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Methods of Microbiology

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Organic salts with positive and negative charges. One ion is ... HEPA hoods & TB rooms. Autoclave. Uses temperature above boiling water. Steam under pressure ... – PowerPoint PPT presentation

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Title: Methods of Microbiology


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
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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

15
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16
Anaerobic Methods
  • Reducing media
  • Anaerobic jar
  • Use both in lab

17
Candle Jar
  • Reduce oxygen levels
  • Provides more CO2
  • 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
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