Practical - Measurement of Stomatal Density (OCR Gateway GCSE Biology: Combined Science)

Measurement of stomatal density on the surface of a leaf

• This is not a required practical but is excellent practice for developing microscope skills and use of mathematical processing of data
• The density of stomata (the number of stomata per unit of area) can be a useful measurement to biologists
  • To assess the plant's likely response to a dry spell of weather
  • To predict its behaviour in windy or wet climates if the plant was being moved for agricultural / horticultural reasons
• This technique can be used to assess how stomatal density varies from species to species

Apparatus

• A plant to sample a leaf from
• Clear nail varnish (ideally solvent based)
• Sellotape
• Microscope
• Microscope slides
• Stage micrometer
• Counting device (clicker/ phone app etc.)
• Calculator

Method

• Select a leaf from a live plant and cut it off the plant
  • Geraniums and spider plants make good subjects for this experiment
• Place the leaf upside down on a flat surface such as a tile or worktop
• Paint clear nail varnish onto the underside of the leaf
• Wait for the nail varnish to dry (approx. 5 minutes)
• Peel off the layer of varnish using sellotape
  • Discard the leaf
• Place the dried varnish impression on a microscope slide
  • A coverslip is not required as this isn't a biological sample, just an impression of one
  • A drop of water is not required either, so long as the sample is laid flat on the slide
• Use the usual steps to focus on the sample
• Adjust the zoom such that a countable number of stomata are visible in the field of view
  • Between 15 and 100 is ideal
  • Even if a stoma is partially visible at the edge, still count it as 1
• Count the stomata in that field of view
  • You may wish to use a clicker or phone app so you don't lose count!
• Move the field of view to another area of the nail varnish layer and repeat
• Count at least 3 separate fields of view and take a mean value
  • Repeat readings allow you to eliminate anomalous results and calculate a reliable mean

Measurements to take

• Use a stage micrometer to measure the diameter of the field of view
  • This has to be at the same magnification power that you used when counting the stomata
• The stage micrometer will be calibrated in micrometers
  • A typical microscope allows the scientist to look at a field of view of about 0.5 mm diameter when on full power (× 400)
• You will have calculated a mean number of stomata per field of view from the previous stage

Step 1: Calculate the radius of the field of view

Radius = Diameter ÷ 2

Radius = 0.46 mm ÷ 2 = 0.23 mm

Step 2:  Calculate the area of the field of view

Area = πr² = π × 0.23²

Area = 0.1662 mm²

Step 3: Divide the mean number of stomata by the area of the field of view to calculate density

Density = 16 ÷ 0.1662 = 96.27 stomata per mm²

Step 4: Round to the required precision (nearest whole number)

Density = 96 stomata per mm²

Limitations

• Not all plant species have easily accessible stomata that create a strong imprint
• Solvent-based nail varnish can destroy some of the cell structure it comes into contact with
• Does the plant grow more stomata (guard cells) according to the conditions in each individual habitat?
• Water-based nail varnish is safer to use but dries more slowly