Required Practical 2

• Preperation of stained squashes of cells from plant root tips
• Optical microscope is used to identify the stages of mitosis in these stained squashes and calculation of a mitotic index
• The apparent size of cells in the root tip should be measured and should calculate the actual size using the formula: actual size = image size/ magnification
• Squashes of cells from plant root tips are preparede by:

  • Cutting a thin slice of root tip using a scalpel and mount onto a slide
  • Soak the root tip in hydrichloric acid then rinse
  • Stain for DNA e.g, with toluidine blue
  • Lower the coverslip using a mounted needle at 45 degrees without trapping air bubbles
  • Squash by firmly pressing down on the glass slip
• Root tips are used because this is where the dividing cells are found and where mitosis occurs
• Stain is used to distinguish chromosomes as chromosomes are not visible without stain
• Pressing down on the cover slip is necessary so it spread out the cells to create a single layer of cells, in order for light to pass through to ake the chromosomes visible.
• The cover slip shouldn't be pushed sideways, this is to avoid rolling cells together and breaking chromosomes
• The root is soaked in acid to seperate the cells and the cell walls, to allow stain to diffuse into cells, to allow cells to be more easily squashed and to stop mitosis.
• An optical light microscope is set up by:

  1. Clipping slide onto the stage and turning on the light
  2. Selecting the lowest power objective lens which is usually x4
  3. Use coarse focusing dial to move stage close to the lens or turn it to move the stage away from lens until the image comes into focus
  4. Adjust the fine focusing dial to get clear images
  5. Swap to higher power objective lens, then refocus it
• Scientific drawings should:

  • Look similar to the specimen or image
  • No sketching or shading
  • Include a magnification scale
  • Label with straight and uncrossed lines
• Mitotic index is the proportion of cells undergoing mitosis with visible chromosomes
• Mitotic index = number of cells undergoing mitosis/ total number of cells in sample
• Determining a reliable MI from observed squashes happens by:

  • Counting cells in mitosis in the field of view
  • Counting only whole cells and only cells on top and right edges -> standardise counting
  • Divide this by the total number of cells in the field of view
  • Repeat with many or at least 5 fields of views selected at random -> representative sample
  • Calculate a reliable mean
• Calculating the time cells are in a certain phase of mitosis by:

  • Identifying proportion of cells in named phase at any one time, number of cells in that phase/ total number of cells observed
  • Multiply by the length of cell cycle