1. Methods of Microbiology Staining Media Microscopy

2. Staining Increase contrast of microorganisms Classified into types of stains –Simple stain: –Differential stain: –Structural or special stains

3. Dyes Organic salts with positive and negative charges One ion is colored –chromophore Basic dye: positive ion is colored –MeBlue + Cl - Acidic dye: negative ion is chromphore

4. Basic Dye Works best in neutral or alkaline pH Cell wall has slight negative charge at pH 7 Basic dye (positive) attracted to cell wall ( negative) Crystal violet, methylene blue, safranin

5. Acidic Dye Chromophore repelled by negative cell wall Background is stained, bacteria are colorless Negative stain-look at size, shape Eosin, India ink

6. Simple Stains One dye, one step Direct stain using basic dye Negative stain using acidic dye

7. Differential Stains More than one dye Gram stain, acid fast Primary dye Decolorizing step Counter stain

8. Special/ Structural Stains Identify structures within or on cells Different parts of cell are stained different colors

9. Media Culture media Inoculum Culture Pure culture Million cells to be visible

10. Living vs Nonliving Viruses, few bacteria Living host-eggs, tissue cells Mycobacterium leprae –armadillos Most microbes grow on nonliving media

11. Synthetic or Chemically Defined Exact chemical composition known Chemoheterotrophs –Glucose-carbon source & energy source –Supplies chemical requirements

13. Complex Media Used for most chemoheterotrophs Bacteria and fungi Cannot write formula for each ingredient C,N,energy, S requirements Vitamins, other growth factors

14. Complex Media Nutrient broth –liquid form Nutrient agar –solid form –Plate –Deep –Slant

16. Anaerobic Methods Reducing media Anaerobic jar Use both in lab

17. Candle Jar Reduce oxygen levels Provides more CO 2 Microaerophilics

18. Selective and Differential Media Selective Differential

19. Filtration Passage of liquid through screen device Pores small enough to retain microbes Sterilize heat sensitive materials Negative Positive HEPA hoods & TB rooms

20. Autoclave Uses temperature above boiling water Steam under pressure Preferred method unless material is damaged Higher the pressure, higher the temperature Need direct contact with steam 15 psi at 121 C for 15 mins

21. Autoclave Prions- protein only Flash sterilization-at 134 C for 3min Packaging Use of indicators

22. Compound Microscope Assigned scope Know parts & functions Proper use & care of scope

23. Microscope Use of light ( visible or UV) or electrons Lenses to magnify Total magnification of compound scope

24. Lenses Parfocal Working distance

25. Resolution Ability to distinguish 2 objects as separate and distinct Dictated by physical properties of light Limit is 0.2um for our light scope

26. Light Scope Simple vs compound Source of illumination Visible light has average wavelength of 550nm or 0.55 um –Enters condenser lenses –Focused into a cone

27. Light Path Passes through opening in stage to slide Light enters objective lens –Image magnified by ocular lens

28. Contrast Improves image detail Difference in light intensity Bacteria are colorless Need to increase artificially by staining Contrast is property of specimen

29. Resolution Distinguish detail within image Property of lens system, measured as resolving power Closest that 2 points can be together and still seen as separate RP = wavelength of light 2 X NA

30. Resolving Power Function of numerical aperture: NA Function of wavelength of light Refractive index of material between objective lens & specimen

31. Oil Immersion Lens Light bends (refracts) as it passes from glass into air Use oil between slide and 100x lens Increases resolution

32. Oil Immersion Lens Lens captures more light Shortest working distance Summary: increased resolution

33. Fluorescent Microscope Used to view antigen antibody reactions Specimen tagged with fluorescent dye Ocular lens fitted with filter that permits longer wavelengths & blocks shorter ones UV light(230-350nm)

34. Electron Microscopy Uses electrons as source of illumination Wavelength of electrons is dependent upon voltage of electron beam