Sampling Criteria

1. Sampling Criteria • Sampling plans will depend on the question that they are designed to answer • Basic criteria • Replication • Representative • Random • Controls • Method Validation

2. Detection of Pathogens in the Environment

3. Detection of Pathogenic Microbes in Environmental Media • Three main steps: • (1) recovery, extraction and concentration, • (2) purification and separation, and • (3) assay and characterization.

4. Assay Methods for Pathogens • culture or infectivity • viability or activity measurements • immunoassays • nucleic acid assays • Protein or other macromolecular/biochemical assays • microscopic examinations

5. Microscopic Methods

6. History of Microscopy • 1595 First Microscopes (tube with lens at each end; 3X to 9X magnification) • Hans and Zacharias Janssen, Dutch eyeglass makers • Improvement on Compound Microcope design and Publication of Micrographia • Robert Hooke; 1665; coined term “cell” • First description of Bacteria (tooth scrapings) and Protozoa (pond water) • Anton van Leeuwenhoek

7. Detecting Pathogens and Indicators in the Environment

8. Microscopy • Goal of microscopy is to improve resolving power • Resolving power is ability to distinguish two points as separate • Function of light and aperture of objective • Resolution=smallest visible distance between two points • Human eye can resolve about 150μm between two points • Most light Microscopes can resolve ~0.2 μm

9. Magnification • The ability to enlarge the apparent size of an image • Function of resolving power of microscope and the eye • Limit of resolution of eye/limit of resolution of microscope = magnification • e.g. 0.15mm/0.0002mm = 750X

10. Microscopic and Imaging Detection of Pathogens • Still widely used for parasites and bacteria • Specific staining and advanced imaging to distinguish target from non-target organisms • Differential interference contrast microscopy • Confocal laser microscopy • Distinguish infectious from non-infectious organisms • Combine with infectivity, viability or activity assays • Overcome sample size limitation due to presence of non-target particles • Flow cytometry and other advanced imaging techniques • Advanced imaging methods require expensive hardware

11. Types of Microscopes • Light microscopes • Compound • Dissection/stereo • Inverted • Confocal • Electron Microscopes • Scanning • Transmission

12. Types of Microscopes • Light microscopes • Compound • Dissection/stereo • Inverted • Confocal • Electron Microscopes • Scanning • Transmission

13. Types of Microscopes • Light microscopes • Compound • Dissection/stereo • Inverted • Confocal • Electron Microscopes • Scanning • Transmission

14. Types of Microscopes • Light microscopes • Compound • Dissection/stereo • Inverted • Confocal • Electron Microscopes • Scanning • Transmission

15. Types of Microscopes • Light microscopes • Compound • Dissection/stereo • Inverted • Confocal • Electron Microscopes • Scanning • Transmission

16. Types of Microscopes • Light microscopes • Compound • Dissection/stereo • Inverted • Confocal • Electron Microscopes • Scanning • Transmission

18. Light Microscopy • Bright Field • Dark Field • Phase Contrast • Differential Interference Contrast • Epifluorescence • Confocal Scanning

19. Kohler Alignment

21. Bright field • Most common of all light scopes • Light is transmitted through specimen • Specimen appears darker than surrounding field • Typical use: Gram Stains

22. Gram Stain

23. Microscopic Detection of Pathogens: Still Widely Used in Clinical Diagnostic Microbiology C. parvum oocysts ~5 um diam. Acid fast stain of fecal preparation

24. Dark Field • Used to increase the contrast of a transparent specimen • Contrast = ability to distinguish an object from surrounding medium • Specimen appears as a bright image against dark background • Often used to observe live non-fixed/stained samples, e.g. observe motility and growth

26. Darkfield Microscopy

27. Phase Contrast • Used to observe fine internal detail • Takes advantage of differences in density of transparent internal cell components • Uses a series of diaphragms to separate and recombine direct versus diffracted light rays

29. DIC • Illuminating light beam is split such that one beam passes through the specimen creating a phase difference with the second reference beam • Beams are then combined so that they interfere with eachother • Allows detection of small changes in in depth or elevation of the surface of the specimen • Thus gives 3d appearance

30. Cryptosporidium parvumDifferential Interference Contrast Microscopy Image courtesy of O.D. “Chip” Simmons, III

31. Fluorescent • Uses UV light source to illuminate fluorescent dyes that then emit visible light • e.g. FITC, Acridine Orange, Rhodamine • Specimens appear as bright colored objects in front of black background • Often used with immunologic procedures

33. Cryptosporidium parvum: Microscopic Analysis of NC field isolate Immunofluorescence Differential Interference Contrast DAPI stain Images courtesy of O.D. “Chip” Simmons, III

34. Electron Microscopy • SEM-Scanning Electron Microscopy • Image is formed as electron probe scans the surface of the specimen • Produces 3d images • TEM-Transmission Electron Microscopy • Image formed as electrons pass through specimen • Specimens must be thin cut • Used to view internal structure

37. Activity Assays/Vital Dyes

38. Detection of Pathogens by Viability or Activity Assays Assay bacteria for viability or activity by combining microscopic examination with chemical treatments to detect activity or "viability". • measure enzymatic activities, such as dehydrogenase, esterase, protease, lipase, amylase, etc. • Example: tetrazolium dye (INT) reduction: 2-[p-iodophenyl]-3-[p-nitrophenyl]-5-phenyltetrazolium Cl (measures dehydrogenase activity). • Reduction of tetrazolium dye leads to precipitation of reduced products in the bacterial cells that are seen microscopically as dark crystals.

39. FISH: DAPI-stained Bacteria Incubated with INT (Tetrazolium Salt) • Enhanced image with artificial colors. • Blue: DAPI stain • Red: INT grains; indicate respiratory active bacteria.

40. Progress in Detection of Bacteria by Viability or Activity Assays • Combine activity measurement and immunochemical assay (for specific bacteria). • Combine fluorescent antibody (FA) (for detection of specific bacterium or group) with enzymatic or other activity measurement • Use image analysis tools to improve detection and quantitation • Flow cytometry • Computer-aided laser scanning of cells or colonies on filters

41. Viability or Activity Assays for Protozoan Cysts and Oocysts • Example: Stain with DAPI (the fluorogenic stain 4',6‑diamidino‑2‑phenylindole; taken up by live oocysts and propidium iodide (PI; taken up by dead oocysts). • Viable Cryptosporidium oocysts are DAPI-positive and PI-negative • Non-viable oocysts are DAPI-negative and PI-positive • Alternative stains may be more reliable • Viability staining is often poorly associated with infectivity Detects cysts and oocysts inactivated by UV and chemical disinfection

42. C. parvum oocysts Dual stain : DAPI (blue) and propidium iodide (red)

43. Immunological Methods

44. Immunoglbulins • 5 Classes • IgA secretory • IgD found in plasma but not serum • IgE involved in allergic reactions • IgG humeral response • IgM humeral response • IgG and IgM most commonly used in immunoassays