Practical skills

• Investigating Mitosis - RP - Preparation

  1. Cut 5-10mm of several tips of a growing root.
  2. Prepare a boiling tube containing 1 mol HCL acid and put in a water bath at 60 °C for 15 mins.
  3. Transfer the root tips to the boiling tube for 5 mins - to incubate.
  4. Use a pipette to rinse the root tips in an observation well containing cold water. Dry the tips on a paper towel.
  5. Place one tip on a microscope slide and add a drop of water. Use a mounted needle to break the tip open.
  6. Add a few drops of acetic orcein stain and leave it for a few mins.
  7. Place a cover slip over the cells and push down firmly.
• Why do we use a stain and a cover slip in the required practical for investigating mitosis? Also, why do we heat the root tips in acid?

  1. The stain adheres to the DNA, so we can view the chromatid sisters under an optical microscope.
  2. A cover slip squashes the tip to create a single layer of tissue for light to pass through. Do not push the cover slip sideways as this could break the chromosomes.
  3. We heat in acid to flood the cells, lowering the pH, and denaturing the protein. This prevents mitosis from occurring any longer. Also, plant cell walls are broken down so the stain can permeate easier
• Investigating Mitosis - RP - Microscopy procedure (after the preparation)

  1. Clip the slide to the stage.
  2. Select the lowest-powered objective lens (e.g. x4)
  3. Look down the eyepiece and use the coarse adjustment knob to move the stage downward until the image is roughly in focus.
  4. Adjust the focus with the fine adjustment knob to get a clear image.
  5. Move to a higher-powered objective lens to see the stages of mitosis
  6. Work out the mitotic index!
• Why do we need to break the tips open with a mounted needle?


  To spread the cells out thinly
• Safety precautions of the mitosis RP

  1. Wear safety goggles, lab coat and gloves
• Osmosis & water potential RP 03 - METHOD

  1. Make a series of dilutions of 1M sucrose sol (0.0, 0.2 etc). Dilute with distilled water.
  2. Measure 5cm^3 of each dilution into separate test tubes.
  3. Use a cork borer to cut out 6 potato chips -ensure they're identically sized.
  4. Dry chips with a paper towel to remove excess water (don't squeeze)
  5. Weigh each chip
  6. Place a potato chip in each test tube & leave for 20 minutes.
  7. Remove, dry gently with a paper towel and weigh each chip.
  8. Calculate the percentage change in mass for each sucrose solution
• Osmosis & water potential RP 03 - GRAPH

  1. Plot a graph of change in mass against concentration of sucrose solution
  2. The point at which the line of best fit crosses the x axis (a 0% change in mass) is where the isotonic solution is
• What is a calibration curve and what is it used for in RP 03?

  A graph used to determine an unknown concentration of a sample by comparing the sample to a set of standard samples with known concentrations (standard curves) made by a dilution series. In RP 03, they are used to determine an unknown water potential in a potato sample
• Cell Membrane Permeability RP 04 - METHOD

  1. Cut beetroot into 6-10 identical cubes using a scalpel and wipe/rinse the cubes to clean off any pigment released from cutting them.
  2. TEMP - place each cube into equal volume of distilled water (5-15ml) & place each test tube in water bath at range of temperatures (30-80°C)
  3. CONC OF SOLVENTS - create a dilution series of ethanol (0-100%) with distilled water
  4. Leave samples for 20 mins so pigment leaks out of beetroot
  5. Set the colorimeter to a blue filter & zero using a cuvette with distilled water. Filter samples into cuvettes + measure absorbance.
• What does a higher absorbance mean in RP 04?

  Higher absorbance = higher pigment concentration = more permeable membrane
• Cell Membrane Permeability RP 04 - GRAPH

  Plot a graph of absorbance against ethanol concentration/temperature
• Stain in mitosis can be iodine (in potassium iodide)