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Principles of Microscopy and Staining



Microbes, such as viruses, bacteria, fungi, algae, and protozoa, are extremely small organisms and their size makes them impossible to see with the naked eye. For humans to see them, the microbes must be magnified. Microscopes magnify the image of microbes by shining light or electrons on them and passing that beam through a series of lenses, which makes the specimen on the slide appear bigger. Organisms may be visualized live in a wet mount or dead after fixing to a microscope slide. Stains, such as crystal violet and safranin, can be used to understand properties of microbes when looking at them through a microscope. Stains contrast the color of a microbe depending on the structure of its cell wall and how it processes nutrients.

At A Glance

  • Microorganisms are very small and range in size from 10 nanometers to 1 millimeter. In general, microbes are only observable with the aid of a microscope.
  • The wavelength of light directly impacts the resolution of a microscope; long wavelengths result in low resolution and short wavelengths result in high resolution.
  • When objects have different indexes of refraction, the light will bend, resulting in a loss of light and blurry images.
  • In a light microscope, light passes through a condenser (converging the beams to pass through the specimen on the slide), the iris diaphragm controls the amount of light that passes into the lens, and total magnification is calculated by multiplying the magnification of each lens.
  • Wet mounts are used to examine live organisms; however, smears fix (kill) the organisms.
  • Besides light microscopes, other types of microscopes are dark field, phase-contrast, fluorescence, confocal, scanning electron, and transmission electron.
  • Stains are used to colorize and contrast different parts of microbes; simple stains use a single color, while differential stains use more than one color.
  • Gram-positive bacteria have a thick peptidoglycan layer and retain the crystal violet stain after being set with iodine. Gram-negative bacteria have a thinner peptidoglycan layer, which is dissolved with alcohol, and do not retain crystal violet but rather stain red with safranin.
  • The most common differential stains are Gram, acid-fast (Ziehl-Neelsen), and Schaeffer-Fulton.