Pollen Preparation Procedure

V. Stefanova
CSD Facility, Department of Earth Sciences, University of Minnesota

Introduction

Pollen analysis is the principal technique available for determining vegetation response to past terrestrial environmental change. The technique has been in use for nearly a century, initially as a method for investigating past climatic changes. More recently, the importance for vegetation change of processes such as human impact, successional change and other biotic and abiotic factors have been recognized. Pollen analysis can be used to examine these factors (Bennet and Willis, 2001).

Pollen grains are microscopic in diameter (most often between 10 and 70 μm) and due to the extremely resistant characteristic of the pollen grain wall, pollen grains can be well preserved in lake sediments, peat deposits, soil, and rocks. The unique morphological characteristic of the pollen grains make them easy to identify to species level. Through proper chemical treatment pollen grains and spores can be extracted from field samples, concentrated, and mounted for identification and quantification.

A basic procedure for the preparation of sediment samples for the analysis of pollen is presented here following Bennet and Willis (2001). It is set out diagrammatically in the table below. More details and some additional techniques are covered in Faegri & Iversen (1989).

Images of sediment samples at different stages were taken to illustrate the processes in the pollen extraction procedure.

Treatment StepResult
Step 1. SpikeTo estimate the concentration of pollen and spores in sediments
Step 2. Hot 10% KOHRemoves humic acids
Step 3. Screening (160μm and 6μm)Removes coarse particles and clay or fine organic particles
Step 4. Hot 10% HClRemoves carbonates
Step 5. Hot 48% HFRemoves silica and silicates
Step 6. Hot 10% HClRemoves colloidal silica and silicates
Step 7. AcetolysisRemoves Polysaccharides (cellulose)
Step 8. DehydrationRemoves water
Step 9. Mounting mediumSilicone oil or glycerol

Figure 1. Steps involved in sediment processing for fossil pollen

 

Step 1: Addition of spike

This is needed in order to obtain estimates of the concentration of pollen and spores in sediments. It must be the first stage, so that any losses of pollen and spores during the processing affect fossil and exotic pollen equally (Figure 2).

Sediment samples under the microscope at the first step in the pollen preparation procedure. Figure 2 A shows pollen of Fagus or beech, Quercus or oak, and the spike. Figure 2 B shows pollen of Tsuga or hemlock and the spike.
Figure 2. Sediment samples at the first step in the pollen preparation procedure. A: pollen of Fagus (beech), Quercus (oak), spike. B: pollen of Tsuga (hemlock), spike.

Step 2: Potassium hydroxide (KOH) treatment

This process removes ‘humic acids’ (unsaturated organic soil colloids) by bringing them into solution, and also disaggregates the sediment. The quantity of humic acid may be considerable in organic material that is highly decomposed (such as some peats) (Figure 3).

Two microscope images of sediment samples after the KOH treatment. Figure 3 A shows pollen of Tilia or basswood. Figure 3 B shows pollen of Artemisia or wormwood
 Figure 3. Sediment samples after the KOH treatment. A: pollen of Tilia (basswood). B: pollen of Artemisia (wormwood)

Step 3: Screening

Coarse screening (160 µm) the sample removes particles larger than most pollen or spores. The residue retained on the sieve can often usefully be examined for smaller identifiable macrofossils (such as trees bud-scales, small seeds of Potamogeton or Betula, conifer needles) (Figure 4). The fine screening (6 µm) removes fine organic particles and clay.

Microscope images of the residue retained on the 160 nano meter screen. Figure 4 A shows a Conifer bud-scale. Figure 4 B shows plant tissue.
Figure 4. Residue retained on the 160 µm screen. A: Conifer bud-scale. B: Plant tissue.

