Required Practical 2 Root Tip Squash
Laura Armstrong & Joe Wolfensohn
Teachers
Recall Questions
This topic requires prior knowledge of mitosis, cell division stages, and light microscopy techniques. You can test your knowledge on these below.
What happens during the S phase of the cell cycle?
DNA replication occurs, ensuring that each daughter cell receives an identical copy of genetic material.
In which phase do the chromsomes become condensed and visible.
Prophase
Why is the lowest magnification used first when using a light microscope?
To have the widest field of view and make it easier to focus
Topic Explainer Video
Root Tip Squash Practical
Aim of the Practical
To prepare and observe a root tip squash in order to identify stages of mitosis and calculate the mitotic index.
Methods
Prepare the root tip sample:
- Cut approximately 1 cm from the tip of a garlic root. Root tips are used as this contains meristem tissue where cells will be actively dividing by mitosis.
- Place in hydrochloric acid (1M HCl) at 60°C for 5 minutes to break down cell walls and allow tissues to spread more easily. (This will help to get a single layer of cells).
- Rinse the root tip with distilled water.
Staining the cells:
- Transfer the root tip to a watch glass and cover it with Toluidine blue stain (or orcein stain) to bind to DNA and make the chromosomes visible.
Squashing the tissue:
- Place the stained root tip onto a microscope slide.
- Cover with a coverslip and apply gentle pressure using the blunt end of a mounted needle or a paper towel to spread the cells into a single layer. (This will allow light to pass through).
Observing under the microscope:
- View the slide under low power first, then increase to high power (×400 magnification).
- Identify cells in different stages of mitosis (prophase, metaphase, anaphase, telophase).
Calculating the Mitotic Index:
- Mitotic Index = Number of cells in mitosis ÷ Total number of cells observed.
- If a cell is in mitosis the chromosomes will be visible, as they have condensed and thickened.
- The mitotic index should be given as a decimal, unless the question asks you for a percentage, in which case you can multiply your answer by 100.
Key Terms
- Mitotic Index: The proportion of cells undergoing mitosis in a given sample.
- Meristem: A region of rapidly dividing cells at the tips of roots and shoots in plants.
- Hydrochloric Acid (HCl): Used to break down plant cell walls, making them easier to squash into a single layer of cells.
- Toluidine Blue/Orcein Stain: Binds to DNA to highlight chromosomes and make them visible.
No answer provided.
Risk Assessment
| Hazzard | Risk | Precaution |
| Hydrochloric acid | Can cause burns or irritation. | Wear gloves and safety goggles. Rinse off skin immediately. |
| Glass slides | Can break and cause cuts. | Handle with care, dispose of broken glass properly into a sharps bin. |
| Stains | Can stain skin and clothing. Can cause irritation. | Wear a lab coat and gloves. Rinse off skin immediately. |
| Sharp blades | Risk of cuts when cutting root tips. | Cut on a suitable hard surface with the blade facing away from the body. |
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Exam Tips
When calculating the mitotic index, ensure you look at several fields of view under the microscope and select them at random. This will ensure a representative mitotic index.
Common Mistake: Confusing interphase with prophase. Look for visible chromosomes as this indicates the cell is in mitosis. During prophase chromosomes will be visible, during interphase, they will not!
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A student prepared a root tip squash slide and counted 20 cells undergoing mitosis in a sample of 200 cells. Calculate the mitotic index and explain what a high mitotic index suggests about the root tissue. (3 marks)
- Mitotic index = 20 ÷ 200 = 0.10 (1 mark)
- A high mitotic index indicates rapid cell division
- Which is expected in root tips as here there is meristematic tissue where cell division and growth occurs. (1 mark)
Explain why the root tips were placed in hydrochloric acid and why the root tip was firmly squashed during this practical? (3 marks)
- HCl will break down cell walls and allow cells to be separated.
- Firmly squashing the tissue will give a single layer of cells.
- This allows light to pass through so chromosomes are visible (and stages of mitosis can be identified)
Practice Questions
Try to answer the practice question from the TikTok on your own, then watch the video to see how well you did!