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Methods of Microbiology Staining Media Microscopy
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Staining Increase contrast of microorganisms Classified into types of stains –Simple stain: –Differential stain: –Structural or special stains
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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
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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
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Acidic Dye Chromophore repelled by negative cell wall Background is stained, bacteria are colorless Negative stain-look at size, shape Eosin, India ink
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Simple Stains One dye, one step Direct stain using basic dye Negative stain using acidic dye
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Differential Stains More than one dye Gram stain, acid fast Primary dye Decolorizing step Counter stain
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Special/ Structural Stains Identify structures within or on cells Different parts of cell are stained different colors
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Media Culture media Inoculum Culture Pure culture Million cells to be visible
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Living vs Nonliving Viruses, few bacteria Living host-eggs, tissue cells Mycobacterium leprae –armadillos Most microbes grow on nonliving media
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Synthetic or Chemically Defined Exact chemical composition known Chemoheterotrophs –Glucose-carbon source & energy source –Supplies chemical requirements
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Complex Media Used for most chemoheterotrophs Bacteria and fungi Cannot write formula for each ingredient C,N,energy, S requirements Vitamins, other growth factors
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Complex Media Nutrient broth –liquid form Nutrient agar –solid form –Plate –Deep –Slant
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Anaerobic Methods Reducing media Anaerobic jar Use both in lab
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Candle Jar Reduce oxygen levels Provides more CO 2 Microaerophilics
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Selective and Differential Media Selective Differential
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Filtration Passage of liquid through screen device Pores small enough to retain microbes Sterilize heat sensitive materials Negative Positive HEPA hoods & TB rooms
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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
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Autoclave Prions- protein only Flash sterilization-at 134 C for 3min Packaging Use of indicators
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Compound Microscope Assigned scope Know parts & functions Proper use & care of scope
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Microscope Use of light ( visible or UV) or electrons Lenses to magnify Total magnification of compound scope
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Lenses Parfocal Working distance
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Resolution Ability to distinguish 2 objects as separate and distinct Dictated by physical properties of light Limit is 0.2um for our light scope
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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
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Light Path Passes through opening in stage to slide Light enters objective lens –Image magnified by ocular lens
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Contrast Improves image detail Difference in light intensity Bacteria are colorless Need to increase artificially by staining Contrast is property of specimen
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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
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Resolving Power Function of numerical aperture: NA Function of wavelength of light Refractive index of material between objective lens & specimen
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Oil Immersion Lens Light bends (refracts) as it passes from glass into air Use oil between slide and 100x lens Increases resolution
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Oil Immersion Lens Lens captures more light Shortest working distance Summary: increased resolution
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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)
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Electron Microscopy Uses electrons as source of illumination Wavelength of electrons is dependent upon voltage of electron beam
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