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Methods of Microbiology

Methods of Microbiology. Staining Media Microscopy. Staining. Increase contrast of microorganisms Classified into types of stains Simple stain: Differential stain: Structural or special stains. Dyes. Organic salts with positive and negative charges One ion is colored –chromophore

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Methods of Microbiology

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  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

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

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

  14. Anaerobic Methods • Reducing media • Anaerobic jar • Use both in lab

  15. Candle Jar • Reduce oxygen levels • Provides more CO2 • Microaerophilics

  16. Selective and Differential Media • Selective • Differential

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

  18. 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

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

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

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

  22. Lenses • Parfocal • Working distance

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

  24. 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

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

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

  27. 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

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

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

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

  31. 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)

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

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