Exploring Mitosis Lab
Today’s lab will give you a chance to get an up-close view of mitosis, the process by which eukaryotic cells
divide to generate identical daughter cells. We’re using garlic (A. sativium) root tip cells because garlic bulbs
grow rapidly, and the root tips of growing plants contain many actively dividing cells. A few days prior to the
experiment you’ll carry out today, one of the two garlic bulbs you “planted” last week was treated with a
chemical that has been observed to alter the rate of mitosis in some cell types. Your goal is to determine
whether this chemical speeds up, slows down, or has no effect on mitosis of garlic root tip cells.
IMPORTANT: In addition to reading this handout, you should review your notes from our discussion of mitosis
in class.
LEARNING OBJECTIVES
• Improve your understanding of mitosis.
• Learn basic cell staining and slide preparation techniques.
• Practice developing hypotheses and predictions.
• Be able to apply data analysis skills developed in prior labs and to draw conclusions based on the results of
your analyses.
INTRODUCTION
Cell Division
Cell division is a carefully controlled process – both when it occurs and the set of steps that ensure each
daughter cell gets one complete copy of the genetic information. In eukaryotic cells (e.g. plant, animal, fungi),
the cell cycle is the ordered series of events from when a cell is produced by division to when it divides. As
shown in the figure below, the cell cycle is divided into two main parts, interphase, and division. During
interphase, cells grow, produce additional molecules and organelles, and replicate their DNA. Cells spend
most of their time in interphase. Actual division is a small part of a cell’s life. The amount of time it takes for a
cell to progress through the cell cycle varies HUGELY between cells. Some cells are produced and then never
divide, while other cells divide rapidly. For example, most cells that transmit nervous system signals (neurons)
never divide. Human skin cells, however, divide about once every 20-24 hours.
Mitosis specifically refers to the process of cell division by which eukaryotic cells divide to produce
genetically identical daughter cells. It involves a series of steps that must occur in a specific order to ensure
that each daughter cell ends up with one, and only one, copy of every chromosome. The very first step in
mitosis involves condensing the snarled mass of replicated DNA in a cell’s nucleus into discrete, organized
“packages.” At this point, each chromosome consists of a pair of sister chromatids – identical DNA molecules
generated by replication. Looking at the images in the figure below, you can see that the chromosomes
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remain condensed until after division is complete. Therefore, condensed chromosomes are a key indicator
that a cell is in the process of mitosis.
Visualizing and Analyzing Cell Division
To visualize the cells in your garlic bulb’s roots, you’ll cut off the very tip of the root and treat the cells with a
fixative. The purpose of the fixative is to “fix,” or stop, the cells in whatever phase of the cell cycle they are in
(interphase or mitosis). As noted above, condensed chromosomes are a sign that a cell is in the process of
dividing, and this is what you’ll use to identify mitotic phase cells. To determine whether or not a cell’s
chromosomes are condensed, you need to be able to see the chromosomes! After you fix your cells, you’ll use
a dye, Carbol-Fuchsin, to stain DNA (it will turn a dark red). Once the cells are fixed and stained, your goal is
to prepare a microscope slide that has a single layer of cells. This will allow you to see individual cells and
count the number of cells that were undergoing mitosis when you harvested your root tip and fixed the cells.
As described in the overview of this lab, one of the two garlic bulbs you planted was treated with a
chemical that has been observed to alter the rate of mitosis in some types of cells. The other garlic bulb is
your control. To determine if, and if so how, this chemical affects the rate of garlic root tip cell mitosis, you
need to determine the mitotic index for your control and treated root tips. The mitotic index is the ratio of the
number of cells in mitosis to the total number of cells, and is calculated using the formula shown below. It’s
basically just the percentage of mitotic cells (cells undergoing mitosis) in a sample. You’ll apply your
knowledge of means, standard deviations, and t-tests to assess whether the chemical has a significant effect
on the rate of mitosis in garlic bulb root tips, and if so, to draw a conclusion regarding whether the chemical
increases or decreases the rate of mitosis. Note that the chemical does NOT inhibit mitosis. If the chemical
inhibited mitosis, your garlic bulb roots would not grow.
mitotic index =
PROCEDURE: Harvest Roots and Fix Cells
IMPORTANT: You MUST wear gloves and lab safety glasses (both are provided in lab) while preparing
your slides. You may remove the safety glasses and gloves when using the microscope.
