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

Table 4.1 Metric Units of Length 8/18/12 MDufilho 2

Microscopy General Principles of Microscopy Wavelength of radiation Magnification Resolution Contrast 8/18/12 MDufilho 3

Figure 4.1 The electromagnetic spectrum 400 nm 700 nm Visible light X rays UV light Infra- red Micro- wave Radio waves and Television Gamma rays Increasing wavelength 10–12m 10–8m 10–4m 100m 103m One wavelength Crest Trough Increasing resolving power 8/18/12 MDufilho 4

Light Air Glass Focal point Specimen Convex lens Inverted, Figure 4.2 Light refraction and image magnification by a convex glass lens-overview Light Air Glass Focal point Figure 4.2 Light refraction and image magnification by a convex glass lens. Specimen Convex lens Inverted, reversed, and enlarged image 8/18/12 MDufilho 5

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 8/18/12 MDufilho

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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 8/18/12 MDufilho

Effect of wavelength on resolution 8/18/12 MDufilho

Figure 4.5 The effect of immersion oil on resolution-overview Microscope objective Microscope objective Lenses Refracted light rays lost to lens More light enters lens Immersion oil Glass cover slip Glass cover slip Slide Slide Specimen Light source Light source Without immersion oil With immersion oil 8/18/12 MDufilho 10

Figure 4.3 The limits of resolution of the human eye and of various types of microscopes Diameter of DNA Typical bacteria and archaea Ribosomes Flea Large protozoan (Euglena) Atoms Proteins Viruses Chloroplasts Chicken egg Amino acids Human red blood cell Mitochondrion 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 Atomic force microscope (AFM) 1 nm–10 nm 8/18/12 MDufilho Compound light microscope (LM) 200 nm–10 mm 11

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 8/18/12 MDufilho 12

Light Microscopy Bright-field microscopes Simple Compound 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) 8/18/12 MDufilho 13

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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 8/18/12 MDufilho