Special Techniques

PHASE CONTRAST MICROSCOPY

Phase contrast microscopy is used for the observation of living cells i.e. unstained material. It is particularly useful for observing motility and for differentiating cells in body fluids such as urine or cerebro-spinal fluid. A special microscope is required with an adapted condenser and objectives. The technique relies on small differences in refractive index between the living cells and the background fluid and hence the microscope must be adjusted correctly for these small differences to be apparent.

SETTING UP THE PHASE CONTRAST MICROSCOPE

1. Set up the microscope for normal illumination. On the condenser is a special “phase plate” with settings that correspond to the objective in use. For normal illumination, set the phase plate to 0.
2. Place a slide with a suitable fluid and coverslip on the stage.
3. Set the phase plate to 20 and the 20x objective in place. This locates the 20x annulus in the light path.
4. Remove one ocular and replace with the phase ocular telescope.
5. Check the slide is still in focus with the other ocular.
6. Focus the telescope such that the phase rings can be seen. Using the condenser plate centering knob adjust the condenser ring such that it corresponds to the objective ring. Clamp into position.
7. Remove the telescope and replace the ocular.
8. Repeat with the 40x objective and oil immersion lens.

USING PHASE CONTRAST

1. Prepare a slide of a fluid specimen by adding a drop of fluid to a slide and covering with a coverslip.
2. Place on the microscope and focus with the phase plate at 0. Note that the microscope has a green filter so the background should be green.
3. For most applications, 40x is sufficient. Swing this objective into place and change the phase plate to 40. Carefully adjust the focus.
4. Objects are seen as dark structures against a lighter green background.
5. After use, return the phase plate to 0 and the objectives to low power. Remove the slide and wipe any excess fluid off the lenses.
   
   

DARK FIELD MICROSCOPY

Dark field microscopy is used to observe organisms that are smaller than the resolving power of an ordinary light microscope. Spirochaetes are an example of organisms in this category. The principle of the technique is that a very intense light source is shone on the specimen and any organisms in the sample will scatter the light which is collected through a special system which excludes the primary light rays. Thus the organisms are seen as bright objects on a dark background (dark field). As the technique relies on light scattering, very clean slides and coverslips are necessary to avoid interference from dust particles. A microscope with a special light annulus and condenser internal mirror system is required. Special objectives are also required as is a powerful light source.

A form of dark field microscopy can be achieved using a phase contrast microscope. This is obtained by using the 20x objective with the 100x setting on the phase plate. As the laboratory does not possess a special dark field microscope, the phase microscope should be used for all dark field work.