Protocol

Authors

Sven-Erik Jacobsen and Mira Bendevis

Author Affliations

Department of Plant and Environmental Sciences, Copenhagen University, Denmark

Overview

Quick and easy procedure to obtain good quality leaf surface imprints for determination of stomata density etc. The slides can be stored for a long time.

Background

Standard operating procedure for student Ecology and Plant Physiology laboratory exercises. This is a very quick and cheap way to determine possible treatment effects on stomata density and index.

Materials/Equipment

• Clear nail polish (Top coat) or Germolene – New Skin (Bayer)
• Clear adhesive tape.
• Microscope slides (with frosted section for labelling)
• Soft paintbrush (for leaves with salt glands)
• Microscope (preferably with grid and camera installed)

Units, terms, definitions

Stomatal index

Stomata density

Procedure

Before you begin, read Notes and Troubleshooting Tips.

1. Apply the nail polish to the upper or lower side (Adaxial or Abaxial) of the leaf (or both) evenly in a narrow and thin strip and allow it to dry. Apply to multiple plants at the same time to make the sampling more efficient.
2. When the polish is dry (several minutes), take a square of tape and stick it on top of the nail polish swath on the leaf. Press down gently to make sure all areas of the swath is attached to the tape. Avoid air bubbles under the tape. Fingerprints can be wiped off with alcohol later.
3. Gently peel the tape from the leaf surface. A cloudy leaf imprint will now be attached to the adhesive tape.
4. Attach the piece of tape with the leaf imprint to a very clean glass slide and label the slide with ID for plant and treatment. Make sure the imprints are pressed as flat as possible on the slide. The slides are easily stored in slide boxes for later use.
5. Focus the imprints under the appropriate magnification on the microscope. Usually 20x or 40x magnification works best. Find an area that appears to have a higher density of stomata. Avoid edge areas, damaged areas, and large veins in the field of view.
6. Counting the number of stomata on a smaller area and then scaling up, rather than counting what is in a full grid or field of view, may give more accurate numbers. This is due to the fact that although it appears flat, the leaf imprint may have tiny variability in elevation levels and therefore not everything in the visible field under the microscope is guaranteed to be in focus, which could result in some stomata not being clearly visible when others are within a very small area. When calculating the stomata density per cm2, refer to a calibrated table for the microscope grid.

Note: Take pictures of each slide, so the stomata can be counted at the comfort of your own desk.

Notes and troubleshooting tips

Before applying to all the leaf samples, test whether nail polish or Germolene works best for the specific leaf material.
Below are a few examples of leaves we have tested with both Germolene- New Skin and nail polish:

Apple – Germolene or nail polish

Quinoa – Nail polish only!

Potato – Germolene or nail polish

• Make sure the nail polish is not of the cheapest quality. It should not reek from solvents or have a yellow tint.
• If the leaves to be imprinted have salt glands, for example like those of quinoa, they should
• Decide in advance which area of the leaf imprints are to be taken from. Try to be consistent with the placement in order to avoid huge variation in numbers. Avoid as many big leaf veins as possible.

Literature references

Related paper:

Yan F, Sun Y, Song F, Liu F (2012) Differential responses of stomatal morphology to partial root-zone drying and deficit irrigation in potato leaves under varied nitrogen rates. Scientia Horticulturae 145, 76-83