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After the sampling period (usually one week, or more often during peak pollen periods), a new drum is assembled in the laboratory and transferred to the sampling site, where it is exchanged for the used drum. The used drum, with the sampling tape, is taken to the laboratory for analysis, using the drum-carrier for this purpose. There, the sample preparation process can start, taking great care to avoid contamination.
The recommended time for the changeover of sampling drums is 12.00 UTM. Since, as indicated below, the sample-mounting process used 24-hour stretches of tape, each sample runs covers 12 hours on one day (from 12.00 UTM to 23.59 UTM) and 12 hours on the next day (from 00.00 UTM to 11.59 UTM).
2-5 Sample preparation
At the Aerobiology Unit laboratories in the REA member Centres, the following items are laid out on a clean table duly prepared for the process:
Wad of blotting paper. To protect the sample mounting surface, absorb any liquids that might be spilt and increase the sample/surface contrast, a white background enhancing sample visibility.
Perspex mounting ruler. Included as an accessory with currently-available commercial samplers. The sampler drum rotates at 2 mm per hour; this transparent perspex ruler, which is over 1 cm thick, has notches every 48 mm. This enables the Melinex® tape, when placed over it (the ruler is wider than the tape) to be readily divided into 48 mm portions, each representing 24 hours’ continuous sampling.
Wad of blotting paper. To protect the sample mounting surface, absorb any liquids that might be spilt and increase the sample/surface contrast, a white background enhancing sample visibility.
Perspex mounting ruler. Included as an accessory with currently-available commercial samplers. The sampler drum rotates at 2 mm per hour; this transparent perspex ruler, which is over 1 cm thick, has notches every 48 mm. This enables the Melinex® tape, when placed over it (the ruler is wider than the tape) to be readily divided into 48 mm portions, each representing 24 hours’ continuous sampling.
Figure 11: Melinex tape over perspex ruler on which each day of the week is marked.
Microscope slide. Before cutting the Melinex® tape into 24-hour portions, place on blotting paper as many slides as there are 48-mm (24-hour) portions of the whole impacted tape (i.e. a maximum of 7).
Each slide is identified by means of a sticky label bearing the name or initials of the sampling station, and the date; since each 48-mm portion contains data for two incomplete natural days, the sample should bear the date of the first of the two days. By this means, a series of samples for successive days is generated.
The procedure used to obtain data for natural days is described in detail below. The portions obtained by cutting up the Melinex tape are placed on the slide, on which a few drops of water have previously been placed to facilitate adhesion. At this stage, it is essential to respect the successive order of dates, both when mounting and at the start and end of sample preparation.
Figure 12: Preparation for a one-day sample, with identification label.
The sample must always be placed on the slide with the start-time to the left and the end-time to the right. To identify these positions, the identification label will always be placed on the left. Microscope sample reading will always be from left to right, i.e. from the first of the two sampled days to the second.
Mounting daily samples. The sample mounting medium should meet the following requirements: 1. It should be water soluble; 2. It should be compatible with the adhesive used; 3. It should allow selective staining of the material of interest (optional); 4. It should allow long-term storage of the material.
The traditionally-used medium is fuchsin-stained glycerin gelatin, whose composition is as follows: 50 ml glycerin, 7 gr gelatin, 1 gr phenol and a small amount of basic fuchsin, diluted in 42 ml distilled water, mixed – using an electric shaker – in an extractor hood due to the toxic nature of phenol. The resulting mixture is pink in colour. This is the medium of choice, since it meets all the above requirements and is compatible with the adhesive used in the REA (silicone fluid). Use of basic fuchsin – a specific stain for plant material – facilitates pollen-grain identification and counting. All chemical products used in the mounting medium and in the silicone fluid should be stored in compliance with current regulations on Health and Safety at Work.
Glycerin gelatin is solid at room temperature, and needs to be liquefied before use. This is most swiftly done in a microwave oven, which takes only a few seconds.
Using a dropper, the liquefied medium is spread in an unbroken line on the slide cover, which is then placed on the slide containing the sample. The line should be continuous, and every effort should be made to avoid air bubbles, which would hinder identification and analysis. Any bubbles noted on the surface should be gently pushed to the edges, using a blunt instrument, before the gelatin resolidifies.
Figure 13: Application of glycerin gelatin to slide.
Sealing medium. Mounted samples should be sealed along the edges of the slide cover using a substance that will remain unaltered over time. Transparent nail varnish-lacquer is used for this purpose, since it is inexpensive, easy to obtain, easy to use, and of low toxicity; it dries rapidly, and is not impaired over time. Since it is transparent, it does not hinder sample identification. After microscopic examination (see below), sealed samples can be stored in a container known commercially as a Combi-box, designed specifically for optical microscopy samples.
Figure 14: Sample sealing. Alongside, collection of daily samples.
Prior to microscopy, mounted samples should be left for a while to allow the glycerin gelatin to solidify fully, and thus act as an adhesive between the slide and the slide cover. This interval also allows the various pollen grains to absorb the stain, thus enhancing their external morphological characteristics. |
