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.

2. Place a slide on the stage of the microscope, move the condenser to its highest position, and focus at 10X magnification.

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

4. 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.

5. Open the field diaphragm until it nearly fills the field, but can still be 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.

6. 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.

7. Completing steps 1 4 establishes Koehler illumination, where the field diaphragm is superimposed onto the object and centers the major...