3.1.1. Microscopic pollen identification and making archival reference slides

Pollen from different plant species can usually be distinguished based on diagnostic traits such as pollen grain size, exine sculpturing and number and size of the apertures (pores or furrows). It is important to keep in mind when working with fresh pollen that the degree of pollen hydration affects external pollen appearance. Transmitted light microscopy is the most widely used technique for pollen identification using fresh, acetolyzed and stained pollen, but scanning electron microscopy (SEM) is also used to study surface details of the exine. In pollen reference collections, pollen grains are usually subjected to acetolysis that removes the protoplasm and leaves the exine (Erdtman, 1969; Kearns and Inouye, 1993). The acetolysis solution contains glacial acetic acid and concentrated sulphuric acid (9:1). According to Dafni et al. (2005) and Kearns and Inouye (1993) the procedure for acetolysis is as follows:

  1. Add pollen sample to a solution of glacial acetic acid for 10 min.
  2. Centrifuge and discard the supernatant.
  3. Add a few mL of acetolysis mixture (glacial acetic acid and concentrated sulphuric acid 9:1).
  4. Heat the solution gently to boiling point in a water bath, stirring continuously with a glass rod.
  5. Cool the solution for a few minutes, centrifuge and discard the supernatant.
  6. Resuspend in distilled water, centrifuge and decant the supernatant. Repeat this step.
  7. Pollen is usually stained to increase the contrast. Several stains (such as methyl-green or fuchsin) can be used, but Safranin O is the preferred stain for most uses in palynology, staining the pollen grains pink to red depending on the amount of stain and type of pollen analysed (Jones, 2012).

After acetolysis, pollen can be preserved for further analyses or to make archival reference slides. A common procedure is to use glycerin jelly slides (Erdtman, 1969):

  1. Prepare a base stock of jelly by combining 10 g gelatin, 30 ml glycerin, and 35 ml distilled water.
  2. On a clean microscope slide add a drop of the prepared jelly and a sample of pollen and stain.
  3. Warm the slide gently, stirring to thoroughly homogenize the mixture.
  4. Add a cover slip, sealing with nail polish or other varnishes around the edges.

Identification keys and atlases with pollen images are available both in general and for specific taxa (Kearns and Inouye, 1993; http://www.geo.arizona.edu/palynology/polonweb.html).