Step 4: Hydrochloric acid treatment

This process removes carbonates. The reaction products are calcium hydroxide, which is soluble, and carbon dioxide, which is released as a gas (often vigorously). Magnesium containing carbonate (which may occur in areas with dolomitic limestones) reacts in the same way, but much slower. If its presence is suspected, longer periods in hot HCl are needed (Figures 5 and 6)

Microscope image of sediment sample after the HCl treatment. Pollen of Tsuga or Hemlock, and pollen of Alnus incana-type or Speckled alder.
Figure 5. Sediment sample after the HCl treatment. A: pollen of Tsuga (Hemlock). B: Pollen of Alnus incana-type (Speckled alder)
Microscope image of sediment samples after the HCl treatment. Figure 6 A shows the pollen of Pinus. Figure 6B shows the pollen of Quercus
Figure 6. Sediment samples after the HCl treatment. A: pollen of Pinus; B: pollen of Quercus

Step 5: Hydrofluoric acid (HF) treatment

This stage removes silica and silicates.

Step 6: Hydrochloric (HCl) rinse

HCl step removes colloidal silica and silicofluorides. Depending on the composition of the sediment, this step may be at least as important as the HF step. The reaction between HF and some minerals may produce an insoluble white precipitate, consisting of fluorides.

Step 7: Acetolysis

This stage removes polysaccharides by hydrolysing the polymer chain into soluble monosaccharide units. Polysaccharides are present on the surface f the grain and in the cytoplasm, so removing them greatly facilitates viewing the grain. Polysaccharides such as cellulose may also be significant components of the sediment, so removing these helps concentrate the pollen in the residue (Figure 7).

Images of pollen after an acetolysis. A, B: Ulmus, or elm; C: Fagus; D: Pinus; E: Tsuga; F: Carya, or hickory; G: Tilia; H: Aster-type Betula, or birch
Figure 7: Images of pollen after acetolysis. A, B: Ulmus (elm); C: Fagus;  D: Pinus; E: Tsuga; F: Carya (hickory); G: Tilia ; H: Betula (birch); I: Aster-type

Step 8: Dehydration

A series of increasingly concentrated alcohols and tertiary butyl alcohol remove water, which does not mix with the silicone oil mounting medium. If the water is not 100% removed, irreversible clumping always occurs.

Step 9: Mounting medium

A good mounting medium should have a refractive index close to sporopollenin (1.48) for sufficient contrast to see features of the grains. Silicone oil and glycerol are preferred mounting mediums (Figure 8).

Images of pollen in silicone oil, A: pollen of Picea mariana with the bisaccate morphology characteristic of many conifers; B: Lilium michiganense showing monocolpate and supra-reticulate morphology; C: pollen of Panicum virgatum showing the monoporate morphology of grasses; D: pollen of Betula papyrifera with 3 pores and psilate morphology; E: pollen of Helianthus annulus represents the tricolporate pollen types with echinate morphology F: pollen of Acer rubrum with tricolpate and striate morphology
Figure 8. Images of pollen in silicone oil, (Pollen Reference Collection, CSD Facility), chosen to illustrate some of the features used in identification. A: pollen of Picea mariana (black spruce), showing the bisaccate morphology characteristic of many conifers. B: Lilium michiganense (Michigan lily), showing monocolpate (one colpus) and supra-reticulate morphology; of C: pollen of Betula papyrifera (paper birch) with three pores (triporate) and psilate morphology; D: pollen of Panicum virgatum (switchgrass), showing the monoporate (single pore) morphology of grasses; E: pollen of Helianthus annulus represents the tricolporate (thee colpi and three pores) pollen types with echinate (spine) morphology (Aster-type); F: pollen of  Acer rubrum (red maple) with tricolpate (3 colpi) and striate morphology.

References

Faegri, K. & J. Iversen, 1989. Textbook of Pollen Analysis (4th ed.). Wiley, Chichester, 328 p.

Bennet, K.D. and Willis, K. J. 2001. Pollen. In: J. P. Smol, H. B. Birks & W. M. Last (eds.) Tracking Environmental Change Using Lake Sediments. Volume 3: Terrestrial, Algal, and Siliceous Indicators. 5-32 p. Kluwer Academic Publishers, Dordrecht, The Netherlands.