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Lab exercises:
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Microscope Instructions
Part I - Preparing an Onion Root Tip Squash
Roots and stems grow from their tips. Actively dividing tissues such as these—referred to as
meristem—are good sources of mitotically dividing cells. You will prepare squashes of onion root tip
(Allium cepa) and observe cells in various stages of mitosis. First observe your TF’s demonstration,
then follow the procedure below.
Safety precautions:
• Wear gloves!
• *********Hydrochloric acid (HCl) is toxic – do not touch, spill, or drink.*********
• Toluidine blue will stain your skin and clothes.
ocular lenses
Procedure:
1. Work in groups of 2 or 3.
stage
2. Using a wax pencil, label one beaker “HCl” and another beaker “Carnoy’s.” Fill with just enough
18% HCl or carnoy solution to cover the tips. A splash in the bottom of the beaker should be
enough.
light source
3. As demonstrated by your TF, cut off (almost) the entire onion root (so that others will not cut off
what’s left and think it has a tip). Make note of which end is the tip. Each group should prepare 56 tips at once.
slide holder
6. Prepare one slide at a time until you get good results. Place a root tip on a slide and cut off all but
the last 1–2 mm of the tip, discarding the rest. Be careful to keep the correct end; the growing tip
should be slightly tapered and slightly whiter in color than the rest of the root.
7. Cover the tips with a few drops of toluidine blue to stain the cells. After two minutes, carefully
blot away the stain by touching a paper towel or Kimwipe to the slide. Try not to touch the root tip
itself.
8. Cover the root tip with one or two drops of water and gently lower a cover slip over the root tip.
Cover the slide with a paper towel and press firmly on the coverslip with your thumb to create a
single layer of cells. Do not let the coverslip twist or slide.
coarse focus
fine focus
stage controls
base
on/off switch
4. Using forceps, place the root tips into 18% HCl for four minutes. HCl softens the tissue in
preparation for staining and squashing.
5. Using forceps, move the tips into Carnoy’s solution for at least four minutes. Carnoy’s solution is
a fixative that preserves the structure of the cells. The root tips can remain in this solution
indefinitely.
arm
objective lenses
brightness
control
Fig. 1. Standard lab microscope
1. Setting up your microscope
a. Microscopes are expensive and delicate instruments. Failure to follow these
instructions may result in poor image quality and expensive damage.
b. If you need to move your microscope, pick it up with two hands, holding the arm and
the base. Check that your microscope is plugged in and turned on.
c. Identify each objective lens by the number printed next to colored line. Most of the lab
microscopes have 4X, 10X, 40X, and 100X objectives. When combined with the 10X
magnification from the ocular lens, the total magnification will be 40X, 100X, 400X, or
1000X.
d. Rotate a low power objective (4X or 10X) into position above the light source.
e. Using the slide holder, clip your slide onto the stage. Using the stage control, center
your sample over the light source.
f. Adjust the distance between the ocular lenses to fit your eyes. This is accomplished by
looking through the ocular lens, then pulling the eyepiece tubes further apart or pushing
them closer together until the two images are satisfactorily fused.
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2. Examine your slide under the microscope.
a. Starting with a low power objective (4X or 10X), focus your sample using the coarse
focus knob, then the fine focus knob. Using the stage controls, move an area of interest
into the center of the field of view.
b. DO NOT use the 100X objective yet; it is an oil immersion lens and requires special
treatment (see below).
c. Once your sample is focused under low power, switch to the 40X objective and focus
using the fine focus knob. NEVER USE THE COARSE FOCUS WITH A HIGH
POWER OBJECTIVE. With the coarse focus, it’s possible to push the slide straight
into a high power objective, potentially scratching the objective and necessitating an
expensive replacement. The objectives use approximately the same focal plan, so once
an image is focused under low power, only minor adjustments to the fine focus are
needed after switching to higher power.
d. If you do not see what you want under high power, switch to a low power objective,
then focus and move your slide as necessary before returning to high power.
e. Before removing a slide or placing a new slide on the stage, rotate a low power
objective (4X or 10X) into place. This prevents the slide from scratching a high power
objective as it is moved.
3.