1) Find your two garlic bulbs, and bring them to your work space.
2) Remove your control garlic bulb from its cup and place it on the petri dish labeled “control” (if your dishes
aren’t labeled, label them!). Carefully pour the liquid in the cup into the liquids waste container, but keep
the cup for later use. Put the toothpick in the Laboratory Waste box.
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3) Repeat step #2 for your chemically-treated bulb, putting the bulb in the petri dish labeled “treated.”
Dispose of the liquid, and put the cup and toothpick in the Laboratory Waste box.
4) Starting with your control bulb, use dissecting scissors to cut approximately one centimeter off the ends of
5-6 of the sprouted roots. Put the root ends in a 1.5 mL microcentrifuge tube labeled “control.” Repeat for
your chemically-treated bulb, putting the root ends in the appropriately labeled microcentrifuge tube.
5) Add 0.5 mL of Fixative (acetic acid/hydrochloric acid) to each tube. WARNING: This solution is caustic and
can burn your skin! Make sure that you are wearing gloves and lab safety glasses when handling.
6) Place the tubes in a floating rack, and put the rack in the hot water bath (50°C) for 6 minutes. After the
incubation period, transfer your tubes to a rack on your bench.
PROCEDURE: Prepare Slides
Your goals now are to stain the root tip cells so you can see the DNA, and spread the mass of tissue out
into a thin layer, by squashing the tissue with a coverslip, so that you can view individual cells. You want to
squash the cells into a thin layer, NOT smear them by moving the cover slip side to side! It takes some
patience, practice, and luck to create a good slide. You may (and likely will) need to repeat this process
several times before you get a good slide.
IMPORTANT:
- You need to make ONE good slide using a tip from control bulb and ONE good slide using a tip from
your chemically-treated bulb.
- Be careful with the used labwipes since they will have both strong acids, and stains on them.
- Clean your slides in the sink with soap and water, NOT ethanol
1) Clean a slide with 70% ethanol by wetting it, and rubbing it dry with a labwipe. Label the slide on the very
edge “C” for control. Obtain a small beaker, and fill it about half way with tap water.
2) Remove a control tip from the tube using a pair of tweezers, (are you wearing safety glasses, and gloves?)
and place it on the slide. Put the tweezers tip down in the beaker of water to rinse off the acids.
3) Using a razor blade, cut away and save the first 1 to 2 millimeters of the root tip. You can use a dissecting
scope to help you cut off the tip. You should be able to tell which end was your cut, and which end is the
growing tip of the shoot since the growing end is rounded. Discard the remainder of the root end in the cup
you kept earlier. IMPORTANT: Be careful using the razor blades and put them back in the container when
you are done!
4) Place one drop of Carbol-Fuchsin stain on the root tip, and allow it to soak for 2 minutes. During this time,
use the dissecting probes to soften up the root tip tissue by lightly poking and working it apart. Be gentle!
5) Being careful not to disturb the tissue, blot away the excess stain using a labwipe.
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6) Carefully place a cover slip over the tissue. Place your thumb squarely on the coverslip, and push down
without sliding side to side. Squash with a medium downward pressure. Holding the edge of the cover slip
with a finger helps keep it steady. Another method is to use the eraser end of a pencil to press gently on
the coverslip. If liquid has leaked out of the coverslip, blot with a labwipe.
7) Observe your squash using the compound microscope at low power (40X – 100X). Do NOT touch the
course focus knob again after leaving 40X. Search carefully for regions of the slide where you can clearly
see a single layer of individual cells. This is important because it is much more difficult to understand what
you see when you are looking through several layers of cells – it’s all a big blur!
8) When you identify a region of the squash where individual cells are clear, move up to 400X and scan your
slide for cells in stages of mitosis. A good slide will have lots of cells visible in various stages of mitosis,
and lots of cells squashed into single cell layers. If you can identify 5 fields of view (the area you see inside
the circular view of the microscope at 400x) that are clear enough to determine if cells are in interphase
versus mitosis (uncondensed vs. condensed chromosomes), it is a good slide for analysis.
