Ch. 3: Observing Microorganisms Through a Microscope

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Units of Measurement

• micrometers (µm)

• nanometers (nm)

  • 1 µm = 1000 nm

  • 1 µm = 10^-3 nm

Total Magnification

objective lens x ocular lens (__x)

Numerical Aperture of Lens

an objective lens with a HIGHER value of NA produces a HIGHLY resolved image

(direct relationship)

Resolution (resolving power)

the ability of the lenses to distinguish two points or the ability to distinguish fine details and structure

• λ / 2NA

  • NA: numerical aperture

  • λ: wavelength

• the SHORTER the wavelength (λ), the better the resolution (indirect relationship)

Compound Light Microscope

• objective lens = 10x

• magnification = up to 1000x

• limit of resolution = 0.2 µm or 200nm

• immersion oil required at 100x

Refractive Index

the measure of light-bending ability of a medium

• for compound light microscopy, light may refract after passing through speciman and may not go through the objective lens

  • immersion oil

Immersion Oil

used to stop light from refracting

Electron Microscope

• uses electrons instead of light

• used for images that are too small to be seen in light microscopes

  • like cell structures or viruses

• has two major types

Transmission Electron Microscopy (TEM)

• a beam of electrons passes through sections of the specimen

• magnification = up to 10,000 - 10,000,000x

  • limit of resolution = 0.2 nm

Scanning Electron Microscopy

• a beam of electrons scans the surface of an entire specimen

• magnification = 1,000 - 500,000x

  • limit of resolution = 0.5 nm

Fluorescence Microscopy

• uses UV (short wavelengths) as a light source of illumination

• fluorescent substances absorb UV lights and emit longer wavelength light (visible)

• cells may be dyed with fluorescent dye

Fluorescence-antibody technique (Immunofluorescence)

• used for rapid and specific detection of pathogens in a patients specimens

  • antibodies specific for a type of microbe are tagged with fluorochrome

  • then, applied to slide with potential microbe

  • if present, the fluorescent will stick to the microbe and cause it to fluoresce under microscopy

Simple Staining

• single basic dye

  • highlights the entire microorganims to visualize cell size, shape, and structure

Mordant

applied to increase the affinity of stain onto a microorganism

• could coat a thin structure to make it thicker and easier to see

Differential Stain

• uses multiple dyes

• used to distinguish between bacteria

Special Stain

• used to distinguish specific parts of microorganisms

Gram Stain

• type of differential stain

• classifies bacteria into gram-negative (pink) or gram-positive (purple)

  • important for identifying unknown bacteria

Acid-Fast Stain

• type of differential stain

• used for the identification of mycobacterium or nocardia

• retain pink/red color from carbolfuchsin b/c it is soluble in lipid cell walls

  • difficult to stain routinely due to high lipid content (mycolic acid) in the walls

Capsule Stain

• type of special stain

  • negative staining

• because capsules are a gelatinous covering that does not accept most dyes, it will appear “halo-like”

Endospore Stain

• type of special stain

• requires heat in order for dye to become trapped inside the endospore coat

• appears green from malachite green

  • however, vegetative cells appear red due to counterstain

Flagella Stain

• type of special stain

• uses mordant and carbolfuchsin to build a wider diameter around the flagella in order to be more visible

  • b/c flagella are thin and small