Lab #5: Mitosis in Onion Root Tip Cells

Biology 160

Lab #5: Mitosis in Onion Root Tip Cells

Objectives:

• Prepare your own specimens of onion root in which you can visualize all of the stages of mitosis.
• Name, identify, and describe the stages of mitosis in plant and animal cells.
• Observe the stages of mitosis in prepared slides of whitefish blastula.
• Compare the process of cytokinesis in animal and plant cells.

General Procedures

• You may work individually or in groups of two, but all students will complete their own lab drawings and questions & turn them in individually.

General Introduction:

Cell division in somatic cells can be broken down into two processes, mitosis and cytokinesis. Mitosis is the process that leads to the equitable distribution of genetic material into the two nuclei of the two daughter cells. Cytokinesis ("cell movement") is the process that leads to the equitable distribution of cytoplasm to the two daughter cells. In short, mitosis is the division of the nucleus, and cytokinesis is the division of the cytoplasm.

In this lab you will study these two processes in onion root tips and whitefish blastulae. The tips of the roots of onion plants are continuously growing to seek out nutrients and water. A blastula is a developmental stage in many animals. It occurs shortly after fertilization and the formation of the zygote. It is the “ball of cells” stage of development.

A quick overview of cell division

The genetic information of plants, animals and other eukaryotic organisms resides in several (or

many) individual DNA molecules, or chromosomes. For example, each human cell possesses 46

chromosomes, while each cell of an onion possesses 8 chromosomes. All cells must replicate their DNAwhen dividing. During DNA replication, the two strands of the DNA double helix separate, and foreach original strand a new complementary strand is produced, yielding two identical DNA molecules.DNA replication yields an identical pair of DNA molecules (called sister chromatids) attached at aregion called the centromere.

DNA replication in eukaryotes is followed by the process called mitosis which assures that each

daughter cell receives one copy of each of the replicated chromosomes. During the process of mitosis,the chromosomes pass through several stages known as prophase, metaphase, anaphase andtelophase. The actual division of the cytoplasm is called cytokinesis and occurs during telophase. During each of the preceding stages, particular events occur that contribute to the orderly distribution ofthe replicated chromosomes prior to cytokinesis.

The stages of mitosis

Prophase. During prophase, the chromosomes supercoil and the fibers of the spindle apparatus begin toform between centrosomes located at the pole of the cells. The nuclear membrane also disintegrates atthis time, freeing the chromosomes into the surrounding cytoplasm.

Prometaphase. During prometaphase, some of the fibers attach to the centromere of each pair of sisterchromatids and they begin to move toward the center of the cell. It is difficult to determine the difference between prophase and prometaphase, so for this lab we will combine the two phases as prophase.

Metaphase. At metaphase the chromosomes have come to rest along the center plane of the cell, called the metaphase plate.

Anaphase. During anaphase, the centromeres split and the sister chromatids begin to migrate towardthe opposite poles of the cell.

Telophase. During telophase, the chromosomes at either end of the cell begin to cluster

together, which facilitates the formation of a new nuclear membrane. This also is when cytokinesisoccurs, leading to two separate cells. One way to identify that telophase has begun in plantsis by looking for theformation of the cell plate, the new cell wall forming between the two cells.

Part 1: Mitosis in onion root tips

Why use onion roots for viewing mitosis?
• The roots are easy to grow in large numbers.
• The cells at the tip of the roots are actively dividing, and thus many cells will be in stages of mitosis.
• The tips can be prepared in a way that allows them to be flattened on microscopes slide (“squashed”) so that the chromosomes of individual cells can be observed.
• The chromosomes can be stained to make them more easily observable. /
Regions of Onion Root tips
There are three cellular regions near the tip of an onion root.
1. The root cap contains cells that cover and protect the underlying growth region as the root pushed through the soil.
2. The region of cell division (or meristem) is where cells are actively dividing but not increasing significantly in size.
3. In the region of cell elongation, cell are increasing in size, but not dividing. / / Region of cell elongation
Region of cell division
Root cap

Viewing Chromosomes

Chromosomes generally are not visible as distinct entities in nondividingcells, since the DNA is uncoiled, but the process of mitosis is facilitated by supercoiling of thechromosomes into a highly compacted form. Supercoiled chromosomes can be visualized in cells,particularly if they are treated with a DNA-specific stain, such as the Feulgen stain.

