Required Practical 2

• Describe 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 on a slide
  2. Soak root tip in hydrochloric acid then rinse
  3. Stain for DNA e.g. with toluidine blue
  4. Lower coverslip using mounted needle at 45 degrees without trapping air bubbles
  5. Squash 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 are 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
  • No sketching/shading - only clear continuous lines
  • Include a magnification scale (e.g. x400)
  • Label with straight, uncrossed lines
• 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:

  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