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1 The 5 I’s of Culturing Microbes Inoculation Isolation Incubation Inspection Identification...

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1 The 5 I’s of Culturing Microbes Inoculation Isolation Incubation Inspection Identification 8/18/12 MDufilho
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Page 1: 1 The 5 I’s of Culturing Microbes Inoculation Isolation Incubation Inspection Identification 8/18/12MDufilho.

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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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