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
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–
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Wavelength of radiation
Magnification
Resolution
Contrast
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Figure 4.1 The electromagnetic spectrum
400 nm
700 nm
Visible light
Gamma rays
10–12m
X UV
rays light
10–8m
Infra- Microred
wave
Radio waves and
Television
Increasing wavelength
10–4m
100m
103m
Crest One wavelength
Trough
Increasing resolving power
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Figure 4.2 Light refraction and image magnification by a convex glass lens-overview
Light
Glass
Air
Focal point
Specimen
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Convex
lens
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Inverted,
reversed, and
enlarged
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image
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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Resolution
Resolution 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
Microscope
objective
Refracted light
rays lost to lens
Microscope
objective
Lenses
More light
enters lens
Glass cover slip
Glass cover slip
Slide
Slide
Specimen
Light source
Light source
With immersion oil
Without immersion oil
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Immersion oil
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Figure 4.3 The limits of resolution of the human eye and of various types of microscopes
Diameter
of DNA Ribosomes
Proteins
Atoms
Viruses
Amino
acids
Typical bacteria
and archaea
Flea
Chloroplasts
Mitochondrion
Large
protozoan
(Euglena)
Chicken
egg
Human red
blood cell
Scanning tunneling microscope
(STM) 0.01 nm–10 nm
Transmission electron microscope (TEM)
0.078 nm–100 µm
Unaided human eye
200 µm–
Scanning electron microscope (SEM)
0.4 nm–1 mm
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Atomic force
microscope (AFM)
1 nm–10 nm
Compound light microscope (LM)
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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 Microscopy
Bright-field microscopes
• Simple
– Contain a single magnifying lens
– Similar to magnifying glass
– Leeuwenhoek used simple microscope to observe
microorganisms
• Compound
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–
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–
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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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