POLLEN PREPARATION PROCEDURE

IntroductionPollen analysis is the principal techniqueavailable for determining vegetationresponse to past terrestrial environmentalchange. The technique has been in use fornearly a century, initially as a method forinvestigating past climatic changes. Morerecently, the importance for vegetationchange of processes such as human impact,successional change, and other biotic andabiotic factors have been recognized. Pollenanalysis can be used to examine thesefactors (Bennet and Willis, 2001).Pollen grains and spores are microscopic indiameter (most often between 10 and70 µm). Due to the highly decay resistantcompounds of the their walls, pollen grainsand spores can be well preserved in lakesediments, peat deposits, soils, and rocks.The unique morphological characteristics ofthe pollen grains and spores make themeasy to identify to species level. Throughproper chemical treatment pollen grains andspores can be extracted from field samples,concentrated, and mounted for identificationand quantification.

Step 2: Potassium hydroxide(KOH) treatmentThis process removes humic acids(unsaturated organic soil colloids) bybringing them into solution, and alsodisaggregates the sediment. Thequantity of humic acid may beconsiderable in organic material thatis highly decomposed (such as somepeats) (Figure 3 and 4).

water, which does not mix with the silicone oilmounting medium. If the water is not 100%removed, irreversible clumping always occurs.

Step 9: Mounting mediumA good mounting medium should have arefractive index close to sporopollenin (1.48) forsufficient contrast to see features of the grains.Silicone oil and glycerol are preferred mountingmediums.

V. Stefanova and J. HeckLacCore, Department of Earth Sciences, University of Minnesota

A basic procedure for the preparation ofsediment samples for the analysis ofpollen is presented here followingBennet and Willis (2001). It is set outdiagrammatically in Figure 1. More detailsand some additional techniques arecovered in Faegri & Iversen (1989) andposted on the LacCore website.Images of sediment samples at differentstages were taken to illustrate theprocesses in the pollen extractionprocedure.

Step 1: Addition of spikeThis is needed in order to obtainestimates of the concentration of pollenand spores in sediments. It must be thefirst stage, so that any losses of pollenand spores during the processing affectfossil and exotic pollen equally (Figure 2).

Figure 2. Sediment samples at the firststep in the pollen preparation procedure. A: pollen of Fagus (beech), Quercus (oak), spike. B: pollen of Tsuga(hemlock), spike.

Step 3: ScreeningCoarse screening (160 µm) the sampleremoves particles larger than mostpollen or spores. The residue retainedon the sieve can be examined forsmaller identifiable macrofossils (suchas trees bud-scales, small seeds ofPotamogeton or Betula, conifer needles)(Figure 5). The fine screening (6 µm)removes fine organic particles and clay,but it is optional because some fungalspores and pollen can be lost as well.

Step 5: Hydrofluoric acid (HF) treatmentThis stage removes silica and silicates.

Step 6: Hydrochloric (HCl) rinseHCl step removes colloidal silica andsilicofluorides. Depending on thecomposition of the sediment, this step maybe at least as important as the HF step.The reaction between HF and someminerals may produce an insoluble whiteprecipitate, consisting of fluorides.

Step 7: AcetolysisThis stage removes polysaccharides byhydrolysing the polymer chain into solublemonosaccharide units. Polysaccharidesare present on the surface of the grain andin the cytoplasm, so removing them greatlyfacilitates viewing the grain.Polysaccharides such as cellulose mayalso be significant components of thesediment, so removing these helpsconcentrate the pollen in the residue(Figure 8).

Step 8: DehydrationA series of increasingly concentrated alcohols and tertiary butyl alcohol remove

Step 4: Hydrochloric acid treatmentThis process removes carbonates. Thereaction products are calcium chloride,which is soluble, and carbon dioxide,which is released as a gas (oftenvigorously). Magnesium containingcarbonate (which may occur in areas withdolomitic limestones) reacts in the sameway, but much slower. If its presence issuspected, longer periods in hot HCl areneeded (Figures 6 and 7).

Figure 8: Images of pollen after theacetolysis. A, B: Ulmus (elm). C: Fagus;D: Pinus. E: Tsuga. F: Carya (hickory). G:Tilia . H: Betula (birch). I: Aster-type.

Quercus

Fagus

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Tsuga

Figure 3. Sediment samples after theKOH treatment. A: Pollen of Artemisia(wormwood).

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Figure 5. Residue retained on the 160 µmscreen. A: Conifer bud-scale. B: Planttissue.

Figure 1. Steps involved in sedimentprocessing for fossil pollen and spores

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Figure 9. Images of pollen in silicone oil,(Reference collection, LRC), chosen toillustrate some of the features used inidentification. A: pollen of Picea mariana(black spruce), showing the bisaccatemorphology characteristic of many conifers.B: Lilium michiganense (Michigan lily),showing monocolpate (one colpus) andsupra-reticulate morphology; of C: pollen ofBetula papyrifera (paper birch) with threepores (triporate) and psilate morphology; D:pollen of Panicum virgatum (switchgrass),showing the monoporate (single pore)morphology of grasses; E: pollen ofHelianthus annulus represents thetricolporate (thee colpi and three pores)pollen types with echinate (spine)morphology (Aster-type); D: pollen of Acerrubrum (red maple) with tricolpate (3 colpi)and striate morphology.References:Faegri, K. & J. Iversen, 1989. Textbook of Pollen Analysis

(4th ed.). Wiley, Chichester, 328 pp.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, TheNetherlands.

Figure 7. Sediment samples after the HCl treatment. A: pollen of Pinus; B:pollen of Quercus

Figure 6. Sediment sample after the HCl treatment. A: pollen of Tsuga. B:Pollen of Alnus incana-type (Speckledalder)

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Figure 4. Sediment samples after theKOH treatment. A: Pollen of Tilia(basswood). B: Pinus bladder. C: Quercus.

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