Required practical 2 Flashcards
Preparation of stained squashes of cells from plant root tips; set-up and use of an optical microscope to identify the stages of mitosis in these stained squashes and calculation of a mitotic index.
Describe how to prepare squashes of cells from plant root tips
- Cut a thin slice of root tip (5mm from end) using scalpel and mount onto a slide
- Soak root tip in hydrochloric acid then rinse
- Stain for DNA eg. with toluidine blue
- Lower coverslip using a mounted needle at 45 degree angle without trapping air bubbles
- Squash by firmly pressing down on glass slip but do not push sideways
Why are root tips used?
Where dividing cells are found / mitosis occurs
Why is a stain used?
● To distinguish chromosomes
● Chromosomes not visible without stain
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 not push cover slip sideways?
● Avoid rolling cells together / breaking chromosomes
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
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 x4)
3a. Use coarse focusing dial to move stage close to lens
b. 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
What are the rules of scientific drawing?
✓ Look similar to specimen / image
✓ No sketching / shading - only clear, continuous lines
✓ Include a magnification scale (eg. x 400)
✓ Label with straight, uncrossed lines
Explain how the stages of mitosis can be identified
Prophase ● Chromosomes visible / distinct → because condensing
● But randomly arranged → because no spindle activity / not attached
to spindle fibre
Metaphase ● Chromosomes lined up on equator → because attaching to spindle
Anaphase ● Chromatids (in two groups) at poles of spindle
● Chromatids V shaped → because being pulled apart at their
centromeres by spindle fibres
Telophase ● Chromosomes in two sets, one at each pole
What is a mitotic index?
● Proportion of cells undergoing mitosis (with visible chromosomes)
● Mitotic index = number of cells undergoing mitosis / total number of cells in sample
Explain 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
Suggest how to calculate the time cells are in a certain phase of mitosis
- Identify proportion of cells in named phase at any one time
● Number of cells in that phase / total number of cells observed - Multiply by length of cell cycle
