Practical: Stomatal Density (SQA National 5 Biology): Revision Note

Investigating stomatal density

• Stomatal density is the number of stomata per unit area, e.g. per mm²; a higher stomatal density will result in:

  • a higher rate of gas exchange

  • increased water loss

• Stomatal density can be estimated by:

  • making clear nail-varnish impressions of the surface of a leaf

  • counting stomata under a microscope

• Comparing densities between species, leaf surfaces (upper vs lower), or plant treatments helps predict how plants may respond to changes in environmental conditions; this is useful in agriculture and horticulture

Apparatus

• A plant, or range of plants, to provide leaf samples

• Clear nail varnish (ideally solvent based)

• Sellotape

• Microscope

• Microscope slides

• Stage micrometer

• Counting device, e.g. clicker/ phone app

• Calculator

Method

1. Select a leaf from a live plant and remove it from the plant

2. Place the leaf upside down on a flat surface, such as a tile or work top

3. Paint clear nail varnish onto the underside of the leaf

4. Wait for the nail varnish to dry (approx. 5 minutes)

5. Peel off the layer of varnish using sellotape

   • Discard the leaf

6. 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, as long as the sample is laid flat on the slide

7. Focus the microscope on the sample and adjust the zoom so that a countable number of stomata are visible in the field of view

   • Between 15 and 100 is ideal

8. Use a stage micrometer to measure the diameter of the field of view

   • Check the stage micrometer’s division size; this is often 0.01 mm, or 10 µm, per division

   • Place the micrometer on the microscope stage and focus clearly on the scale

     • This should be at the same magnification you will use for counting stomata

   • Move the slide so that a division line sits exactly on the left edge of the circular field of view

   • Count the number of full divisions that span from the left edge to the right edge of the field

     • Estimate a fraction if the edge falls between lines

   • Calculate diameter: number of divisions × length of each division

     • Example: 46 divisions × 0.01 mm = 0.46 mm

   • The diameter can be used to calculate area: area of a circle = πr²

9. Count the stomata in that field of view

   • You may wish to use a clicker or phone app so you don't lose count

10. Move the field of view to another area of the nail varnish layer and repeat

11. 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

12. Calculate the mean stomatal density: number of stomata ÷ area of field of view

13. Repeat with, e.g.:

    • the upper epidermis of the same plant

    • a plant of the same species grown in different conditions

    • a different plant species

Limitations

Safety

• Solvent-based nail varnish is flammable and produces fumes:

  • Keep varnish away from flames

  • Use in a well-ventilated room

  • Cap bottles promptly

  • Dispose of used tape and leaf material appropriately

  • Wash hands after the practical