Why test for starch?
Glucose made by photosynthesis is quickly used or converted into other molecules, so it does not build up enough in the leaf to be tested directly. Starch, however, is stored in chloroplasts as the plant's main reserve. So testing a leaf for starch is the standard indirect way to show that photosynthesis has been happening.
Why de-starch the plant first?
If a leaf already contains starch from the previous day, that would mask the result of the experiment. To de-starch a plant:
- Place the plant in a dark cupboard for 24 to 48 hours.
- With no light, the plant cannot photosynthesise, so any existing starch in the leaves gets broken down and used up in respiration.
After de-starching, the leaves should give a negative starch test. Then the experiment can begin.
The iodine test for starch in a leaf
- Pluck the leaf and drop it into a beaker of just-boiled water for 30 seconds. This kills the cells and breaks down the cell walls so iodine can enter.
- Turn off the Bunsen burner. Transfer the leaf into a boiling tube of ethanol, and stand the tube in a beaker of hot water (water-bath heating, not direct flame) for about 5 to 10 minutes. Ethanol is highly flammable, so keep it well away from any naked flame. The hot ethanol dissolves the green chlorophyll out of the leaf, making colour changes easier to see.
- Dip the now-pale leaf in warm water to soften it (the ethanol made it brittle).
- Spread the leaf out on a white tile and cover it with iodine solution.
- Observe the colour. Areas where the leaf has been photosynthesising contain starch and will turn blue-black. Areas without starch stay yellow-brown.
Investigating the need for light
After de-starching:
- Cover part of one leaf (still attached to the plant) with aluminium foil or a piece of black card.
- Place the plant in bright light for several hours.
- Pick the leaf and test it for starch.
Result: the area that received light turns blue-black; the area covered by foil stays yellow-brown. This shows that light is needed for photosynthesis.
Investigating the need for carbon dioxide
After de-starching:
- Enclose one leaf inside a conical flask containing potassium hydroxide solution (KOH absorbs all the carbon dioxide from the air inside the flask). Seal the flask around the leaf stalk with cotton wool and tape so air cannot enter.
- Enclose another leaf inside an identical flask without KOH as a control.
- Place the plant in bright light for several hours.
- Test both leaves for starch.
Result: the leaf in the KOH flask stays yellow-brown (no CO₂, no photosynthesis, no starch). The control leaf turns blue-black. This shows that CO₂ is needed for photosynthesis.
Investigating the need for chlorophyll
Use a variegated leaf (one that is patchy green and white). The white patches contain no chlorophyll.
- De-starch the plant.
- Place it in bright light for several hours.
- Remove the variegated leaf, sketch the pattern of green and white areas before testing.
- Test the leaf for starch.
Result: the green areas turn blue-black (chlorophyll was present, photosynthesis happened, starch was made). The white areas stay yellow-brown (no chlorophyll, no photosynthesis, no starch). This shows that chlorophyll is needed for photosynthesis.
Safety notes for the leaf-starch tests
- Ethanol is highly flammable. Heat it in a water bath, never directly over a Bunsen flame. Switch off the Bunsen before transferring the leaf into the ethanol.
- Iodine and potassium hydroxide are irritants. Wear safety goggles and wash off any splashes immediately.
- Hot water and hot apparatus. Handle with tongs; let glassware cool before putting it away.