A window on the past: Measuring stomatal density
Class practical
This technique is straightforward, but requires care to achieve good results.
Stomatal density usually varies between the upper and lower epidermis of any leaf, will vary from species to species, and may change with carbon dioxide concentration in the surrounding atmosphere and with light intensity. Any change will take several weeks to occur, so there is an opportunity here for a long-term, self-directed investigation.
Mathematical analysis of the data gathered may be a challenge for some students – but is simplified with a spreadsheet such as the one to be downloaded below.
Lesson organisation
Demonstrate the technique and give students an opportunity to practise the skill of making epidermal peels or impressions. Supply students with plants kept for several weeks under controlled conditions (such as varying CO2 level, water supply, humidity, temperature or light intensity) or offer this as the groundwork for a longer-term self-directed investigation for students.
This 
stomatal density calculation spreadsheet (15 KB) calculates the area of the field of view for an input radius, and calculates stomatal density. This shows all students how spreadsheets can help with this kind of number work in science, and will support students whose mathematical skills are weak.
Apparatus and Chemicals
For each group of students:
Microscope with eyepiece graticule (calibrated for different magnifications) or stage graticules on microscope slides to allow simple measurement of field of view (Note 6)
Nail varnish and sellotape
or Germolene ‘New skin’ to take impressions from the epidermis
For the class – set up by technician/ teacher:
Plants to investigate (Notes 1 and 2)
Health & Safety and Technical notes
Plant sap can be irritating to the skin. Students may have allergic reactions to nail varnish or Germolene ‘New skin’.
1 Plants with hairy leaves plants are not suitable for taking impressions. Spider plants (Chlorophytum comosum) work well in this procedure. Red hot poker (Kniphofia) is a common garden plant; this monocotyledonous example is easy to study, because you can peel its epidermis and view it directly under a microscope. Grey willow (Salix cinerea) has been studied in modern and fossil forms. Also useful are the Mexican hat plant (Kalanchoe diagremontiana), and Arabidopsis thaliana - this is a popular model organism for plant biology and genetics; it has one of the smallest plant genomes and was the first plant genome to be fully sequenced. Pot geranium (Pelargonium) is also easy to study and you can tear sections of epidermis for direct viewing. Variegated Tradescantia leaves can be used for direct viewing of stomata in the regions with little or no chlorophyll.
2 Grow plants under clear domes taped to a bench or tray so you can control the atmosphere around them. You could use a large plastic water container, a plastic garden bell cloche or a laboratory bell jar. Include a thermometer in each dome so you can monitor temperature. Reduce humidity levels by including a desiccant such as silica gel or calcium chloride (Note 3). Increase humidity levels by including an open vessel of water (such as a deep Petri dish), and topping it up regularly before the liquid evaporates fully. Reduce light levels by covering domes with increasing numbers of layers of fine fabric or horticultural fleece. The level of carbon dioxide in normal atmospheric air is low, but plants will produce some from their metabolic processes. You could increase the carbon dioxide level by breathing into the dome, or adding a boiling tube with a few marble chips (calcium carbonate) and 10-20 cm3 of dilute hydrochloric acid (approximately 0.1 M). You could reduce carbon dioxide levels by including a carbon dioxide absorber such as soda lime (Note 4).
3 Desiccants: Silica gel (see CLEAPSS Hazcard), for users in year 7 advises wearing gloves and to beware of dust from blue (self-indicating) silica gel. The advantage of blue moisture-indicating gel (containing < 0.5% cobalt(II)chloride) is that it can be reheated and used again. The risk to health from cobalt(II)chloride is very low. Calcium chloride (see CLEAPSS Hazcard) is an IRRITANT as a solid. Wash hands immediately if any gets on the skin and wear eye protection when handling the powder or granules.
4 Carbon dioxide: Calcium carbonate is described as LOW HAZARD on its CLEAPSS Hazcard. Hydrochloric acid (See CLEAPSS Hazcard) at low concentrations presents a minimal hazard to students, and will be neutralised as the marble chips dissolve. Soda lime is described as CORROSIVE on its CLEAPSS Hazcard. Ensure that students do not handle the soda lime. Students should wear eye protection if there is any risk of getting soda lime in their eyes. Be aware of the first aid response should any soda lime get into anyone’s eyes.
5 Make an epidermal impression by spreading a thin layer of nail varnish on the leaf and leaving it to dry. Remove the layer of varnish by attaching clear sticky tape to it, peeling it from the leaf surface and sticking it to the slide. You could try Germolene ‘New skin’ as an alternative to nail varnish. It has slightly different elastic properties and may make a clearer impression. You won’t need to use sticky tape with ‘New skin’.
6 Graticules printed on plastic are available at reasonable prices – around £20 for 10 – if your microscopes are not already equipped with them.
Ethical issues
There are no ethical issues associated with this procedure.
Procedure
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 = πr2, 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.
Studies of stomatal density of plant samples up to 300 years old in botanical libraries have been used alongside evidence of recent changes in carbon dioxide levels in the atmosphere. Studies of stomatal density in fossils has been correlated with information from ice cores to give evidence of how carbon dioxide levels in the atmosphere have affected plants in the past. (See Annals of Botany.)
There are commercial implications when stomatal density changes in plants grown under artificial light or with additional atmospheric CO2 to improve crop production. This is because stomatal reaction to high light intensity or high CO2 levels alter their water requirements, and the cost of supplying water to the plants.
Health & Safety checked, October 2008
Downloads
Download this stomatal density calculations spreadsheet (15 KB) to help your students manage the calculations in this procedure.
Web links
www.saps.org.uk/students/supporting-materials/543-seeing-without-eyes-how-plants-learn-from-light-an-article-for-post-16-students
SAPS 'Seeing without eyes – how plants learn from light' by Stephen Day is an 8-page booklet explaining how phytochromes in plants react to light intensity and provide a mechanism for plants to respond to light.
http://www.plantscienceimages.org.uk
SAPS plant science images database with images of stomata from dicotyledonous plants such as Kalanchoe lower epidermis and Arabidopsis thaliana.
http://aob.oxfordjournals.org/cgi/content/abstract/76/4/389
Abstract of Annals of Botany 76: 389-395, 1995. 'Stomatal density and index of fossil plants track atmospheric carbon dioxide in the Palaeozoic' by Jennifer C. McElwain and William G. Chaloner. This research correlates the stomatal density of forest tree species collected over the last 200 years with changes in carbon dioxide levels in the atmosphere. It goes on to link stomatal density in fossil specimens from the Palaeozoic with carbon dioxide levels as deduced from ice cores.
(Websites accessed October 2011)