Exercise 1.9 - The Phase Contrast Microscope

LEVEL II

Materials

• Phase contrast microscope
• Telescopic ocular for centering phase rings
• Culture of Amoeba proteus
• Transfer pipettes, slides, coverslips
• Prepared, prestained slide of Amoeba proteus

Procedure

1. Establish Koehler illumination on the microscope. If the instructor approves, center the phase annular ring and its corresponding phase plate.
   • Place a slide on the stage of the microscope, move the condenser to its highest position and focus at 10X magnification.

   • Open the condenser diaphragm to its maximum setting, and close the field diaphragm completely

   • Using the condenser movement control, move the condenser until a sharp image of the field diaphragm is observed. To determine that the focus is indeed the field diaphragm, slightly open and close the field diaphragm to see if its movement can be detected in the field of view. When focused, there will be a slight blue haze on the edge of the diaphragm.

   • Open the field diaphragm until it nearly fills the field, but can be still seen. Center the field diaphragm in the field of view using the centering screws on the substage condenser. Open the field diaphragm to completely fill the field of view.

   • Remove one of the oculars from its tube and while peering down the tube, open the condenser diaphragm until it just fills the field of view at the bottom of the tube. Replace the ocular in the tube.

     Completing steps a-d establishes Koehler illumination, where the field diaphragm is superimposed onto the object and centers the major optical components of the microscope.

     To check on the phase annulus and its corresponding phase plate, remove an ocular and replace it with a telescopic ocular designed to focus on the rear lens of a phase objective. Match the phase objective with its corresponding setting on the phase condenser and visually verify that the phase annulus (a clear ring) is perfectly matched to the phase plate (a darker ring). If it is not, ask the instructor for assistance in centering the phase annulus. This is most often accomplished by adjusting a second set of centering screws attached to the phase condenser. Replace the normal ocular before using the phase contrast optics. Return the phase condenser setting to the normal bright field position.

2. Make a simple wet mount of the amoeba and observe under bright field microscopy at 10X.

3. Locate an active amoeba and center it in the field of view. Rotate the condenser phase ring to match the 10X phase with the 10X objective.

4. Observe the difference in the appearance of the amoeba between normal bright field and phase contrast.

5. Draw the amoeba viewed under phase contrast. Label organelles which are more clearly visible with phase contrast than with bright field microscopy.

6. Return the phase control on the condenser to the normal bright field setting, switch to a higher magnification (20X or 40X) and observe the amoeba at the higher magnifications with and without phase enhancement.

7. Compare the view of the amoeba under phase contrast, normal unstained bright field and darkfield (Exercise 1.8) with the view of the prestained commercial preparation of Amoeba proteus. List the organelles and/or structures which are more clearly demonstrated by each optical technique.

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Drawing of amoeba observed with phase contrast optics

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