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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 103m 10–4m 10–8m

Increasing resolving power

Trough

10–12m

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Figure 4.2 Light refraction and image magnification by a convex glass lens-overview

Convex

lens

Inverted, reversed, and enlarged image

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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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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Calculating Resolving Power

Formula D = ________wavelength (nm)_________

NA (condenser) + NA (objective)

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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

Glass cover slip

Light source Specimen

Slide

Without immersion oil

Glass cover slip

Light source

Slide

Microscope objective

More light enters lens

Lenses

With immersion oil

Immersion oil

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Chicken

egg

Human red

blood cell

Large

protozoan

(Euglena) Chloroplasts

Flea Typical bacteria

and archaea

Diameter

of DNA

Viruses Proteins

Ribosomes

Amino

acids

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 eye

200 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 Microscopy Bright-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)

– Video on Microscopy

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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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