Beekeeping - Pollinic analysis

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Pollinic analysis

by Paul SCHWEITZER

with the kind authorization of

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Collecting samples as references

Pollinic analysis requires two stages. The first is to identify the pollen grains that are under observation. The second to count them.

Identification can only be done by comparing the morphology and size of the observed pollen grains with identified grains of pollen which serve as a reference. These can be microphotographs, either hard copy or digital: they will make up database which can be consulted to make comparisons. Photographs can only be used if they are of good quality and taken from several angles to ensure a complete view of the pollen. Unfortunately, many documents are of poor quality and therefore of little use. A word of advice: never use drawings from old-fashioned books on beekeeping as a reference point. They lack in precision and often contain flagrant mistakes.

No photographic document, however good it may be, can ever replace the real thing. Melissopalynologists have to put together their own pollen collection from pollen gathered on plants and then put between slides. A lengthy undertaking for sure. In fact, one that never ends. It does however present numerous advantages: a pollen collection allows for precision in identifying grains and furthermore during the field work required to collect pollen specimens, nectar producing flowers can be identified in their natural environment, which will at be the very origin of the pollen spectrum present in the subsequently produced honey.

Pollen analysis of honey is not simply scientific laboratory research, field work is necessary to provide a global view to ensure a true understanding of the nature of honey and the possibilities offered by pollinic analysis in research aimed at identifying the botanic and geographic origin of honey types.

To meet all these requirements, it is obvious that to undertake pollinic analysis, a good knowledge of botany and plant taxonomy is needed. It is indispensable that flowers be identified correctly. The best way to start is probably with the highest nectar producing plants, as beekeepers are usually fairly familiar with them. In any case, it is indispensable to start by referencing the pollens that are most frequently encountered in honey. Then, during forays to different areas, other species (considered less productive) can be gathered. By collecting the widest possible range of pollen as a reference, this exploration should lead to an inventory of plants listed under their different plant groups.

When making reference plates of flowers, the flowers should be picked just before the buds open. It is better to let the flowers open in the laboratory to avoid contamination by wind-borne pollen (frequent at certain times of the year) or even by insect-borne pollen.

The plant should be identified: family, genus, species. The date and location where the flowers were picked should also be recorded.

Preparation of reference slides

Palynology is above all the study of fossilized pollen. The outer membrane of the pollen spore, the extine, is made up of an extremely resistant substance which is capable of fossilization. The study of fossilized pollen sheds light on the relation between plant groups as well as climatic changes through time. As palynologists only have the extine for identification purposes, their reference pollen spores are prepared by using the extine. The spore case is emptied by a method known as acetolyzation. The method was perfected by ERDTMAN in 1960. It is a delicate operation because it requires the use of highly corrosive chemical products (concentrated anhydrous and acetic sulfuric acid) as well as a centrifuge. This technique gives a high definition to all the details and patterns on the extine.

Pollinic analysis can be done without acetolyzation, with pollen that has been cleaned of its fat bodies. The patterns on the extine are not as clear but despite this most pollen can still be identified. This method saves time and is much simpler. However, the pollen must be cleaned. Some pollen grains contain numerous globules of yellow liquid which can obstruct the view if they are not removed.

Ether or chloroform can be used for this operation. Place the ripe anthers either on a slide or in a watch-glass. Pour a few drops of chloroform on them, this will free the pollen grains from the anthers. Discard the rest of the anthers once the solvent has evaporated.

If using a watch-glass the residual pollen is taken up by glycerin gelatin, melted beforehand in a Bain Marie (not over 40°C). The pollen is placed in the middle of a glass microscope slide which is then carefully covered with a second slide, making sure no air is left between the slides. Once cooled the gelatin solidifies. The preparation can then be sealed with luting varnish and labeled This is done by covering the edges of the slide with varnish to protect the contents from air. There are a number of different varnishes (Canadian Balm is an example). Otherwise, nail varnish will do the job. You can also use paraffin as a seal. The label should mention:

The two systems can also be combined: classification by family first, then by size and morphology.

The pollen is prepared without coloring agents, therefore it will show its true aspect exactly as it would be observed in honey. It is indispensable to prepare each pollen grain in this way. Pollen grains prepared like this are usually turgescent as in honey. However, they will not show all their morphological characteristics. As a result you may wish to color them. For this purpose, the glycerin gelatin should be colored with basic fuchsine. This is a fairly delicate operation as all the pollen grains do not take color in the same way. By trial and error, you have to find the right intensity for each grain. A range of dye can be obtained by adding from 0.2 to 0.5 ml of fuchine alcohol solution to 10 ml of liquid glycerin gelatin.

The prepared reference slides will alter over time. In all prepared slides, an increase in volume and dimension of the pollen grains can be observed. Despite this, old slides are not without value as they sometimes show certain details very clearly. However, they cannot be used as a reference for size. Therefore, the collection, needs to updated with new slides every few years. In hot climates, the collection should be kept in cool storage to prevent the gelatin from melting.

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	slides (for microscope)
	slide covers
	watch-glasses
	small dissection equipment (needles, scissors)
	labels
	box for storing the slides
	small Bain Marie
	ether or chloroform
	glycerin gelatin
	luting varnish
	and perhaps coloring agents such as basic fuchsine in a 0.1% alcohol solution

	the simplest consists of the family name: cruciferae, leguminosae, labiatae etc.
	a more complicated, longer but more practical way uses a classification by size and morphological characteristics of the pollen grains.