Techniques for the Extraction of Pollen Grains

The following points highlight the two laboratory techniques for the extraction of pollen grains. The techniques are: 1. Pollen/Spore Extraction Pro­cedure 2. Absolute Pollen Counts.

1. Pollen/Spore Extraction Pro­cedure:

It is not possible to use the entire core systematically for pollen extraction, hence samples are taken at selected depth intervals along the core. The selection of depth intervals is entirely based on the several factors such as length of core, rate of sedimentation, frequency of pollen assemblage and the dating of the samples.

Firstly the core surfaces are cleaned properly, then the samples are taken by cutting the core in a regular intervals. Each sample is then treated chemically to retrieve pollen assemblage.

The most essential prerequisite to study fossil spores and pollen grains of Quaternary deposits is a sound knowledge of the regional vegetation and flora and a well-stocked pollen Herbarium. The pollen/spores slides of the extant flora are prepared from a reference collection to facilitate identification of the dispersed fossil grains. Single grain preparations are also made for ready reference of index pollen types and to identify the fossil grains.

i. Permanent Reference Slide Preparation:

Both Herbarium and fresh materials are acetolysed and a part of the material are chlorinated after acetolysis.

The Acetolysis method as suggested by Erdtman (1960) briefly is as follows:

Dry polliniferous materials from local Herbarium specimen are carefully crushed on a finely-meshed brass sieve (0.11 sq.mm.) resting on a funnel set on hard glass centrifuge tube. After each treatment the brass sieve is exposed to a flame until it becomes red hot in order to avoid sample to sample contamination. Acetolysis mixture is prepared in a measuring cylinder by slowly adding one volume of concentrated sulphuric acid to nine volume of acetic acid anhydride.

Then 10 ml of acetolysis mixture is added drop by drop to test tubes containing powdered material and stir carefully with a glass rod. After thoroughly mixing, the mixture is heated to boiling point (100°C) in a water bath for 2-3 minutes until it becomes slightly brown in colour. The mixture is allowed to stand for a few minutes, then centrifuge and finally decant. After adding a little distilled water, the sediment is shaken thoroughly, again centrifuge and decant.

Process is repeated for two to three times. Then the foam of the mixture is removed by adding a few drop of alcohol (95%) or acetone and the mixture is then sieved twice through a finely meshed brass net to eliminate foreign particles and again centrifuge. After decanting, distilled water is added to the sediment. Half of the mixture is kept for chlorination and 50% glycerine is added to the other half.

To effect chlorination 5 ml of glacial acetic acid, a few drops of concentrated sodium chlorate solution and few drops of concentrated hydrochloric acid are added and the mixture is stirred by a glass rod. The mixture is then centrifuged, decanted and washed thoroughly to remove all traces of chlorine in it. After centrifuging and decanting, 50% glycerine is added to it.

Then the two parts (non-chlorinated and chlorinated) are mixed, centrifuged and decanted. Then the tubes containing acetolysed pollen/spore materials are kept in inverted condition on filter paper for a few hours in order to dry the material. Chlorination is avoided in case of thin-walled pollen grains.

For preparation of permanent slides, a minute piece of glycerine jelly is taken on a clean platinum needle (sterilizing after each treatment burning in a flame), touch carefully to the bottom of the centrifuge tube (where the pollen/ spores are precipitated) and transfer on a clean slide.

Then the slide is heated gently and after that the jelly spread evenly by the platinum needle. A round cover glass is placed on the material and sealed off with chips of paraffin (melting point 60°C -62°C). In case of air-bubble encroachment within the paraffin, the slides are kept in an incubator (temperature not more than 60°C) for a few hours to get rid of the air-bubbles.

For fresh materials, the flowers or anthers containing pollen are fixed in glacial acetic acid, then transfer directly to the brass sieve, crush and wash down to the centrifuge tube with 70 per cent alcohol.

The mixture is then centrifuged, decanted and acetolysis mixture is added to the residue and the remaining procedure is adopted as in case of Herbarium material. The delicate pollen grains (e.g., Cannaceae) which do not survive the acetolysis procedure, are refrained from acetolysis procedure. Such grains are placed in 70% alcohol, stain with safranine and mount in glycerine jelly.

ii. Preparation of Fossil Samples:

The peat or soil samples are prepared by conventional acetolysis method as was proposed by Erdtman (1960). The 1 to 10 gm samples are first boiled with 10% KOH to get rid of the humic substances and also to allow disintegration of the compactness. The boiled samples are sieved through the finely meshed brass screen resting on a funnel to avoid the unwanted materials and finally taken in a centrifuge tube.

Each sieving sample is centrifuged with distilled water and decanted. The process is repeated five to six times. After decanting the samples are transferred to a polythene centrifuge tube and treated with 40 per cent hydrofluoric acid for overnight or more as are required to facilitate digestion of siliceous matters as most of the samples contain a good amount of sand particles.

After digestion of the siliceous matters, the sample is centrifuged for three to four times and decanted. The test tubes(glass) containing precipitates are treated with 10 ml of acetolysis mixture and stirred thoroughly with glass rod. The rest of the procedure is adopted as in the case of Herbarium material. Finally, treated samples containing pollen/spores are kept in 50% glycerine in small vials for future reference.

iii. Single Grain Preparation:

Single grain preparation is made to facilitate identification of the fossil pollen grains of unknown types under oil immersion and photomicrography for documentation.