MATERIALS (per group)

safety goggles / ruler / pipette (1)
gloves / scissors / discard flask (small glass)
micro-tube (1) & styrofoam holder / forceps / water in dropper bottles
onion root tip (1-2) / dissecting probe / Fuelgen stain(1/bench)

MATERIALS (classroom station)

HCL & pipettes / slides
large discard container / cover slips
Heat block

PROCEDUREs

You will work ingroups of two for this lab exercise. Each group of two will be assigned either an onion that was growing in water or one growing in rooting hormone. The onions we will be using have been previously treated with a fixative to stabilize the cells.

Important: We will be using HCL, so you will need to wear goggles and gloves while preparing the slides.

“Softening’’ the roots (so that they later can be spread on amicroscope slide).

1. Mark the micro-tube with your initials.

2.Using scissors, cut off one entire root from the onion (either A or B as assigned). Be sure to know which end is the root tip and which end is the cut end.

3. Next cut the root so you have a piece approximately 1cm long that includes the root tip. Using forceps, transfer the root tipinto a plasticmicro-tube.

4. Fill the tube about 2/3 full with 1M HCl, using a pipette.

*** Caution: Work with the HCl carefully, it is a strong acid. ***

5. Place the tube in a heat block set to 60o C, and allow the roots to incubate for 12 minutes.

6. After the 12 minute incubation period, remove the tube from the heat block.

Removing HCL & rinsing roots

Important: During these steps,Do Not put HCL, Feulgen stain or the water rinses down the sink! Use the discard flask.

7. Using a plastic ‘squeeze’ pipet, carefully remove the HCl from the micro-tube and transfer it to the discard flask.

8. Add water from the dropper bottle to the micro-tube to rinse the root tip, and thenuse the pipette to remove the rinsing water and transfer it to the discard flask. Repeat 2 more times.

9. After removing the water from the third rinse, you are now ready to stain the root tip.

Staining the chromosomes

10. Add enough Feulgen stain from the dropper bottle tocover the root in the micro-tube.

*** Caution: the Feulgen stain will strongly stain skin and clothing. ***

11. Return the micro-tube to the heat block and incubate the roots in the stain for 12 minutes. During this time the very tipof the root will begin to turn red as the DNA stains the numerous smallactively dividing cells at the tip.

Removing the Feulgen stain rinsing roots

12. Using a plastic ‘squeeze’ pipet, carefully remove the Feulgen stain from the micro-tube and transfer it to the discard flask.

13. Add water from the dropper bottle to the micro-tube to rinse the root tip, then use the pipette to remove the rinsing water and transfer it to the discard flask. Repeat 2 more times.

14. After removing the water from the third rinse, you are now ready to prepare the slide.

Preparing the slide
15. Transfer the root to the center of a clean microscope slide and add a drop of water.
16. Using a razor blade cut off most of the unstained part of the root, and discard it. Note – if entire sample is stained, do not cut off anything.
17. Cover the root tip with a cover slip, and then carefully push down on the cover slide with the wooden end of a dissecting probe. Push hard, but do not twist or push the cover slide sideways – the goal is to flatten the root, not twist it. The root tip should spread out to a diameter about 0.5 – 1 cm. /
Interphase Early-Prophase Mid-prophase
Observations of onion root tip slide.
Scan the microscope under the 10x objective. Look for the region that has large nuclei relative to the size of the cell; among these cells will be found cells displaying stages of mitosis. Examples are shown in the figure to the right.
Switch to the 40X objective to make closer observations. Since prophase and prometaphase are difficult to distinguish, classify all these cells as prophase. /
Find representatives of each stage (prophase, metaphase, anaphase, telophase) and make a drawing of each on the data sheet. / Metaphase Anaphase Telophase
& Cytokinesis

Part 2: Mitosis in animal cells

Materials:

Prepared slide:Cross sections of whitefish blastula

Procedures

1. Obtain a prepared slide of cross sections of whitefish blastula and observe using the 10x objective. Look for cells undergoing each of the stages.

2. Switch to the 40X objective to make closerobservations. Find representatives of each stage (prophase, metaphase, anaphase, telophase) and make a drawing of each on the data sheet.

Cleanup

• Pour group discard flasks into large discard flask. Then rinse the group discard flasks in the sink and return to the cart.
• Discard micro-tube and pipettes in garbage.
• Put cover slips in broken glass jar.
• Rinse slide, wipe dry with a tissue and return to the slide box. Please take care to make sure the slides are placed in the correct box and properly aligned!
• Return all other equipment to the carts in the front of the room.
• Return whitefish slides neatly to the correct box.
• Put microscope away correctly.

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