Exercise 1.9 - The Phase Contrast Microscope
- Phase contrast microscope
- Telescopic ocular for centering phase rings
- Culture of Amoeba proteus
- Transfer pipettes, slides, coverslips
- Prepared, prestained slide of Amoeba proteus
- 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
- 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
- Make a simple wet mount of the amoeba and observe under
bright field microscopy at 10X.
- 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.
- Observe the difference in the appearance of the amoeba
between normal bright field and phase contrast.
- Draw the amoeba viewed under phase contrast. Label
organelles which are more clearly visible with phase
contrast than with bright field microscopy.
- 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.
- 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
Drawing of amoeba observed with phase contrast optics
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Cell Biology Laboratory Manual
Dr. William H. Heidcamp, Biology Department, Gustavus Adolphus College,
St. Peter, MN 56082 -- firstname.lastname@example.org