3 - Calculating mitotic index

Where in plants can cells undergoing mitosis be found?

Meristem tissue at the shoot & root tips

What is the mitotic index?

The ratio of cells undergoing mitosis to the total number of cells in a sample:

• only prophase, metaphase & anaphase → must have visible chromosomes

What is the equipment required for this practical?

• Light microscope

• Microscope slides & coverslips

• Water bath (50°C)

• White tile & paper towels

• Stop clock & scalpel

• HCl & acetic orcein stain

• Distilled water & dissecting needles

• Scissors

• Root tip (garlic)

• Small bottle

What is the method for this practical?

1. Heat 1.00 moldm^-3 of HCl at 55°C in a water bath for 15 minutes

2. Place a garlic clove in a bottle of HCl & leave for 5 minutes

3. Cut a small sample of root tip (5-10mm) using a scalpel

4. Transfer the root tip to acetic orcein stain & heat in a water bath for 5 minutes

5. Place the tip on a microscope slide & macerate with a needle

6. Lower the coverslip down carefully onto the slide (ensure there are no air bubbles)

7. Wrap in a paper towel & gently squash the slide

8. Place the slide under a microscope & set the objective lens on the lowest magnification, then use the coarse adjustment knob to move the lens down to just above slide

9. Use the fine adjustment knob to re-adjust the focus until image is clear

10. Count the cells in various stages of mitosis & divide by total number of cells

Why is the root tip treated with HCl?

HCl dissolves & breaks down the middle lamellae of cell walls

Why is acetic orcein added to the root tip?

Acetic orcein binds to the chromosomes to make them more visible

Why is the coverslip squashed?

To form a single, thin layer of cells

Why is the root tip macerated with a needle?

To spread the cells out & make the chromosomes visible, therefore showing which cells are undergoing mitosis

What is the risk, safety precaution & in emergency for the hazard: HCl

Risk:

• may cause harm/irritation to eyes or in cuts

Safety precaution:

• wear eye protection

• avoid contact with skin

In emergency:

• wash off skin immediately

• flood eye/cuts with cold water

What is the risk, safety precaution & in emergency for the hazard: acetic orcein

Risk:

• may cause harm/irritation to eyes or in cuts

Safety precaution:

• wear eye protection

• avoid contact with skin

In emergency:

• wash off skin immediately

• flood eye/cuts with cold water

What is the risk, safety precaution & in emergency for the hazard: scalpel

Risk:

• cuts from sharp object

Safety precaution:

• cut away from fingers

• use forceps to hold sample whilst cutting

In emergency:

• elevate cuts & apply pressure

• don’t remove glass from wound

• seek medical assistance

What is the risk, safety precaution & in emergency for the hazard: broken glass

Risk:

• cuts from sharp objects

Safety precaution:

• take care when handling slides & coverslips

• keep glassware away from edge of desk

In emergency:

• elevate cuts & apply pressure

• don’t remove glass from wound

• seek medical assistance

What is the equation to calculate the mitotic index?

Mitotic index = number of cells with visible chromosomes / total number of cells in sample

Why is onion/garlic usually used in this practical?

They have a small genome, so have a small number of chromosomes which means they are more easily seen