Investigating Mitosis

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Preparation of Stained Squashes of Cells from Root Tips

Only a few cells are able to continue dividing in a multicellular organism. In plants, the growing tips of roots and shoots contain meristem tissue that can divide by mitosis for growth.

1) Sample preparation

Wear gloves and use forceps to handle the tips.
Root tips must be sprouting (actively growing).
Place into 5 M hydrochloric acid.
After 5 minutes, rinse the tips in cold water in a watch glass.

2) Cut the root tips

Using a sharp scalpel, cut root tips that are 2 mm long.
Place a root tip onto a microscope slide. Ensure the slide is clean to reduce the chances of artefacts.

3) Staining

Carefully add 2-3 drops of stain and leave for two minutes.
Use a mounted needle to spread out the root tips into a thin layer.
Place a coverslip over the top of the tips.

4) Squashing

Squash down by applying force to the cover slip. This could be with the flat end of a pencil, or the slide could be covered with a paper towel and pressed.
Force must be vertical or the cover slip may break and cause injury.

5) Viewing the sample

Place the slide on the microscope stage using the lowest power lens.
Focus the lens on the sample using first the coarse control and then the fine control.
Move the slide to see the range of cells. The cells closer to the tip will be those more actively dividing.
On a lens power of 400x, it should be possible to clearly see the chromosomes in the dividing cells.

Mitotic Index

The mitotic index is used to work out the proportion of cells in the sample that are in mitosis.

Equation

The equation for mitotic index for a particular field of view is:
- Mitotic index = number of cells in mitosis ÷ total number of cells

E.g. Mitotic index

In a field of view, 29 cells are counted in total.
22 of the 29 cells have visible chromosomes and are actively dividing.
- 22 ÷ 29 = 0.7586
- About 76% of the cells in the sample are undergoing mitosis.

Cell Size

The size of a cell can be calculated by using a micrometer and graticule.

Calculating cell size

The eyepiece graticule is a glass disc in the eyepiece of a microscope.
Cells can be seen through a microscope and a micrometer can be used to calculate the actual size of the cells.

Calibrating the eyepiece

In order to calculate cell size, the eyepiece graticule must be calibrated.
The graticule is calibrated by comparing the length of each division in the micrometer to the length of each division in the graticule.

E.g. Graticule calibration

E.g. 100 divisions on the eyepiece graticule are equal to 25.9 divisions on the micrometer.
- In the micrometer, 100 divisions = 1mm.
- This means that 1 division = 10µm and 100 eyepiece divisions = 25.9 × 10µm = 259µm.
- This means that 1 eye piece division = 259 ÷s; 100µm = 2.59µm.
- The ratio of micrometer division to eyepiece division is 10µm to 2.59µm.

Calculating actual cell size

Once the eyepiece graticule has been calibrated, actual cell size can be calculated using the equation:
- Actual size = size of image ÷ magnification

E.g. Actual cell size

The length of a cell is measured to be 33mm in an image drawn at a magnification of 400x.
- Size of image = 33mm = 33000 µm.
- Magnification = 400x.
- Actual size = 33000 ÷ 400 = 82.5µm.