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The 5 I’s of Culturing Microbes
• Inoculation• Isolation• Incubation
• Inspection• Identification
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Table 4.1 Metric Units of Length
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Microscopy
• General Principles of Microscopy– Wavelength of radiation– Magnification– Resolution– Contrast
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Figure 4.1 The electromagnetic spectrum
Visible light
Micro- wave
Infra- red
UV light
X rays
Radio waves and Television
One wavelength
400 nm 700 nm
Gamma rays
Increasing wavelength
Crest
100m 103m10–4m10–8m
Increasing resolving power
Trough
10–12m
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Figure 4.2 Light refraction and image magnification by a convex glass lens-overview
Convexlens
Inverted,reversed, andenlargedimage
Focal point
Specimen
Glass
Light
Air
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Principles of Light Microscopy
• Magnification occurs in two phases – – The objective lens forms the magnified real image– The real image is projected to the ocular where it is
magnified again to form the virtual image
• Total magnification of the final image is a product of the separate magnifying powers of the two lenses
power of objective x power of ocular = total magnification
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ResolutionResolution defines the capacity to distinguish or
separate two adjacent objects – resolving power– Function of wavelength of light that forms the
image along with characteristics of objectives• Visible light wavelength is 400 nm–750 nm• Numerical aperture of lens ranges from 0.1 to 1.25• Oil immersion lens requires the use of oil to prevent
refractive loss of light• Shorter wavelength and larger numerical aperture will
provide better resolution• Oil immersion objectives resolution is 0.2 μm• Magnification between 40X and 2000X
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Effect of wavelength on resolution
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Figure 4.5 The effect of immersion oil on resolution-overview
Microscopeobjective
Refracted lightrays lost to lens
Glass cover slip
Light sourceSpecimen
Slide
Without immersion oil
Glass cover slip
Light source
Slide
Microscopeobjective
More lightenters lens
Lenses
With immersion oil
Immersion oil
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Chickenegg
Human redblood cell
Largeprotozoan(Euglena)Chloroplasts
Flea Typical bacteriaand archaea
Diameterof DNA
Viruses Proteins
Ribosomes
Aminoacids
Atoms
Scanning tunneling microscope(STM) 0.01 nm–10 nm
Scanning electron microscope (SEM) 0.4 nm–1 mm
Transmission electron microscope (TEM) 0.078 nm–100 µm
Atomic force microscope (AFM)
1 nm–10 nm
Compound light microscope (LM) 200 nm–10 mm
Unaided human eye200 µm–
Mitochondrion
Figure 4.3 The limits of resolution of the human eye and of various types of microscopes
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Contrast
• Differences in intensity between two objects, or between an object and background
• Important in determining resolution
• Staining increases contrast
• Use of light that is in phase increases contrast
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Light MicroscopyBright-field microscopes
• Simple– Contain a single magnifying lens
– Similar to magnifying glass
– Leeuwenhoek used simple microscope to observe microorganisms
• Compound– Series of lenses for magnification– Light passes through specimen into objective lens – Oil immersion lens increases resolution– Have one or two ocular lenses– Total magnification (objective lens X ocular lens)– Most have condenser lens (direct light through specimen)
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Care and Use of Microscope
• Moving microscope from storage • Storing microscope
– Short objective in place, center stage– Clean, cord wrapped correctly and covered
• Cleaning microscope– Lens paper, swabs and cleaning solution
• Learn components• Focusing the microscope on specimen• Use of Oil Immersion lens
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