3. Make a temporary squash preparation of a root tip to show stages of mitosis in the meristem under the light microscope.

Question 1

What is mitosis?

Answer 1

The process of cell division resulting in two identical daughter cells.

Mitosis is used in growth, repair, asexual reproduction.

Prophase, Anaphase, Metaphase, Telophase.

Question 2

What organism is used when observing mitosis in root tips?

Answer 2

Garlic bulb roots.
The root tip of plants contain regions known as the MERISTEM, where cells are actively dividing. Each garlic cell only has eight chromosomes.

In order to see the chromosomes, cells need to be separated to give a one-cell-thick layer.

Question 3

How are individual plant cells held together?

Answer 3

Plant cells are glued together by a MIDDLE LAMELLA of PECTINS.

Question 4

How are the links between plant cells broken down?

Answer 4

Hydrochloric Acid, (HCl) breaks down pectins that hold cells together. Aceitic Orcein stains the chromosomes dark red and fixes cells, stopping mitosis.

Question 5

What is the hypothesis?

Answer 5

The greatest proportion of cells will be in interphase. Out of the cells undergoing mitosis, most will be in Prophase, as this is the longest stage. The fewest number of cells will be in anaphase as this is the shortest phase.

Question 6

Method:

Answer 6

1. Fill a small bottle with HCl and place it in a thermostatically controlled water bath set at 55 degrees C. Leave for 15 minutes to let the acid warm to the temp of the water bath.
2. Place a garlic clove in the top of the bottle so roots are submerged In HCl. Leave for 5 mins.
3. Take clove out and rinse roots thoroughly in tap water. Use a pair of sharp scissors to cut off several root tips at 5-10cm in length. Let them fall into a small vial of acetic orcein stain standing on a white tile. Use scissors to ensure root tips are immersed in stain. Place lid onto vial. (Lids should have a pin-prick hole or should be slightly loose if they are screw caps, to prevent the ejection of liquid when heating.)
4. Place the vial containing root tips in acetic orcein in the 55 degrees C water bath for 5 mins to intensify staining.
5. Use forceps to take tips out of vial and place them on a microscope slide. Add a drop of water to root tip o slide. Tease root tip apart with needles (MACERATION) to spread out cells. Cover with a coverslip. Replace vial lid.
6. Wrap slide in layers of paper towel and press gently on the paper to squash tissues.
7. Examine under microscope on low power to identify the areas of dividing cells. Position cells in the centre of the field of view.
8. Move to high power (X400). Identify the stage in the cell cycle of each of the cells in the field of view.
9. Count the number of all cells. Count the number of cells in each stage. Record results in table.
10. Draw and annotate one cell from each stage.

Question 7

Why is staining necessary?

Answer 7

Organelles are transparent usually, so would not be visible without staining.
In order to see the chromosomes.

Question 8

Risks:

Answer 8

Burns from acetic orcein:

• wear goggles, lab coats, disposable gloves.
• avoid contact with skin.
• if contact occurs, wash area thoroughly with water for 10 mins.

Broken glass:

• avoid touching
• inform a teacher

Damage to eyes if sunlight strikes mirror of microscope.
-be cautious when using microscope

Question 9

Mitotic index calculation:

Answer 9

Mitotic index= Number of cells in mitosis/ Total number of cells observed.

Question 10

Analysis of results:

Answer 10

• Calculate mitotic index
• Calculate an estimate for percentage of time spent by cells in each phase by dividing the number of cells in each phase by the total number of cells visible.

Question 11

Why is the plant heated with acid?

Answer 11

To break up the tissues into individual cells. The cellulose walls of plant cells are held together by pectins such as calcium pectate. Hydrochloric acid breaks this down.

Question 12

What is the effect of maceration and pressing the slide?

Answer 12

Separates cells in the meristem tissue into individual cells in a single layer-easier to see chromosomes and identify the stages of division.

Question 13

What information do cell counts show?

Answer 13

The relative duration of each stage inn the cell cycle.

The longer a phase, the more cells are likely to be going through that phase at any time.

Question 14

What does Acetic Orcein do?

Answer 14

• It stains chromosomes a dark red colour.

- Kills cells without damaging them or changing their structure, allowing us to study them.