2-6 Sample analysis and counts.

Sample analysis is performed at a magnification of 40x10. Lower magnification would not enable identification of certain pollen types, while higher magnification would reduce the visual field. In principle, any microscope ensuring a good image and high resolution is suitable.

Figure 15: Microscopic reading of samples

Microscopic examination of aerobiological samples is essential for obtaining reliable results; it is also one of the most time-consuming stages in the process, due to the abundant material sometimes present on sample tapes.

As indicated earlier, the high-quality image obtained at the magnification recommended is ideal for identifying the various pollen types, largely distinguished by their external morphological features. The microscope should be well focussed, and a low-scatter white beam enables greater precision when identifying pollen types, thus minimising confusion of similar types.

Counting method:

Since it would take too long to count all the pollen-grains and spores on a slide, sub-sampling is essential in order to ensure the provision of timely information. The area selected should represent at least 10% of the whole slide (according to European Aeroallergen Network rules)

Figure 16: REA counting method: four continuous horizontal sweeps.

During these sweeps, the number of each pollen grain type identified is counted; this provides information on the pollen count throughout the day.

Figura 18: Disposición de reglilla de acetato bajo el portaobjetos correspondiente a una muestra diaria.

Pollen or spore counts for each hour can now be noted. The first hour is from 12.00a.m. to 13.00p.m. on a given day. The last hour is from 11.00a.m. to 11.59a.m. on the following day. The number of pollen grains per hour is noted on a data collection form, together with the sampling date and site. Each table represents one pollen or spore type, and comprises 4 horizontal rows, each containing 24 cells.

Figure 18: Pollen-type data collection form.

2.7 Expressing the results.

Pollen counts should be expressed as the daily mean count per cubic metre of air, thus ensuring standardisation of data from all sites. For this purpose, the number of pollen grains counted is multiplied by a factor that takes into account the volume of air sampled (10 litres/minute), and the size of the microscope field of vision used (magnification 40x10). These measurements are included in the REA protocol. However, as indicated earlier, this factor will vary depending on the brand of microscope used. 

- Calculating the correction factor:

The first step is to measure of the microscope field of vision at a magnification of 40x10.

Example: Let the diameter of the field of vision be 0.45 mm:

Air sampling rate: 10 l/min = 600 l/hour = 14400 l/day = 14.4 m3
Mean diameter of the microscope field of vision: 0.45 mm
Area of one horizontal sweep = 48 mm x 0.45 mm = 21.6 mm2
Surface analysed = 21.6 x 4 sweeps = 86.4 mm2
Total surface sampled = 48 mm length x 14 mm width = 672 mm2

Particle content per cubic metre of air = (672 mm2/86.4 mm2) x (1/14.4) x N
N = number of pollen grains in four sweeps.
Particle content per cubic metre of air = N x 0.54

2.8 Spanish Aerobiology Network database.

nce the total number of pollen grains per cubic metre of air has been ascertained for each identified and unidentified pollen type, for the whole 7-day week, these data are entered on standardised weekly data-recording sheets. This ensures availability on paper of a list of the minimum pollen types analysed. These weekly data sheets are then used as the basis for entering data into the computerised database.

CITY: ________________________ RESPONSIBLE _______________________________
PHONE Nº __________________  FAX: ___________________ E-MAIL: _______________
TYPE OF SAMPLER: _______________ DATE: From ____/____/200__ to ____/____/200__

Figure 19: Daily data form for each pollen type.

The database designed by the REA Coordinating Centre is accessible to the various Monitoring Units belonging to the REA. Although Spain has a national Pollen Data Bank, located at the IT department of the REA Coordinating Centre, it is advisable for each Aerobiology Unit to have its own local database, since the Unit is responsible for managing and updating information at local level. The availability of a set of historical records not only ensures thorough knowledge of the incidence of local pollen types; it also facilitates the interpretation of results and the preparation of pollen forecasts, since mean-count curves can be drawn up for each pollen type, enabling inter annual variations from the mean to be analysed.

Essentially, any computer software incorporating a database function (e.g. Excel, Access Microsoft Ò) can be used to create and maintain such a data storage system. The use of a statistical software package and of land-use maps is also recommended, as a basis for preliminary and advanced studies of airborne particle behaviour.

The spreadsheet of the database created by the Coordinating Centre is active in both Excel and Access, and contains a sheet for each day together with a list of potential pollen types. This application facilitates the entering of the day’s pollen-grain counts. Days without records due to sampler malfunction will be deleted before statistical analysis.

Figure 20: Pollen Data Acess Sheet.

Every week, each Regional Aerobiology Unit sends the latest update to the Coordinating Centre, by e-mail addressed to rea@uco.es. From there, data are automatically entered into the National Database, using a file created for each sampling site.

Figure 21: Introduction of pollen data in the application created.

Authorised staff at the REA Coordinating Centre are issued with passwords enabling them to enter data from the various sampling stations into the European Pollen Data Bank run by the European Aeroallergen Network (EAN http://www.univie.ac.at/ean/), based at the HNO-Klinik, University of Vienna, Austria. Information on the aerobiological situation in several European countries is updated weekly at www.polleninfo.org

A PC is therefore an essential tool for all Aerobiological Monitoring Units, in order to ensure the smooth operation of the Unit. The PC is a means of connecting the various Units and the Coordinating Centre, providing a fast channel for communicating data, news, general information and operating instructions for Units which have just joined. The Units use e-mail to send the weekly data obtained from aerobiological analysis to the Coordinating Centre, using the established format. The REA e-mail address is: rea@uco.es.

Bibliography:

1. Domínguez, E., C. Galán, F. Villamandos & F. Infante. 1992."Handling and evaluation of the data from the aerobiological sampling".  Monografías REA/EAN Nº 1. Editado por el Departamento de Biología Vegetal y Ecología, Universidad de Córdoba. (D.L.: CO-476-1992).
2. Hirst, J.M. 1952. “An automatic volumetric spore-trap”. Ann. Appl. Biol., 39:257-265