Required Practical 2 Flashcards
Preparation of stained squashes of cells from plant root tips; set-up and use of an opitcal 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 using scalpel and mount onto a slide.
- Soak root tip in hydrochloric acid.
- Stain for DNA (e.g. with toluidine blue).
- Lower coverslip using a mounted needle at 45° without trapping air bubbles.
- Squash by firmly pressing down on glass slip but do not push sideways.
Why is a stain used?
To distinguish chromosomes which are not visible without a stain.
Why are root tips used?
This is where dividing cells are found/mitosis occurs here.
Why squash/press down on coverslip?
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.
- Clip slide onto stage and turn on light.
- Select lowest power objective lens.
- Use coarse focusing dial to move stage close to lens. Turn coarse focusing dial to move stage away from lens until image comes into focus.
- Adjust fine focusing dial to get clear image.
- 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.
- Label with straight, uncrossed lines.
What stage of mitosis is shown in the diagram below? Explain how you know.
PROPHASE
- Chromosomes visible/distinct because they’re condensing.
- Randomly arranged because no spindle activity/not attached to spindle fibres.
What stage of mitosis is shown in the diagram below? Explain how you know.
METAPHASE
- Chromosomes lined up on equator because attaching to spindle.
What stage of mitosis is shown in the diagram below? Explain how you know.
ANAPHASE
- Chromatids at poles of spindle.
- Chromatids V-shaped because being pulled apart at their centromeres by spindle fibres.
What stage of mitosis is shown in the diagram below? Explain how you know.
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 mitotic index from observed squashes.
- Count cells in mitosis in field of view.
- Count only whole cells - standardise counting.
- Divide this by total number of cells in field of view.
- Repeat with at least 5 fields of view selected randomly - representative sample.
- Calculate a reliable mean of mitotic index.
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.
