Required practical 4: Investigation into the effect of a named variable on…

The idea

Beetroot is used because its vacuoles contain the red pigment betalain, which stays inside while the tonoplast and cell-surface membrane are intact and leaks out once they are damaged. Cut identical cylinders with a cork borer and wash them thoroughly to remove pigment released by the cells cut at the surface; without that step the first colour you measure is an artefact of cutting rather than an effect of your variable. Expose each cylinder to a different level of the named variable, usually temperature or ethanol concentration, for a fixed time, then remove the beetroot and measure the colour of the surrounding solution in a colorimeter, using a filter of the complementary colour and calibrating with distilled water as the blank. More pigment gives a higher absorbance, and so indicates greater permeability.

What earns the marks

• Washing the cut cylinders to remove pigment released by cells damaged during cutting.
• Controlling the volume of solution, the exposure time, and the size and number of beetroot pieces.
• Using a colorimeter calibrated with distilled water, with a higher absorbance meaning more pigment and so greater permeability.
• Explaining increased permeability above about 45 degrees Celsius by denaturation of membrane proteins, with bonds broken and tertiary structure changed.
• Higher temperature also increasing phospholipid movement so that gaps appear in the bilayer.

Common mistakes and how to put them right

• Saying the membrane melts, or that the phospholipids denature.

  Put it right: Only proteins denature.

• Omitting the washing step, so pigment from cutting damage is counted as leakage caused by the variable.

• Reporting absorbance and transmission the wrong way round, so greater leakage appears as a lower reading.

• Leaving the beetroot in the tube when taking the colorimeter reading.

• Describing ethanol as denaturing the bilayer rather than dissolving the lipid out of it.

Exam tips

• Say which quantity your colorimeter measures and in which direction it changes, because examiners expect the link from reading to permeability to be explicit.
• Give a full explanation of temperature effects in two parts: phospholipid movement below the denaturing point, protein denaturation above it.
• List controlled variables specifically, naming volume, time and tissue size, rather than writing keep everything else the same.