Required Practical 4

• Describe a method to investigate the effect of a named variable (eg. temperature) on the permeability of cell-surface membranes

  1. Cut equal sized / identical cubes of plant tissue (eg. beetroot) of same age / type using a scalpel

  2. Rinse to remove pigment released during cutting or blot on paper towel

  3. Add same number of cubes to 5 different test tubes containing same volume of water (eg. 5cm3)

  4. Place each test tube in a water bath at a different temperature (eg. 10, 20, 30, 40, 50oC)

  5. Leave for same amount of time (eg. 20mins)

  6. Remove beetroot and measure intensity of colour of surrounding solution:

  ● Semi-quantitatively

  ○ Use a known conc. of extract & 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 colorimeter

  ○ Draw a calibration curve → plot a graph of absorbance (y) against conc. of extract (x) and draw a line / curve 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?

  ● Matching to colour standards is subjective

  ● Colour obtained may not match any of colour standards
• why wash the beetroot before placing it in water?

  ● Wash off 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 in colorimeter than expected

  ● So results are comparable
• how could you ensure beetroot cylinders were kept at the same temperature throughout the experiment?

  ● Take readings in intervals throughout experiment of temperature in tube using a digital thermometer / temperature sensor

  ● Use corrective measure if temperature has fluctuated
• what does a high absorbance suggest about the cell membrane?

  ● More permeable / damaged

  ● As more pigment leaks out making surrounding solution more concentrated (darker)
• explain how temperature affects permeability of cell-surface membrane

  ● As temperature increases, permeability increases

  ○ Phospholipids gain kinetic energy and fluidity increases

  ○ Transport proteins denature at high temperatures as H bonds break, changing tertiary structure

  ● 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 membrane

  ● High or low pH increases permeability

  ○ Transport proteins denature as H / ionic bonds break, changing tertiary structure
• explain how lipid-soluble solvents affect permeability of cell-surface membranes

  ● As concentration increases, permeability increases

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