Measuring Stomatal Density

Stomata control the movement of gases in and out of a leaf, making carbon dioxide available for photosynthesis, and controlling the loss of water from the leaf through transpiration.

Stomatal density varies between monocots and dicots, between plant species, and between the underside and top side of the leaves on a plant.

There are a number of ways to measure stomatal density, and the different techniques are explored here. One popular method has been to use clear nail varnish to make an impression of the epidermis. Other methods include using Germolene New Skin and using a water-based varnish from DIY shops.

We also suggest some potential investigations for students to carry out.

Because of the size of stomata, you will need a reasonably good microscope for this. Your choice of magnification will depend on the leaf material that you are using, and the size of the stomata.

Background Information

The image shows a surface view of the lower epidermis of Kalanchoe (Kalanchoe sp.), a dicotyledonous plant. Three stomata and their associated guard cells are shown.

Each pore or stoma is surrounded by two sausage-shaped guard cells, which change shape to control the size of the stomatal aperture. In the majority of leaves with an upper and lower surface (dorsiventral) like this dicot, most stomata occur in the lower epidermis. They are usually evenly distributed in the leaves of monocots.

The stomata of most species open in daylight and close in the dark. Those plants that use CAM photosynthesis (an adaptation to reduce water loss in arid conditions), stomata close during the heat of the day, to reduce evapotranspiration, and open at night to absorb carbon dioxide for use in photosynthesis.

The guard cells contain chloroplasts (visible in this image), but in most plant species they are not able to carry out the full process of photosynthesis.

The wavy blue lines, looking rather like a jigsaw puzzle, are the cell walls of the epidermal cells. Guard cells develop and differentiate from epidermal cells.

Update your subject knowledge

Update your own knowledge of stomata in under 5 minutes, with two short videos from one of the UK’s leading biologists.

In this 3 minute video, Prof Alistair Hetherington introduces ‘The Mystery of the Evolution of Stomata’.

In this 4 minute video, Alistair explains why fundamental research, such as his own on stomata, helps us respond to climate change.

These videos would also be suitable for post-16 biology students.

Selecting your plants

One of the best plants for doing epidermal peels is the red hot poker plant Kniphofia. Being a monocot its stomata are highly ordered in rows, but they are big and great for stomatal opening and closing using solutions of different concentrations.

Almost as good is the Elephants Ear Saxifrage Bergenia. This also peels very easily, but the stomata are smaller although clearly visible at x100 magnification. This is a dicot so the distribution is more random.

Geraniums (a dicot) and spider plants (another monocot) also make excellent stomatal peels.

Using Clear Nail Varnish

Using clear nail varnish is a traditional method to measure stomatal density, since making the impression and viewing it under a microscope can be completed in one lesson. However, some leaves are prone to damage from the solvent in the nail varnish. The leaves absorb it, turn brown, and fail to produce any impression. Pupils lose interest and get frustrated because their leaves ‘aren’t working’. Also, for a GCSE class, several pots of nail varnish are needed so that no one is left waiting, thus adding to expense.

• Prepare an epidermal impression by coating the leaf surface with nail varnish. Peel off the dried layer of nail varnish by using sellotape and stick this onto a slide.
• Alternatively, with some plants you can peel off an epidermal strip directly, which you can mount in water on a slide and place under the microscope.
• If you have an eyepiece graticule which you can use, you can work at a relatively low power, and you can count the number of stomata within different squares to act as replicates.
• If you do not have an eyepiece graticule, you can work at a higher magnification and count a number of different fields – the area visible under the microscope – at any one time.

Make sure that:

1. You get enough counts to be able to analyse your results statistically,
2. You calculate the area of leaf which you are counting in order to give a quantifiable result e.g. stomata per square mm. You will need to calibrate the size of the field of view, or the size of individual squares within a field, using a stage micrometer to do this.

Using ‘New Skin’

A good alternative is to use Germolene ‘New Skin’ to take the impressions. There is an excellent protocol on Measuring Stomatal Density on the Practical Biology website.

Using Water Based Varnish

A successful alternative to nail varnish is to use a clear water based varnish (available at ‘Do it Yourself’ supermarkets). A half litre tin is cheap, and can be divided up into smaller amounts for ease of use. Paint the opaque varnish thinly on to the leaf to produce a clear film. Leave it to dry as usual. These water based varnishes take longer to dry, so if the leaves are coated during one lesson, the impressions can be peeled off and examined the next. The varnish is non toxic, so can be used on living plants without removing the leaves – this means that school plants do not have to be denuded for this experiment! In addition to revealing the stomata, the cell walls also show up.

Other Methods

Other suggestions include producing impressions on acetate film, by placing a leaf in propanone and then pressing it onto the acetate. This does not work for some plant leaves, especially those that have an uneven surface – and the leaf still has to be removed from a plant. Another method is to rub a board pen over the surface. The solvent-based ink permeates the leaf, showing up the stomata. However, this seems to work only with certain types of pen – probably related to the strong solvent in the pen. This also raises health and safety issues.

Suggestions for investigations

As well as studying stomatal patterns and densities in a variety of plants, the following questions may be posed to students:

• Does the density vary over a leaf surface?
• Does the density vary between different leaves of the same plant? . . . or between different plants of the same species? (I have looked at a number of the Brassicaceae: B. oleracea v. capitata (cabbage), v. gemmifera (brussels sprout), v. italica (broccoli), v. botrytis (cauliflower). All these leaves are available from greengrocers – and you can eat the rest of the vegetable afterwards!
• Does the density vary between plants from different habitats. (I have used a number of cacti and succulent plants to do this.)
• For plants that reproduce vegetatively, is there any difference between parent and offspring? (Kalanchoe and begonias are useful here).

Acknowledgements to Barry Meatyard and Mary MacDonald.