifugal samplers

17. Sampling microbes from surfaces Swabs –cotton, dacron, calcium alginate, sponge Swipes/Wipes –cotton, nitrocellulose membranes, polyester bonded cloth, velvet or velveteen Vacuum Filtration –Hepa bag vac, wet vac Contact Plates and Paddles (RODAC) New Methods –Adhesive Strips and Paddles –Scraping/Aspiration Yamaguchi, et al. 2003; Cloud, et al. 2002; Lemmen, et al, 2001; Poletti, 1999; Craythorn, et al. 1980; Osterblad, et al. 2003; Taku, et al. 2003

19. Purification/re-concentration PEG (polyethylene glycol) Organic Flocculation IMS (Immunomagnetic separation) Ligand capture BEaDs (Biodetection Enabling Device) Capillary Electrophoresis Microfluidics Nucleic Acid Extraction Spin Column Chromatography Floatation Sedimentation Enrichment

20. Immunomagnetic Separation Y Y Y Y Bead Antibody Microbe

21. Immonomagnetic separation assay

22. Summary (Sampling) 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

23. Detection methods

24. Light microscope

25. Electron microscope

26. Sizes of microorganisms

27. Cultural methods (bacteria) Traditional approach 1 st step –pre-enrich and/or enrich using non-selective and then selective broth media –grow colonies on membrane filters 2 nd step –Transfer to differential and selective agars –Recover presumptive positive colonies –Biochemical, metabolic and other physiological testing –Serological or other immunochemical typing –Other characterization: phage typing, nucleic acid analyses, virulence tests

28. Enrichment Cultures Observe for growth by turbidity, clearing, gas production, color change, etc. Score as presence- absence (positive or negative) (sometimes) Quantify using replicate and different volumes to compute a Most Probable Number Left: negative Right: positive (color change)

29. Cultural methods (bacteria) Plating methods –Spread plating technique –Pour plating technique Most Probable Number (MPN) technique

31. Different bacterial colonies on general media

33. Cultural methods (viruses and protozoa) Animal infectivity assays –Mouse –Gerbils –Champagnes –Human Cell-culture infectivity assays –Primary cell lines –Established cancer cell lines

35. Immunological methods

36. Nucleic acid-based methods

37. Antigen and antibody reaction

38. Structure of DNA

39. Immunological methods Immunoprecipitation assays Immunoblotting assays Enzyme-Linked Immunosorbent assays (ELISA)

41. Nucleic-acid based methods Gene probing –Southern and northern hybridization –Microarray Polymerase Chain Reaction (PCR)

42. Gene probe detection

44. Real-Time PCR and Quantitative Fluorogenic Detection Molecular beacon. Several 5' bases form base pairs with several 3' bases; reporter and quencher in close proximity. – If reporter is excited by light, its emission is absorbed by quencher & no fluorescence is detected. Detection of PCR product by molecular beacon. – Beacon binds to PCR product and fluoresces when excited by the appropriate light. – [Fluorescence] proportional to [PCR product amplified]