Site author Richard Steane
The BioTopics website gives access to interactive resource material, developed to support the learning and teaching of Biology at a variety of levels.
Before proceeding to the next stage, a leaf is removed and checked for the presence of starch (See under the section "Testing a leaf for starch").
If no starch is present, then it is possible to proceed to the next stage. If it is, then it must be returned to the dark.
Leaves of the plant are enclosed within glass flasks, supported at an angle by stands and clamps (250/350ml wide-mouthed flasks will usually do). Each flask will contain small amounts of various liquids, which should not be allowed to touch the leaves.
B1 Sodium hydroxide solution or potassium hydroxide solution - CAUTION - CORROSIVE!
B2 Sodium hydrogencarbonate solution or simply carbonated water
B4 no liquid
After the insertion of the leaf, 2 wads of absorbent cotton wool are placed in the neck of the flask, one above and one below the petiole (leaf stalk). These are then saturated with water (from a washbottle). This may need to be repeated a couple of times a day.
Alternatively, split corks or bungs can be used, together with lots of petroleum jelly (messy!).
The plant must be kept well watered and exposed to good light.
After a couple of days or more, the leaves are removed for testing. It may be useful to mark them with cuts to identify them later.
Strict safety precautions must be observed, because ethanol vapour is very flammable.
This procedure might be best demonstrated by the teacher.
Depending on the facilities available in the lab, there are two main methods. Each has 2 steps (a & b).
Using Bunsen burners:
a) A beaker containing water (supported by a tripod and gauze) is brought to the boil by heating it with a bunsen burner set to a non-luminous flame.
Check that the beaker is stable on the tripod, then:
Place the leaf in the boiling water for 1-2 minutes or until soft and flabby.
b) Turn off your burner and ensure that all burners on your bench are out before moving to the next step.
Transfer the leaf to another container of Industrial Methylated Spirit (initially colourless). Do not use purple meths or pure ethanol.
The container is then heated gently - preferably by floating it in the hot water in the same beaker as before, and left for several minutes.
Do not re-warm the beaker using a lit Bunsen burner.
Using electrically heated waterbaths:
a) Place the leaf directly into a waterbath at 100 °C/maximum setting.
b) Transfer to a beaker of IMS in a waterbath set to 78 °C.
The same precautions apply as above - no naked flames.
Immerse the whole leaf briefly in cold water - tap water will do.
Spread out the leaf in a plastic (Petri) dish.
Add a few drops of iodine solution (I/KI), and wait a few minutes for colour to develop.
Grey-blue-black areas represent starch (e.g. B2 above).
Yellow-orangey-red areas represent no starch (e.g. B1 above).