The slide containing the specific grain is placed under low power microscope and then the object is brought at the center of the focus. A fine needle is used with a minute cube of glycerine jelly (not liquid) at the tip of the needle and after ‘fishing’ the grain it is transferred on another clean slide under low microscopic control. Then the slide is heated gently on a microflame. A circular coverglass is placed on it, slightly heated and finally sealed with paraffin in the usual method.

Care should be taken so that the grains do not shrink or crumple while being transferred. The glycerine jelly should be soft and the process has to be carried deftly with certain acquired skill.

iv. Preparation of Glycerine Jelly:

Gelatin 50 gm, glycerine 150 ml, distilled water 175 ml, phenol crystals 7 gm are the requisite for the preparation of the jelly. The first three constituents are mixed thoroughly and boil in a water bath for one to two hours. The phenol crystals should be added and mixed thoroughly. While still warm and molten, the glycerine jelly should be poured on a petridish making a thin, uniform layer of about 0.5 cm thick. It is then cooled and preserved in refrigerator.

2. Absolute Pollen Counts:

The techniques of pollen extraction as described earlier are used for determination of relative frequency of pollen grains only. But that data cannot provide us the density of pollen types with the deposits.

The rate of sedimentation of a deposit can be known by making radiocarbon dating of several sediments. This is complemented with the introduction of absolute pollen counting method to determine the pollen density of a particular sample.

The different methods are proposed for pollen density counts:

(a) Volumetric method:

In this method the extracted sample of known volume is dehydrated in benzene or tertiary butyl alcohol and is suspended thoroughly in a known volume of liquid. A known volume of a aliquot is taken by a pipette and mounted in silica oil. The quantities of materials used in the preparations are adjusted to produce a convenient concentration of pollen on the final slides so that all the pollen grains are counted.

(b) Weighing method:

A known weight of sample is prepared which is thoroughly suspended in a known weight of glycerol. A weighted subsample of the suspension is mounted in slide and all pollen grains are counted. So the concentration of pollen in terms of the weight of the original sediment can be determined.

(c) Exotic marker grain method:

This method was first employed by Benninghoff (1962) and subsequently modified by several other authors. In this technique a suspension of known concentration of an exotic pollen/spore type (Lycopodium is conventionally used), other than that naturally occur in the sediment, homogenously disperses in the suspension medium (e.g., glycerol) and the concentration of pollen is determined accurately.

The final pellet obtained from the processed sediment samples is weighed and a weighed quantity of exotic suspension is added. The precise quantity of pollen types is determined by the consideration that the final concentration of exotic pollen as a proportion of total pollen should be 20-40%.

The concentration of each pollen type relative to the exotic concentration is calculated from the sample counts. From those data one can calculate back to the original concentration of pollen types in the sediment.

The advantage of this technique is that it is not necessary to count all pollen grains on a slide. Several modified system of this method are described by different scientists. Sometimes it is possible to assess the proportional relationship between the locally over-represented type and a non-local pollen type by means of absolute pollen count.

This method is found to be very useful if the sedimentation rate is constant that can be determined by radiocarbon dating method. Here pollen counts are expressed as pollen influx which represents as numbers of grains of a species per square centimeter per year.

Pollen diagram Construction:

The pollen analytical technique has been applied to wider problems of vegetation history. The pollen count should be sufficient to give a good approximation of the relative abundance of the pollen types which are a greatest interest to the study. In this respect minimum of 200- 400 pollen sum may be adequate for such study and each pollen type could be expressed as a percentage of the total pollen.

The result may be reproduced in the form of a table but that is not sufficient to evaluate the vegetation and climatic changes of a particular area. So the results are represented in the form of a diagram. There are several ways to construct a diagram. It was Lennart von Post (1935) who first published his result in the form of a ‘Composite diagram’ and this has been universally accepted with slight modifications.

In general, the vertical axis of the diagram represents depth and the horizontal axis depicts the proportional abundance of the pollen types in which each sample is represented by a horizontal line at the corresponding level (Fig.13.2).

In the sample line, percentages are recorded and each species is indicated by means of conventional symbols which are now quite well standardized. Now each horizontal sample line with its symbols is called a Pollen spectrum, hence the diagram consists of a number of spectra from different levels. To demonstrate the change of vegetation the symbols for each species are connected from one spectrum to another by Pollen lines, thus form the curves of the diagram.

The area of the main diagram should always extend to 100 per cent and preferably comprise all the pollen types that form a part of basic sum. In some cases it is advised not to take all pollen types in one diagram which may create complicacy and also loose clarity and comparability with other diagrams.

So the entire diagram maybe divided into two diagrams namely, AP (Arboreal Pollen) diagram, including forest trees only, and NAP (Non-Arboreal Pollen) diagram depicts non-forested areas like heaths, steppes, meadows, cultivated lands, vegetation under forest canopy etc. NAP diagrams are represented in the form of histograms. More recently both AP and NAP diagrams are constructed in the form of histograms.

In general, the extreme left column of the composite pollen diagrams represent the lithostratigraphic units in sequential order (Fig.13.2). On its right is the column giving the dates of radiocarbon dating. On its further right vegetation/climatic zones are indicated based on the incidence of pollen grains. The total pollen sums are represented in the extreme right column of the diagram (Fig.13.2).