In The Laboratory

The Use of Lycopodium Spores as a Spike
It has been suggested that those investigators working in regions where Lycopodium forms a component of the local vegetation may wish to use an alternative spike e.g. Eucalyptus, though this is not available in convenient tablet form. The Lycopodium tablets contain the spores of Lycopodium clavatum. In the pollen preparations they are usually smaller and have a more crumpled appearance than fresh Lycopodium and are distinguishable on this basis, hence a different spike should not be necessary.

The Number of Lycopodium Spore Tablets Added to Each Trap Sample
The question that needs addressing is "What is a statistically sound number of Lycopodium spores in relation to the amount of pollen counted?" The statistical confidence with which pollen influx can be calculated (see calculating of results) increases with the number of Lycopodium spores counted. This means that the more tablets that are added to the trap contents the more accurate, statistically, the pollen influx calculations will be. However, using Mosimann's equations (Maher, 1972) it can be demonstrated that once the figure of 500 Lycopodium counted is reached there are only very small increases in the reliability of the count. Whereas a count of 500 Lycopodium will give a minimum error in the influx calculations, a count of less than 50 is largely meaningless, as the statistical error is so great. Obviously it is difficult to predict beforehand what the pollen content of a trap will be. Experience has shown, however, that in many areas it is very difficult to achieve the recommended 500 Lycopodium without adding vast quantities of tablets (up to 40!). Only in very sparsely vegetated parts of the north of Europe is the recommended sum achievable with a realistic number of tablets. Physical and economic restrictions are such that it is only practical to add up to a maximum of 5 tablets per trap. This is taken as a maximum requirement, with the proviso that pollen counting is continued until the total of Lycopodium spores counted exceeds 50.

Removal of Water From the Trap Sample
Three alternative methods have been employed which are presented here in their order of preference:

  1. Pass the whole sample through a paper filter using a vacuum pump and then acetolyse the filter paper in a small beaker using the normal acetolysis mixture (1 part conc. H2SO4 and 9 parts acetic anhydride, heated for 2 minutes in a water bath). For this the filter paper must be of a type which dissolves on acetolysis (see original guidelines). A potential problem arises if the sample contains a lot of particulate matter and the filter paper becomes clogged. In this case method (2) could be considered.

  2. Sieve using a 10 micron sieving cloth so that the pollen is retained on the sieve (sieving cloth can be supplied by Lockertex, P.O. Box 161, Church Street, Warrington, Cheshire WA1 2SU, UK). The sieving cloth can be made into a simple sieve using two pieces of plastic tubing (drainpipe!) which fit one inside the other. The cloth is spread over the smaller diameter tube, a rubber solution adhesive (of the sort used by plumbers) is then used to seal the joint between this and the larger diameter tube which is pressed over the smaller one, thus keeping the sieving cloth taut. This method is quicker than using a vacuum pump when the sample has a large amount of fine particulate matter. Experiments have shown (Tinsley, pers. com.) that with this mesh size of cloth there is no loss of pollen. This should be rechecked, however, when working in areas where extremely small pollen types are important.

  3. Centrifuge the whole sample using a centrifuge which holds 250 or 500 ml tubes and amalgamate the centrifugate. The disadvantage with this is the potential loss of any saccate grains which may float on the surface. This can be overcome if the supernatant is then filtered as in (1) above.
There is still the possibility of pollen floating on the surface of the liquid during later stages of centrifugation in connection with the laboratory preparation. At this stage the addition of alcohol releases the surface tension and helps the pollen to spin down.

Removal of Clay
There should really be NO clay in the pollen trap collections since the presence of such mineral material suggests that soil from the surroundings has been washed into the trap potentially bringing with it fossil pollen. It is important to prevent such soil entering the trap; a ring of tightly wedged stones around the top of the trap should help to stabilise the immediate ground surface. Some fine mineral particles may, however, be blown into the trap by wind and these will need to be removed in order to produce a good pollen preparation. Such fine particles can be removed by heavy liquid separation using cadmium or by the zinc chloride method (Bates et al., 1978). The cadmium method is outlined below:
Mix together:

Stir, allow to cool and then filter. The resulting solution must have a density of exactly 2.25
Dry the pollen sample (any water in the sample could change the density of the solution) and mix it with the prepared solution in the ratio 1:3, stirring carefully. Centrifuge at 2500 rotations/min for 16 minutes. The organic matter is concentrated on the top of the liquid while the mineral material falls to the bottom. The uppermost part of liquid (containing the pollen) can then be carefully decanted and washed with distilled water.

Laboratory Preparation of the Moss Samples
Although the moss polsters will not be able to provide pollen influx data, it is, nevertheless often useful to have an idea of the amount of pollen contained in such a sample, particularly in the case of the single 5 cm diameter sample which is comparable to the pollen trap in surface area. For this reason, Lycopodium tablets should be added at the very beginning of the preparation process. As with the trap collection, the tablets should be dissolved overnight in water before being added. To separate the pollen grains from the moss plants, the sample (be it the single 5cm diameter sample or the amalgamated 10 sub-samples) should be boiled for up to 10 minutes in 10% KOH or NaOH, stirring occasionally. It may be necessary to do this in a beaker. The sample should then be shaken thoroughly. The moss can be removed using a coarse kitchen sieve and washed using distilled water to ensure that all the pollen has been extracted. The moss can then be disposed of. The pollen is now suspended in the alkaline solution. If the sample still contains remnants of moss, it may be convenient to pass it through a finer sieve (c. 0.25 mm mesh). If there is a high concentration of pollen in the sample, a sub-sample of the solution can be extracted for preparation. The sample can be condensed by centrifuging the solution. The sample should be washed in distilled water and centrifuged again. It should then be prepared using the standard laboratory techniques described in the original guidelines, beginning with acetolysis.

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