SAFETY: Be alert to any students suffering allergic responses to the materials handled. Offer gloves as skin protection if necessary, and make sure students wash their hands thoroughly at the end of the procedure.
Preparation
a Grow plants in different conditions for 4-6 weeks. Useful varieties of plants are suggested in Note 1. Ideas for creating different conditions for the plants are suggested in Note 2.
b Make sure students know what stomata look like, and understand something about their function in the plant.
Investigation
c Collecting epidermal evidence: the epidermis will peel from some leaves quite readily. First cut the leaf. Use your fingernails to catch hold of and peel off the epidermis, or use a sharp razor blade to peel off the epidermis. Mount the peel in a drop of water on a microscope slide with a coverslip. Alternatively, make an epidermal impression with nail varnish or another clear substance, and place that on a microscope slide to view it (Note 5).
d Discuss and decide how many impressions or epidermal samples should be taken and from where on each plant to get a representative sample. Try to be consistent about which part of the leaf to use.
e View the epidermal impressions using a calibrated microscope fitted with an eyepiece graticule (Note 6).
f Calculate the true area of the field of view. You can calculate this using the formula area = \pir2, when you have measured the true radius of the field of view (r).
g Count the stomata visible in each of three areas of the impression.
h Calculate the stomatal density for each area of the impression sampled.
i Analyse average density for each impression and for each plant.
j Download this stomatal density calculation spreadsheet (15 KB) to help you calculate area of field of view, stomatal density for each impression, and average stomatal densities.
Teaching notes
Stomata control the movement of gases and vapours into and out of a leaf. They are often discussed primarily in the context of controlling loss of water from a leaf – as shortage of water is a common stress experienced by plants. The stomata of wilting plants close which minimises further water loss from the leaf.
However, closed stomata will also reduce the availability of carbon dioxide for a photosynthesising leaf. So at low concentrations of carbon dioxide, in light conditions, stomata are stimulated to open wide which permits photosynthesis to continue. In low light conditions, carbon dioxide concentration is not a limiting factor for photosynthesis and stomata can be closed without affecting carbon dioxide uptake.
Over short time scales, reducing the size of the aperture in each stoma reduces the loss of water vapour from a leaf, but also reduces the amount of carbon dioxide that the plant can absorb. Therefore, at high light intensities (which are often accompanied by high temperatures and low water levels) reducing water loss has the concomitant effect of limiting photosynthesis.
Some species of plant have stomata on both sides of the leaf, and others have stomata only on the lower epidermis. The shape of stomata (and the mechanisms for controlling the size of the aperture) differ between monocotyledonous plants (such as grasses) and dicotyledonous plants.
Some species seem to respond to prolonged ambient levels of light intensity and carbon dioxide by developing leaves with an altered density of stomata. For example, at high CO2 levels, a lower density of stomata will not limit the rate of photosynthesis, but will reduce water loss. At higher light intensities, a higher density of stomata will maximise the rate of photosynthesis, but with the risk of enhanced water loss.
Interaction between factors is complex and varies from species to species. A literature search will find many suggestions of factors already investigated which could promote ideas for further work in the school/ college laboratory.
