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3. Examine eggs using your
microscope. Put 2-3 drops in well of depression slide and
add coverslip. Examine under lowest power first. ALWAYS
FOCUS BY MOVING OBJECTIVE TO LOWEST SAFE POSITION WHILE
WATCHING FROM THE SIDE TO MAKE SURE THAT YOU DON'T HIT THE
SLIDE. Then look through the eyepieces while moving the
objective AWAY from the slide. Follow the microscope
instructions to adjust the eyepieces, field diaphragm and
condenser. Rotate the nosepiece to change to higher
magnification. Take a sample of the fertilized eggs and
examine under 10X magnification. Compare them to the
unfertilized eggs, and identify those with a fertilization
envelope. Try looking at them under dark field on the
microscope set up on the instructor's bench. Rinse
depression slides with dH2O and gently wipe dry.
4.
Measure rate of fertilization. Make sure that you have a
clean depression slide and coverslip ready. Transfer a
sample of eggs to a fresh beaker, and dilute a sample of
sperm. Add sperm to eggs, stir, NOTE TIME and transfer 2-3
drops to your depression slide. Examine preparation for
presence of fertilization envelopes. When do you see the
first one? How long does it take for most to be raised? What
is the approximate percentage of the eggs that are
fertilized in your batch? You can also combine the sperm and
eggs directly on the microscope slide so that you can begin
observing immediately.
5.
Examine sperm using oil-immersion. Place a drop of diluted
sperm on a regular (flat) microscope slide and coverslip.
Focus using lower power objectives, then rotate nosepiece to
position between 100X and next highest power objective. Add
one small drop of immersion oil to the center of the field
of view, then carefully rotate 100X objective into position
and fine adjust focus. BE CAREFUL, do not get oil on the
other objectives. When you are done, CAREFULLY blot all the
oil from the objective using lens paper. Use several regions
of the sheet, do not wipe back and forth as this may scratch
the lens.
At
the end of class, all depression slides should be cleaned
and returned to the benches.
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