1. Microbiological Methods Scientific Research and Medical Diagnostics

2. Overview Of Microbiological Methods Phenotypic tests –Microscopy –Culture techniques –Immunological techniques Genotypic tests

3. Methods for Studying Microbes By phenotype (expressions of genes, physiology) –Cell morphology, colony morphology, ‘behavior’ –Growth conditions (aerobic, anaerobic) –Selective/differential media (utilization of specific nutrients, resistance to chemicals) –Test for various enzymes e.g. oxidase, catalase –Serology, Antigen/antibody binding By genotype (genetic sequence or structure) –Based DNA profiles based of RE digestion –Based on sequence of DNA or PCR

4. Compound Microscope A variety of lenses can be used to magnify small objects from 40X to 1000X Only used for thin, transparent objects <1mm thickness

5. Light microscopes Image absorbs light and appears dark to the observer

6. Fluorescence Microscopy Dye or labeled antibody binds to object and fluoresces under UV light May provide greater resolution for small bacteria Dye may indicated a specific physiological state of the bacterium

7. Electron Microscopy Specimen coated with gold or other metal that will be stimulated by electron beam electron microscopes: SEM,TEM, STM, AFM

8. Preparations for light microscopy Used to identify phenotypic characteristics of certain taxa Usually performed on microscope slides using sample of pure culture Some tissue samples may be examined directly (CSF) or after staining Common staining procedures for bacteria include: Gram stain, acid fast stain, endospore stain, capsule stain Does not usually identify to the species level but is useful in combination with other methods of identification

9. Binary Fission Most common method of bacterial reproduction Allows for vary fast population growth 8 cells after 3 generations 64 cells after 6 generations 512 after 9 generations….

10. Bacterial Growth curve lag log stationary decline Limited nutrients etc…

11. Culture Media Culture- maintenance of a lineage, usually implies in vitro Culture medium- the substrate on/in which the culture is maintained –Natural or Synthetic –Selective and/or Differential –Enrichment –Solid or Liquid

12. Isolation streak on agar in a petri dish Microcolony on growth medium Bacterial Culture and Isolation

13. Test tube culture Agar slants Broth

14. Selective and differential medium

15. Culturability Most microorganisms are difficult to culture or not culturable Culturable does not mean ALWAYS culturable –VBNC or VNC (viable but non-culturable) Some organisms such as (E. coli) are very easy to culture Metabolic factors are proximate factors that influence an organism’s culturability. Remote factors include environmental conditions, chemicals, pH etc…

16. Variation in growth conditions Aerobic-utilizing oxygen Anaerobic-not using oxygen Facultative-means growth will occur under certain conditions if necessary Obligate- strictly limited to specified conditions Fastidious organisms are difficult to grow in lab Organisms with highly specialized lifestyle utilizing unusual compounds for growth and survival Obligate Intracellular symbionts require growth in living growth medium (e.g. lab animal or their tissues, HeLa cells)

17. Methods of Enumeration Yields information about growth, risks etc.. Only an estimate of actual population density Several methods Direct microscopic counts Spread plate Membrane filtration Most probable number Spectrophotometry Flow cytometry

18. Direct microscopic counts Known volume of sample added to microscope slide Slide is marked with special grid to aid with counting the number of observed cells per unit area Does not usually allow for inferring that cells are viable; however some chemicals can be added that indicate viable cells only Glass slide grid

19. Spread Plating Sample spread evenly over surface of medium Only works for samples with density of ~300 CFU/ml or greater Colonies appear after incubation

20. Membrane Filtration Sample Liquid sample passed through porous membrane which is then placed on agar Used for concentrated or dilute samples

21. Dilutions Sample with unknown density of bacteria test tubes, each with 9ml of sterile buffered water Transfer 1ml from sample to first tube, then 1ml from first to second etc… 10X Final dilution factor? Dilution factor for each step

22. Dilutions.1ml from tubes onto plates Incubate plates Count the dilution that yields between 30-300 colonies Take average of three plates

23. Identifying organisms by the presence of certain biochemical reactions Many tests revolve around the observation or measurement of bacterial enzymes, which are phenotypic characteristics Enzymes can be detected by adding a substrate either in vitro or in vivo to a bacterial sample and observe reactions (e.g. catalase test) Growth media may allow biochemical reactions to be tested Commercially available kits allow for multiple tests to be performed simultaneously

24. Immunological (serological) tests Can be used to detect specific antibodies or specific antigens Either the antibody or antigen will be hypothesized the other will be known Antibodies from patient’s serum mixed with known antigen sample Y Y Y Known antibodies mixed with unknown antigen Y Y Y

25. Detection The binding of antibodies and antigens may be detected or visualized in several ways: –Precipitation –Agglutination –Fluorescence –RBC lysis (Complement fixation) –Enzymatic color reaction –Electrophoresis and staining

26. Precipitin Tests Y Y YY Y Y Y Y Y Y Y Precipitated (out of solution) antibody- antigen complex, will appear cloudy while rest of tube is clear

27. Agglutination Y Y Y Y YY Y positivenegative Can be performed on glass slide or in plastic microtitre (microwell) plates

28. Coomb’s antiglobulin test Antibodies may be formed against other antibodies The resulting complex allows for ‘amplification’ of agglutination First antibody X made in animal A then injected into animal B Animal B produces antibodies Z to first antibody Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y

29. Immunofluorescence Antibody with fluorescent label binds to specific antigen from sample Can be done on slide and viewed through microscope or in Can be done in microwell plates Ψ Ψ Ŧ § § Ŧ § YY

30. Y Y

31. ELISA Enzyme Linked Immunosorbent Assay Usually performed in microwell plate Can be used to detect specific antibody or antigen Y Y Y Y

32. Complement fixation test Serum sample taken which is hypothesize to contain antibodies to specific antigen Known antigen added to serum Complement added to serum/antigen mixture If serum contains antibodies compliment will be fixed at this point otherwise, it will remain free in the serum/antigen mixture and will be fixed at next step RBCs bound to antibodies added to mixture and: –If lysed then serum did not contain suspected antibodies(-) –If not lysed, then serum did contain antibodies (+)

33. Gel Electrophoresis Gel made of translucent, porous matrix through which molecules can move when exposed to an electric field Samples added to wells in matrix - +

34. Western Blot Antigens separated by gel electrophoresis Paper placed on gel Proteins diffuse from gel to paper Labeled antibodies applied to paper and cause color change where specific binding occurs Y + -