Required Practical 2: Root Tip Squash
How to prepare a stained root tip squash, identify stages of mitosis under a microscope, calculate the mitotic index and work out actual cell size. Includes the 5-mark exam question, a worked model answer and the examiner trap on dividing by the total number of cells.
Chapters (10)
Exam question [5 marks]
Describe how you would prepare a root tip squash and use it to find the mitotic index.
Try it on paper first, then check the mark scheme below. The question is original and was written for this lesson.
Mark scheme
| Mark | Mark point | Also accept | Do not accept |
|---|---|---|---|
| 1 | Cut the tip of the root, because the meristem there has cells dividing by mitosis | "growing region", "cells dividing", "meristem" | "the tip is softer"; "cut the root" with no tip or no reason |
| 2 | Warm in (hydrochloric) acid to separate / soften the cells | "hydrolyse the material between cells", "break down cell walls / middle lamella" | "kills the cells" with no separation; "to stain" |
| 3 | Add a stain so the chromosomes are visible | "stain binds to chromosomes / DNA", "makes chromosomes dark" | "stain the cells" with no mention of chromosomes or visibility |
| 4 | Squash under a coverslip to make a thin layer, one cell thick, so light can pass through | "single layer", "no overlapping cells", "press with thumb" | "flatten it" with no reason; "slide the coverslip" |
| 5 | Mitotic index = number of cells in mitosis / total number of cells observed | "cells in mitosis divided by all cells counted", percentage form | "cells in mitosis / cells in interphase"; "cells in mitosis / number of fields" |
The five mark points are the method steps in order, each with its reason. Marks 1 and 4 need the reason as well as the action. The wrong denominator in mark 5 is the common error. The actual-size calculation (actual size = size of image / magnification) is taught in the video as part of the practical but is not marked here; it is tested separately as a calculation.
Model answer (5/5)
Cut a few millimetres from the tip of a growing root, because the meristem there has cells that are dividing by mitosis. Warm the tip in hydrochloric acid to separate the cells, then rinse it. Add a stain that binds to the chromosomes so they show up. Lower a coverslip and squash it firmly with filter paper, to make a layer one cell thick so that light can pass through. Under the microscope count the cells in mitosis and the total number of cells in several fields. The mitotic index is the number of cells in mitosis divided by the total number of cells observed.
Examiner trap
- Calculating mitotic index with the wrong denominator (it is cells in mitosis / total cells)
- Not squashing to a single cell layer
- Not saying why root tip is used (meristem dividing)
Key terms
- root tip squash
- stain
- mitotic index
- optical microscope
- actual size
- magnification
- acid hydrolysis
Lesson notes
A written version of the video, chapter by chapter, for revision. Times link to the video.
0:22Choosing the root tip
Step one: choose the right tissue. We cut a few millimetres from the very tip of a growing root. Why the tip? Because that is where the meristem is. The cells there are dividing by mitosis, so we have a good chance of catching cells in every stage. If you cut from higher up the root, the cells have stopped dividing, and you would find almost none in mitosis.
0:46Softening with acid
Step two: soften the tissue. Warm the root tip in hydrochloric acid for a few minutes. The acid breaks down the material holding the cells together, so they separate and are easier to spread out. Then rinse the tip in water, so the acid does not interfere with the stain.
1:04Staining the chromosomes
Step three: stain. Cells are almost transparent, so we add a stain that binds to the chromosomes. Chromosomes then show up dark against the pale cytoplasm, which lets us tell the stages of mitosis apart. Leave the stain for a few minutes to soak in.
1:21Squashing the tissue
Step four: the squash. Lower a coverslip carefully, to avoid trapping air bubbles. Then press down firmly with your thumb, through filter paper, without sliding the coverslip sideways. The aim is a thin layer of cells, ideally one cell thick, so light passes through and nothing overlaps. Thick clumps are the classic reason a squash fails.
1:45Using the microscope
Step five: look. Start on low power to find the cells, then switch to high power and focus carefully. Now identify the stage of each cell from how its chromosomes look. Condensed and scattered is prophase. Lined up in the middle is metaphase. Pulled apart is anaphase. Two new nuclei forming is telophase. Count the cells in each stage, and count the total in the field. Then move to other fields and repeat, to get a bigger sample.
2:18Calculating mitotic index
Step six: the mitotic index. The mitotic index is the number of cells in mitosis, divided by the total number of cells you counted. Say twelve cells are in mitosis, out of sixty cells in total. Twelve divided by sixty is nought point two, or twenty percent. It tells you how much of the tissue is dividing. A high index means a lot of cell division, as in a growing root tip or a tumour.
2:47Actual size of a cell
The practical also asks you to work out how big a cell really is. Actual size equals the size of the image, divided by the magnification. Measure the cell in the image in millimetres, say thirty, with a magnification of four hundred. Thirty divided by four hundred is nought point nought seven five millimetres. Multiply by a thousand to convert to micrometres. That is seventy five micrometres.
3:49Examiner trap and recap
The trap. The mitotic index divides by the total number of cells, not just the cells that are not dividing. Recap. Root tip, acid, stain, squash, count. Next time, we move from the inside of the cell to its boundary, the fluid mosaic model of the membrane.
Watch first
This lesson builds on:
- Magnification vs Resolution Calculations · AQA 7402 · 3.2.1.3
- Cell Fractionation and Ultracentrifugation · AQA 7402 · 3.2.1.3
- Mitosis and the Cell Cycle: Stages · AQA 7402 · 3.2.2
Keep going
More from 3.2 Cells
- Mitosis and the Cell Cycle: Stages · AQA 7402 · 3.2.2
- Cancer: Uncontrolled Cell Division · AQA 7402 · 3.2.2
- Binary Fission and Virus Replication · AQA 7402 · 3.2.2
- Nucleus, Mitochondria and Chloroplasts · AQA 7402 · 3.2.1.1
Independent revision resource, not affiliated with or endorsed by AQA. The exam question is original and was written for this lesson; it is not an AQA question. Spotted an error? Email info@medservatory.com with the lesson code AB-033.