Understanding and mastering the techniques for studying various aspects of pollen, both modern and fossil, is very important (Faegri and Iversen 1964). The primary purpose of using various palynological techniques is to undertake microscopic examination of pollen and spores. Essential pollen morphological characters can be studied only if proper techniques are employed by the investigators.
In case of fossil pollen and spores, maximum caution has to be taken to recover them in an undamaged condition from sediments in which they are embedded. Specialized techniques used in the study of pollen trapped in honey samples and airborne pollen are described in Chapter 12 on Melissopalynology and Chapter 13 on Aerobiology respectively.
In this chapter, first the techniques used for modern pollen are described, followed by techniques used for microfossils including fossil pollen and spores.
A: TECHNIQUES OF PREPARING MODERN POLLEN AND SPORES FOR MICROSCOPIC EXAMINATION
Fresh pollen grains when observed under a light microscope appear as dense objects and reveal only the colour, size, shape and faint outline of sculpture patterns and pollen wall excrescences. In order to study them more closely under a light microscope to reveal other important diagnostic pollen morphological characters, it is necessary to make some special pollen preparations.
Fresh or preserved pollen/spore materials are used for preparing type slides which may be used for detailed pollen morphological studies or for comparisons with slide material of airborne pollen/spores to confirm or otherwise identification. Such collection of slides over time enables 'back tracking' of taxa of pollen and spores for research purposes. Appropriate stains acetocarmine or basic fuchsin for pollen, cotton blue/methylene blue for fungal spores are used.
Wodehouse (1935) had suggested treatment of fresh pollen grains with absolute alcohol to dissolve the waxy coating of the outer pollen wall and staining them with methyl green. This way only exine is stained while the intine and cell contents remain unstained. In order to get a better contrast, use of weak aqueous cosine or fuchsin was proposed. This method is in use for preparation of index (reference) fresh pollen slides useful for comparison and identification of airborne pollen, which are essentially untreated. A revolutionary method of pollen preparation namely acetolysis was developed by Gunnar Erdtman and his chemist brother in 1933.
Acetolysis involves acetylation of pollen grains during which the carbohydrate fraction of grains (protoplasmic contents) is removed or dissolved, thus leaving only the exine with its diagnostic features such as ornamentation, apertures and exine stratification. The process of acetolysis can be referred as acid hydrolysis wherein cellulose; a polysaccharide is removed effectively. The function of acetolysis is to dissolve cellulose, hemicellulose, and chitin. A secondary effect of the procedure is the darkening of the pollen grains, which allows one to more easily examine aspects of pollen wall layers, apertures and surface patterns. The assets of acetolysis of pollen grains include bringing transparency and expansion of pollen to facilitate examination of pollen morphological characters.
Acetolysis really consists of two processes: The initial acid hydrolysis reaction is carried out by concentrated sulphuric acid. The secondary esterification process is carried out by the acetic anhydride. Acetylation, on the other hand refers only to the second process of esterification, yet in today's literature it has become synonymous with acetolysis. In general, the effectiveness of the acetylation process is increased directly as the temperature and time is increased. Thus far, there is no evidence to suggest that it has any harmful effect upon sporopollenin even after long periods of duration.
Some of the sulphur ions attach to the pollen grains and thus tend to stain them brown. An oxidant will effectively remove excessive sulphur ions from the grains. The sulphuric acid acts first as the components for the acid hydrolysis step and then secondly to hold the ester components in solution. The acetic anhydride carries out the actual acetylation step by breaking the cellulose into esters and ester derivatives
Acetylation works much more rapidly since it attacks the OH (hydroxyl) group along the periphery of the cellulose molecule thereby breaking it down. Also, acetylation does not produce the water-soluble compounds found in the acid hydrolysis method but does produce compounds, which must be removed with glacial acetic acid. This is one reason why after acetylation is completed; the material is washed in glacial acetic acid.
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