Wet mounts using Phase Contrast Microscopy

Principles


Principle: Phase contrast microscopy takes advantage of fact that structures with different refractive indexes bend the light differently. With special phase optics, this difference in the ability to bend light translates into a difference in contrast.    For example, light rays traveling through cellular material get bent and retarded.   We probably could not detect this bending in bright field.   In phase contrast, a phase plate slows down the highly refracted light rays and puts them "out of phase".   This is seen as a difference in contrast between the cell and its background. The background is generally seen as dark, with the organism in sharp contrast.    This is extremely useful for microbiologists because we can view microorganisms without physically or chemically altering the cell. Bacteria (and other microorganisms) can be viewed alive.   Simple wet mounts can be made so such properties as motility and growth can be observed.

Procedure

  1. Place small amount of sample on a microscope slide; cover with cover slip.
  2. View under phase contrast microscope .

Advantages

  • Can view live samples and observe motility and responses to stimuli

Disadvantages

  • Phase optics are more expensive than bright field, and must be properly aligned.
 Fluorescent in Situ Hybridization (FISH) method

 

 

 

Examples

iron deposits and Leptothrix