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SOS 4307

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SOS 4307

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    1. SOS 4307/5308 Ecology of Water-borne Pathogens Lecture 5: Microbial Sample Collection Microscopic techniques 16 January 2009

    2. Questions to ask oneself: What am I looking for? How many samples? What kinds of samples?

    3. Soil Sampling: tools

    4. Soil Sampling: where to sample and how many samples to take

    5. Soil is a very heterogeneous habitat…

    6. Human pathogens colonize rhizosphere

    7. Water Sample Collection: when, where and how much?

    8. Coupons for collecting biofilms

    9. Environmental Sample Collection Problems and limitations Cross-contamination is almost always an issue Redox/oxygen sensitive samples The Scale issue If a microbe were as big as a human, then surface area of a gram of soil is like the surface of all continents combined One gram of soil contains 1010 microbial cells, approximately 4,000 species Heterogeneity of [micro] environments VBNC

    10. What can a microbiologist extract from a soil/water sample? Microbes (fungi, bacteria, viruses) DNA RNA Proteins/enzymes Other “signature” molecules (fatty acids, metabolism by-products, etc)

    11. Techniques for separation of chemicals

    12. Isolation of nucleic acids from environmental samples Nucleases are present in soil/water samples add EDTA to chelate cation cofactors Steps in nucleic acid extraction: Concentrate dilute samples Lyse cells (lysozyme, detergents, solvents, glass beads or freezing/thawing) Isolation (buffer in pH 8), then wash with organic solvents. Precipitation: lower pH, add alcohol.

    13. Protein isolation Proteases in environmental samples Various chromatography techniques are involved in extraction Size exclusion Ion exchange

    14. You have your soil/water samples, now what?

    15. The [elusive] Microbial Diversity What is a microbial species? is there a genetic/phenotypic marker? does allopatric speciation apply? Allopatric (geographic) speciation occurs when geographically isolated populations evolve intrinsic (genetic) isolation.

    16. The [elusive] Microbial Diversity Biogeographic differentiation: two models

    17. The [elusive] Microbial Diversity

    18. Testing Methods

    19. Microscopic Detection Direct Microscopic Observations cyanobacteria, eukaryotes (e.g. Giardia) and particles (OK) viruses (useless) bacteria (tricky) ID is problematic, especially for motile rods aided by fluorescent tags (immunological procedure) Gram stain

    20. Types of light microscopy Bright field Image seen is the result of light transmission through the specimen Dark field Some light is prevented from reaching the objective Phase-contrast Different cell components have different densities, which interact differently with light creating contrast Differential interference The light beam is split into two: one goes through the specimen, the other is a reference beam. The beams interfere, create a 3D image

    21. because of differences in cell wall composition, different classes of bacteria interact differently with Gram stain components Gram-negative stain red/pink Gram-positive stain purple Gram Staining

    22. Gram Staining

    23. Flagella stain Contains “mordant” so that other coatings stick

    24. Fluorescent microscopy UV light as a source of illumination For cells expressing fluorescent proteins or fluorescent stains

    25. FISH Fluorescent in-situ hybridization

    26. Electron microscopy Electrons, not light, form images a beam of electrons goes through the sample in a vacuum Scanning EM Image is formed as electrons scan the surface Transmission EM Image is formed when electrons pass through the image requires fixation, dehydration,embedding

    27. Confocal scanning microscopy Two lenses are used to focus the sample Can take images at successive planes

    28. Optical Methods In-line detectors is coupled with immunoassays depending on the immunoassay, may detect bacteria themselves (faster method, min-hrs) or induced proteins (slower method, 4 hrs) chlorination interferes with detection (10-100x lower detection limit, 40% false-positives when using fiber-optic waveguide) chloramine or chlorine-neutralized work OK

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