Using the 100X oil immersion lens
a. When light moves from air to glass or from glass to air, it bends and results in loss of
resolution above 400X magnification. Magnification up to 1200X can be achieved if
the air between the lens and the slide is replaced with oil. Oil has the same refractive
index as glass, so light will not bend and become distorted. The upper limit of 1200X
magnification with light microscopy is dictated by the wavelength of light.
b. Once your sample is focused under the 40X objective, rotate the objectives so that the
sample is between the 40X and 100X objective. Add a small drop of immersion oil to
the slide and then carefully rotate the 100X oil immersion lens into place. The objective
will essentially touch the slide, separated only by a thin layer of immersion oil. Focus
using the fine focus knob.
c. Do NOT get oil on the other lens, it will obscure the image. Be especially careful that
the 40X lens does not come in contact with excess immersion oil on the slide.
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Results (Part I – Mitosis):
1. From your slides, diagram a cell in each stage of mitosis (interphase, prophase, metaphase,
anaphase, and telophase). Briefly describe what is happening during each stage.
Tips:
o The DNA should be dark blue and the cytoplasm light blue.
o To quickly pick out mitotic nuclei under low power, look for “spiders” - nuclei with
dark, condensed chromosomes that look like legs.
o Document what you see on your slide. Don’t try to recreate the tidy cartoons that often
appear in textbooks.
2. Count the number of chromosomes in two different cells. One cell should be in prophase. The
other cell can be in any phase of your choice (including prophase). For each cell, indicate whether
you see one or two chromatids per chromosome. Make additional notes, as appropriate.
Tips:
o The cells and chromosomes are not flat. Use the fine focus to move up and down
through the cell so you can better count the chromosomes.
o A cell is defined by the surrounding plasma membrane. During mitosis, a single cell
may have two physically separate groups of chromosomes. Be precise in your
language.
3. Choose a representative field of view and count the number of cells in each stage of mitosis
(including interphase). Count at least 100 cells and record the number of cells in each stage.
Part II - Observing Meiosis in Lily Bud Anthers
pistil
anthers
Fig. 2. Lily flower
4. Clean up
a. When you are done with the microscope, use lens paper to wipe the oil from the 100X
objective. DO NOT use a paper towel or kim wipe, these will scratch the lens. Do not
wipe oil onto any of the other lenses.
b. Turn off the power. If necessary, unplug and return to the storage cabinet.
In flowering plants such as Lilium, meiosis occurs in the reproductive organs of the flower. Pollen
(sperm) is produced in the anthers, and ovules (eggs) are produced in the pistil. Inside the anther,
thousands of diploid cells called meiocytes undergo meiosis to produce spherical tetrads, each
containing 4 haploid cells. These haploid cells will then mature into pollen grains. The lab staff has
already taken lily buds at different stages, removed the anthers and fixed them. Watch as you TF
demonstrates how to dissect the meiocytes from these anthers, then follow the procedure below.
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Procedure:
1. Work in groups of 2 or 3.
2. Place an anther on a clean slide and add a small drop of acetocarmine stain. Using a dissecting
needle, smash and chop the anther with the side of the needle in the pool of stain. The meiocytes
will leak out into the stain drop on the slide. Using dissecting needles, remove the empty anther
sac, leaving the meiocytes in the pool of stain. Do not let the cells dry out.
3. Carefully place a coverslip over the sample.
4. Using a paper towel or Kim Wipe, gently blot the excess stain from the sides of the coverslip. Lily
microspores are delicate and can be seen without squashing. Too much pressure will lyse the cells
and make the stages of meiosis difficult to discern.
5. Examine your slide under the microscope, using the instructions on p.28.
6. Each group will need to dissect several anthers from Meiosis I and Meiosis II in order to observe
all the stages. All the meiocytes within an anther will be at or around the same stage. See if your
technique improves with practice. You may also exchange slides with other lab groups.
Results (Part II – Meiosis):
4. From your slides, diagram a cell in each stage of meiosis (prophase I and II, metaphase I and II,
anaphase I and II, telophase I and II). Briefly describe what is happening during each stage.
Tips:
o All the daughter cells of a single meiocyte are held together in a sac.
o Document what you see on your slide. Don’t try to recreate the tidy cartoons that often
appear in textbooks.
5. Choose two non-sister cells and count the number of chromosomes in each cell. For each cell,
indicate the stage of meiosis, whether you see one or two chromatids per chromosome, and
whether you see synapsed homologs. Make additional notes, as appropriate.
Tips:
o A cell is defined by the surrounding plasma membrane. All the daughter cells of a
single meiocyte are held together in a sac. Be precise in your language.
o The cells and chromosomes are not flat. Use the fine focus to move up and down
through the cell so you can better count the chromosomes.