9) If you can’t find regions of the slide with individual cells, your squash was unsuccessful. Try re-squashing
by pressing on the coverslip again. If that fails, start back at step #1 and make a new slide using one of
your other root ends! Now that you know how to make a slide, you can make 2-3 at the same time.
10) Once you have one good slide for your control garlic bulb, repeat the process to prepare one good slide for
your chemically-treated bulb. Label this slide “T” for treated.
PROCEDURE: DATA COLLECTION
As described in the introduction, the mitotic index is the ratio of the number of cells in mitosis to the total
number of cells. For the purposes of your experiment, you will make counts of cells within several fields of
view of your microscope at 400X. Again, the field of view (FOV) is everything that you can see inside the
circular view of the microscope. For every FOV you select for observation, you will count the total number of
mitotic cells and the total number of interphase cells (the sum of the two is the total number of cells). When
you analyze your data, you will compare the mitotic index of untreated garlic root tips to chemically-treated
garlic root tips to determine if, and if so how, the chemical alters the rate of mitosis in garlic root tip cells.
1) In the space provided under “Data Collection” (in the lab questions handed out in lab), make TWO tables to
collect your data – one for your control garlic bulb and one for your chemically-treated garlic bulb. You
need rows for five FOV (see below) and columns to record the number of cells in mitosis, the number of
interphase cells, the total number of cells, and the mitotic index. Make sure you label your tables as control
or chemically-treated samples!
2) Starting with your control garlic bulb slide, identify a good FOV where you can clearly determine whether
cells are in interphase vs. mitosis.
3) Count all the cells that you can identify as being in mitosis, and record the number in your table (this maybe
a very small number!). Count all the interphase cells, and record that data in the table.
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4) Repeat the step above for four additional FOV (five total). Make sure that your FOV do NOT overlap so
you don’t double count cells.
5) Use the same process to count five FOV for your chemically-treated garlic blub slide, recording the data in
the appropriate table.
6) After you have completed your counts, be sure to clean all your slides well in the sink, disposing of the
cover slips in the Lab Waste bin.
PROCEDURE: DATA ANALYSIS
To determine whether the chemical altered the rate of mitosis in garlic root tip cells, you need to determine
the average mitotic index and standard deviation (SD) for each of your conditions, and use a t-test to compare
your control and chemically-treated samples to obtain a p-value. You can then use your p-value and the
predictions you developed at the beginning of lab to draw conclusions from your data. Use the table provided
under “Data Analysis” to record the results of your data analysis.
1) Open Excel. Using the knowledge you’ve developed in previous labs, copy the data (number of mitotic
cells and number of interphase) from the tables you created for your control and chemically-treated garlic
bulb root tips into the spreadsheet.
2) Use Excel to calculate the total number of cells and mitotic index for each FOV for each condition. Record
the values in the table in your lab handout.
3) Use the Excel formulas below to calculate the average mitotic index and standard deviation for each
condition. Record the values in the table provided under “Data Analysis.” Note that cell range in the
formulas refers to the range of cells that contain your data (e.g. B3:B7).
“=AVERAGE(cell range)”
“=STDEV(cell range)”
4) Use the Excel formula “=T.TEST(cell range,2,3)” to compare your two conditions and obtain a p-value.
IMPORTANT: For your cell range, you need to select ALL the cells that contain mitotic index values for
BOTH conditions. If you’re not sure how to do this properly, please ask your lab TA! Record the p-value in
your lab handout.
5) Answer the remaining questions in your handout based on the results of your data analysis. When you are
certain that your data tables are complete and you have finished all of the questions, TURN IN your lab
questions. Each person must turn in their OWN lab questions.
REFERENCES
http://www.nuffieldfoundation.org/practical-biology/investigating-mitosis-allium-root-tip-squash
Bonner, JM. (2010) A Scenario-Based Study of Root Tip Mitosis. 31: 36-49, in Tested Studies for Laboratory
Teaching (K.L. Clase, Editor).
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