rp2 -> Preparation of stained squashes of cells from plant root tips

how to prepare squashes of cells from plant root tips

1. Cut a thin slice of root tip (5mm from end) using scalpel and mount onto a slide

2. Soak root tip in hydrochloric acid then rinse

3. Stain for DNA eg. toluidine blue

4. Lower coverslip using a mounted needle at 45 degrees without trapping air bubbles

5. Squash by firmly pressing down on glass slip but do not push sideways

Why not push cover slip sideways?

Avoid rolling cells together / breaking chromosomes

Why squash / press down on cover

slip?

● (Spreads out cells) to create a single layer of cells

● So light passes through to make chromosomes visible

Why soak roots in acid?

● Separate cells / cell walls

● To allow stain to diffuse into cells

● To allow cells to be more easily squashed

● To stop mitosis

Why is a stain used?

To distinguish chromosomes

Why are root tips used?

where mitosis occurs

Describe how to set-up and use an optical microscope

1. Clip slide onto stage and turn on light

2. Select lowest power objective lens (usually x 4)

3a. Use coarse focusing dial to move stage close to lens

3b. Turn coarse focusing dial to move stage away from lens until image comes into focus

4. Adjust fine focusing dial to get clear image

5. Swap to higher power objective lens, then refocus

Explain how the stages of mitosis can be identified

In interphase (not mitosis), chromosomes aren’t visible but nuclei are. In mitosis, chromosomes are visible.

mitotic index?

● Proportion of cells undergoing mitosis (with visible chromosomes)

● Mitotic index = number of cells undergoing mitosis / total number of cells in sample

how to determine a reliable MI from observed squashes

● Count cells in mitosis in field of view

● Count only whole cells / only cells on top and right edges → standardise counting

● Divide this by total number of cells in field of view

● Repeat with many / at least 5 fields of view selected randomly → representative sample

● Calculate a reliable mean

how to calculate the time cells are in a certain phase of mitosis

1. Identify proportion of cells in named phase at any one time

● Number of cells in that phase / total number of cells observed

2. Multiply by length of cell cycle