Required Practical 4

• Describe a method to investigate the effect of a named variable on the permeability of cell-surface membranes:

  1. Cut equal sized/identical cubes of plant tissue (e.g. beetroot) of same age/type using a scalpel
  2. Rinse to remove pigment released during cutting or blot on a paper towel
  3. Add same number of cubes to 5 different test tubes containing same volume of water (e.g. 5cm3)
  4. Place each test tube in a water bath at a different temperature (e.g. 10, 20, 30, 40, 50 degrees)
  5. leave for the same amount of time (e.g. 20 minutes)
  6. Remove beetroot and measure intensity of colour of surrounding solution
• How to measure colour intensity of surrounding solution?

  Semi-quantitatively:

  • Use a known conc. of extract and distilled water to prepare a dilution series (colour standards)
  • Compare results with colour standards to estimate conc.

  Quantitatively:

  • Measure absorbance (of light) of known concentrations using a colourimeter
  • Draw a calibration curve - plot a graph of absorbance (y) against conc. of extract (x) and draw line of best fit
  • Absorbance value for sample read off calibration curve to find associated extract conc.
• What are the issues with comparing to a colour standard?

  • Subjective
  • Colour obtained might not match any of the colour standards
• Why wash the beetroot before placing it in water?

  • Wash of any pigment on surface
  • To show that release is only due to named variable
• Why regularly shake each test tube containing cubes of plant tissue?

  • To ensure all surfaces of cubes remain in contact with liquid
  • To maintain a concentration gradient for diffusion
• Why control the volume of water?

  • Too much water would dilute the pigment so solution will appear lighter/more light passes through colourimeter than expected
  • So results are comparable
• How could you ensure beetroot cylinders were kept at the same temperature throughout the experiment?

  • Take readings of temperature in tube in intervals throughout the experiment using a digital thermometer/temperature sensor
  • Use corrective measures if temperature has fluctuated
• What does high absorbance suggest about the cell membrane?

  • More permeable/damaged
  • As more pigment leaks out making surrounding solution more concentrated
• Explain how temperature affects the permeability of cell surface membranes:

  1)As temperature increases, permeability increases

  • Phospholipids gain kinetic energy and fluidity increases
  • Transport proteins denature at high temperatures as hydrogen bonds break, changing tertiary structure

  2)At very low temperatures, permeability increases

  • Ice crystals can form which pierce the cell membrane and increase permeability
• Explain how pH affects permeability of cell surface membranes:

  • High or low pH increases permeability

  - Transport proteins denature as H/ionic bonds break, changing tertiary structure
• Explain how lipid soluble solvents e.g. alcohol affect the permeability of cell surface membranes:

  • As concentration increases, permeability increases

  - Ethanol (a lipid soluble solvent) may dissolve phospholipid bilayer (gaps form)