This practical protocol is designed for students doing an Extended Project Qualification, Advanced Higher investigation, or IB investigation in biology.
They are linked to our Sweet or sour – what’s in our supermarkets? project starter.
Protocol – Starch Grain Assay
The following procedure can be used to determine the mass of solid starch grains present in fruit or vegetable tissues. The procedure as described is for analysis of a potato, but it can be applied to any vegetable or fruit. The blender is not essential, and can be replaced with a mortar and pestle, or the latest small hand-blender (usually used for pureeing vegetables for soups) is very effective at disrupting tissues. The violence of the blending process is effective at liberating the maximum number of starch grains from the cells.
The method depends upon the insolubility of starch in saline and the resulting sediment gives a measure of starch grains present in the material.
• Peel and cut the potato into small pieces (less than 1 cm cubes).
• Weigh the pieces, and put them in a blender, adding 2 cm3 1% NaCl for each gram of potato. Blend the mixture, to produce a loose slurry. Try blending for about 30 seconds, or more if needed.
• Pour the slurry through two layers of muslin, stretched over the mouth of an appropriate sized beaker. Dispose of the solids left in the muslin.
• Leave the filtrate in the beaker to stand for some minutes, to allow the grains of starch to form a solid sediment at the bottom of the beaker.
• When the sediment has formed, carefully pour away the liquid above it, and re-suspend the sediment in fresh 1% NaCl. Once again, allow time for the starch grains to settle to the bottom.
• Repeat the re-suspension / re-washing procedure twice more.
• Finally rinse the starch layer in a similar fashion, using 0.01 M NaOH.
• Then assess the ‘amount’ of sediment (containing the starch grains), either by its volume or by its mass. (Volume could be useful in terms of simplicity.)
To measure volume of sediment, pour a known volume of the starch suspension into a tapered, calibrated centrifuge tube. Allow it to stand vertically for a few minutes. The volume of starch grains can then be directly read off the side of the tube. If you do not have a pre-calibrated tube, then make one by adding small aliquots of water to a suitable tube, and using a marker pen to indicate the volumes on the side of the tube.
As an alternative, to assess the sediment in terms of mass, weigh to find the wet mass, or dry to a constant mass and then weigh to find the dry mass of starch grains obtained.
Remember you can always check the quality of your extract by adding iodine solution, which should colour the grains blue-black, and/or examining a sample under the microscope. You might be able to distinguish the unique features of starch grains belonging to particular species of vegetables. Oh, and don’t forget to add a drop of amylase solution to such a sample, and see the effects of amylase on the structure of the grains.