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Chapter 2

CHAPTER 2 - A Brief Journey to the Microbial World.docx

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Biology 2581B
Chris Brandl

MICROBIOLOGY CHAPTER 2: A BRIEF JOURNEY TO THE MICROBIAL WORLD 2.1 SOME PRINCIPLES OF LIGHT MICROSCOPY • Light microscope: examine cells at relatively low magnification • Electron microscope: look at cells at very high magnification • Resolution: ability to distinguish two adjacent objects as distinct and separate  Governs our ability to see small things  Function of wavelength of light • Magnification can be increase infinitely, but resolution cannot b/c it’s a function of the physical properties of light The Compound Light Microscope • Resolution: 2 micrometers • Uses visible light to illuminate cell structure • Bright field, phase-contrast, differential interference contrast, dark-field. fluorescence • Bright-field: see specimen b/c of contrast between specimen and surrounding  Contrast is created by cell absorbing/scattering light to various degrees • Made of objective and ocular lenses • Light source focused by condenser • Bacteria cells are difficult to see in light microscope b/c the cells lack significant contrast with surroundings • Pigmented MO are good b/c colour adds contrast Magnification and Resolution • Total magnification = objective x ocular • Resolution does not improve above 2000x mag • Numerical aperture: light-gathering ability; determines resolution  Higher mag = higher numerical aperture • Diameter of smallest object resolvable = 0.5λ/numerical aperture  Blue light is highest resolution b/c it’s the shortest wavelength • After 1000x mag, can only improve resolution  100x = oil-immersion lens  Oil increases light-gathering ability 2.2 IMPROVING CONTRAST IN LIGHT MICROSCOPY Staining: Increasing Contrast for Bright-Field Microscopy • Use dyes to stain cells to increase contrast • Each class of dye has affinity for specific cellular materials • Basic dyes: positively charged (ex. methylene blue, crystal violet, safranin) MICROBIOLOGY  Bind to –vely charged (ex. nucleic acid, acidic polysaccharides, cell surface) • To prepare: smear  air dry  flame to fix  stain for a few minutes  rinse w/ water  blot to dry  place under high-power/oil-immersion lens Differential Stains: The Gram Stain • Differential Stains: stains that render different colours • bacteria can be gram-positive (purple/violet) or gram-negative (pink)  Happens b/c of gram +/- cell walls  After staining w/ crystal violet, ethanol decolourizes gram-negative  Counter-stain w/ a different colour (ex. safranin) and the two types of cells can be distinguished • Gram +/- fluoresce differently under fluorescent microscope • To prepare: flood heat-fixed smear w/ crystal violet for a min  Add iodine for 1 min decolourize w/ alcohol for 20 sec  counterstain w/ safranin for 1-2 min Phase-Contrast and Dark-Field Microscopy • Staining kills/distorts cells; use phase-contrast / dark-field • Phase contrast: cells differ in refractive index  Light passing through cell is of different phase than surroundings  Phase ring on objective lens results in dark image on light background  Has phase plate that amplifies phase differences • Dark field: light reaches specimen from sides only  Specimen scatters light – results in light on dark background  Better resolution  Good for observing motility – can resolve flagella Fluorescence Microscopy • Fluoresce: t
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