Calculating Mitotic Index using Plant Cells (AQA A-Level Biology): Revision Notes
Calculating Mitotic Index using Plant Cells
Purpose and principle
This practical investigates cell division in plant tissues to calculate the mitotic index. Plant cells actively divide at specific growth regions called meristems, which are found at shoot and root tips. These meristematic cells are totipotent, meaning they retain the ability to differentiate into any cell type.
The mitotic index represents the proportion of cells actively undergoing mitosis within a tissue sample. It provides a measure of how rapidly cell division is occurring in that particular tissue.
Root tips are ideal for this investigation because they contain high concentrations of dividing cells, making mitotic figures easier to observe and count. The actively growing meristematic tissue at root tips provides the best opportunity to observe cells in various stages of mitosis.
To visualise the different stages of mitosis, cells must be treated with acid to soften the tissue and stained to make chromosomes clearly visible under an optical microscope.
Apparatus and materials
The following equipment is required for this practical:
- Optical microscope
- Microscope slides and cover slips
- Water bath
- Hydrochloric acid (1 mol dm⁻³)
- Toluidine blue O stain
- Distilled water
- Scalpel
- Forceps
- 100 ml beaker
- Fresh root tip (e.g. from onion or garlic)
Method
Preparation of the sample:
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Heat 1 mol dm⁻³ HCl to 60°C in a water bath. This temperature optimises the acid treatment without destroying cellular structures.
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Using a scalpel, carefully cut a small sample from the root tip (approximately 5mm from the very tip where cell division is most active).
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Transfer the root tip sample to the heated HCl and incubate for 5 minutes. This acid treatment softens the tissue by breaking down the cell walls and middle lamella.
Remove the sample from HCl using forceps and wash thoroughly in cold distilled water. Remove the very tip of the root using a scalpel, as this contains the root cap which has fewer dividing cells.
Slide preparation:
- Place the treated root tip on a microscope slide and add several drops of stain (such as toluidine blue O). The stain binds to DNA and makes chromosomes visible during mitosis, allowing identification of cells undergoing division.
Carefully lower a cover slip onto the slide, ensuring no air bubbles form as these distort the image. Avoid sliding the cover slip sideways as this could damage the chromosomes and cellular structures.
Microscopic observation:
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Place the slide under an optical microscope and begin with the objective lens on lowest magnification to locate the tissue.
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Use the coarse adjustment knob to bring the lens close to the slide surface.
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Use the fine adjustment knob to carefully focus until the image becomes clear. Higher magnification may be needed to distinguish individual cells and identify mitotic stages.
Data collection and processing
To determine the mitotic index, systematically count cells across multiple fields of view. Count both cells showing visible chromosomes (indicating active mitosis) and the total number of cells observed.
The mitotic index is calculated using the formula:
Worked Example: Calculating Mitotic Index
Step 1: Count the cells
- Cells with visible chromosomes (dividing): 45
- Total cells counted: 520
Step 2: Apply the formula
Step 3: Convert to percentage (if required)
Record results in a table showing the number of dividing cells, total cells counted, and calculated mitotic index. Count at least 500 cells across several fields of view to ensure statistical reliability.
Risk assessment
Always follow proper laboratory safety procedures when handling chemicals and sharp instruments. Wear appropriate protective equipment and work in a well-ventilated area.
| Hazard | Risk | Safety precaution | In emergency | Risk level |
|---|---|---|---|---|
| Hydrochloric acid | May cause harm/irritation to eyes or cuts | Wear eye protection; avoid skin contact, tie up long hair | Wash skin immediately; flood eyes/cuts with cold water | Low |
| Toluidine blue O stain | May cause harm/irritation to eyes or cuts | Wear eye protection; avoid skin contact | Wash skin immediately; flood eyes/cuts with cold water | Low |
| Scalpel | Cuts from sharp blade | Cut away from fingers; use forceps to hold sample; keep away from desk edge | Elevate cuts; apply pressure; seek medical assistance | Low |
| Broken glass | Cuts from sharp fragments | Handle slides and cover slips carefully; keep glassware away from desk edge | Elevate cuts; apply pressure; do not remove glass from wound; seek medical assistance | Low |
Key takeaways
Key Points to Remember:
- Mitotic index measures the ratio of dividing cells to total cells in a sample
- Root tips contain meristematic tissue where active cell division occurs
- HCl treatment at 60°C softens cell walls to allow chromosome observation
- Staining makes chromosomes visible so mitotic cells can be identified
- Count cells with visible chromosomes and divide by total cell count for accurate results
- Count at least 500 cells across multiple fields of view for statistical reliability