Collection and Sample Preparation Obtaining Results
		Methods of Capture

2.1 Type of sampler

Various monitoring methods, based on differing principles, have traditionally been used in aerobiological research.

Before selecting a method for capturing airborne biological particles, the aim(s) to be pursued must be clearly identified. These may include:

1. To obtain a continuous record, or to take short, intermittent samples
2. To obtain hourly, daily or weekly data
3. To make a count of the total number of airborne particles, or simply of viable airborne particles
4. To study either a particular taxonomic group or all airborne particles
5. To obtain either a value for pollen rain or a value for the number of particles per volume of air

From the outset, the over-riding aim of the REA has been to create a database of airborne pollen and spore counts by continuous recording, using a sampler that facilitates detection of these particles. 

As a standard, the REA uses volumetric suction samplers based on the impact principle (Hirst, 1952). These samplers enable standard data to be obtained regardless of the biogeographical and bioclimatic characteristics of the sampling area; they also allow hourly data to be recorded throughout the day. The suction flow rate is 10 litres air per minute, similar to the volume of air inhaled by the human lung.

This monitoring system is used by all the working groups in the various member countries of the European Aeroallergen Network (EAN), of which the REA forms part.

Hirst-type samplers offer a range of advantages: the robustness of the apparatus itself, which has to remain outdoors in adverse weather conditions; ease of use; efficiency; and minimum requirements – once in place, it requires only a permanent electricity socket and an anchoring system.

The two brands of Hirst-based equipment commercially available at present – the VPPS 2000 made by Lanzoni s.r.l., Italy, and the Burkard 7-day recorder spore-trap, by Burkard Manufacturing Co. Ltd., UK – can function continuously for one week without attention, providing daily and hourly records. Detailed technical specifications are provided with the equipment.

2.2 Volumetric suction sampler units

As indicated above, all REA sampling stations currently use the volumetric suction sampler based on the impact principle, as initially designed by Hirst (1952). This is an essential requisite of the REA working protocol (Domínguez et al., 1992). Although these samplers were specifically designed for the capture of fungal spores, later modifications have enabled the highly-efficient capture of all solid airborne particles, of both biological and non-biological origin, whose diameter ranges between 1 and 100 micrometers.

The sampler consists essentially of three components: an impact unit, a wind vane and a vacuum pump.

The impact unit comprises an entrance orifice measuring 14 x 2 mm, and a circular support (drum) to which particles adhere. The drum is driven by a clockwork mechanism, enabling it to rotate 2 mm every hour, thus ensuring continuous air sampling and the provision of both hourly and daily data.

A length of Melinex® tape, coated with an adhesive substance, is wound around the drum; particles sucked in from outside at a certain speed adhere to the tape, minimising as much as possible the rebound effect.

The vane, fixed to the outer metal casing of the impact unit, ensures that the entrance orifice is always positioned according to the prevailing wind direction, thus enhancing the efficiency of capture of particles borne on air currents..

The vacuum pump is fitted with a mechanism to regulate air input volume. Throughput for airborne particles is 10 litres/minute, similar to volume of air inhaled by human lung.

Figure 3: A. Hirst-type volumetric sampler (Hirst, 1952); B.  Impact Unit.

 2.3 Conditions for sampler positioning

In addition to the requirements outlined above, the installation of traps must comply with certain minimum requirements for aerobiological studies. The following regulations governing sampler positioning and installation have been adopted for all REA samplers:

1. The sampler should be placed on a readily-accessible, flat, horizontal surface.
2. Care should be taken to ensure that adjacent buildings do not screen the sampler or impede the flow of air. The sampler should be placed on the roof of a building; the height above ground level will depend on the city and on the height of neighbouring buildings.
3. The sampler itself should be elevated with respect to the installation surface, in order to avoid friction between air layers, which some studies have found to cause ground air turbulence. A tripod or small tower can be erected for this purpose.
4. Wherever possible, the sampler should not be placed in the vicinity of a fixed or mobile source of mass emission of biological or non-biological particles. The presence of single-species plant populations in the immediate surroundings of the sampler can lead to over-representation of a given pollen type, and thus to distorted data not representative of the species within the geographical range of the sampler. Proximity to non-biological particle sources may favour the massive presence of residues in samples, which will considerably hinder identification
5. The sampler should not be placed at the edge of a building, in order to avoid the turbulence generated by the impact of wind against the side of the building.

Once in position, the sampler should be securely anchored to the ground, since it may at times be exposed to very strong winds.

Once installed and anchored, the sampler is connected to a permanent electricity socket, since the suction mechanism requires a constant electricity supply. The connecting cable should be well insulated.

Figure 4: Sampler installed on the flat roof of the Educational Sciences Faculty, about 15 metres above ground level. Raised from the ground using a metal tower. University of Córdoba.

As soon as the apparatus is plugged into the socket, the suction pump will start to function, and the sound of aspiration will be heard.

The first step in preparing the sampler for operation takes place in the laboratory: a length of adhesive-coated Melinex tape is wound around the drum. The drum can function continuously for one week.

The adhesive used to trap the particles should meet the following requirement: 1. It must be water-insoluble;  2. It should not dry up or evaporate; 3. The thickness of the spread film should not change over time, and should not be affected by temperature or humidity; 4. It should be highly retentive, thus ensuring that impacted particles do not bounce off again; 5. It must not support the growth of fungi or bacteria; 6.  It should not be opaque under microscopy light; 6. It should be easy to use.

The REA working protocol uses LANZONI s.r.l. ® silicon fluid. This substance, a solution of pure silicone diluted in carbon tetrachloride has the advantage, amongst others things, that its physical properties remain unaltered over a range of temperatures from –20 to +150ºC, making it suitable for all the country’s bioclimatic zones.

 The adhesive is spread onto the Melinex tape using a soft brush whose diameter is similar to that of the tape. This operation should be performed in an extractor hood due to the volatile and toxic nature of the carbon tetrachloride.

Figure 5: Application of silicone fluid to the Melinex tape wound around the drum

Once the adhesive-coated has been applied to the tape, the drum is carried to the sampler, in a hermetically-sealed metal drum-carrier, to avoid potential contamination in transit. This also minimises the risk of the Melinex tape rubbing against anything else.

Prior to operating the apparatus, the vane should be secured to ensure problem-free set-up

Figure 6: Securing the vane.

With the head of the impact unit closed, the suction rate should be regulated to10 litres/minute, adjusting the flowmeter to the suction slit. Alternatively, the regulating nut (outside on the Lanzoni sampler, inside on the Burkard sampler) can be used for this purpose. The flow rate should be checked every week.

Figure 7: Checking the flow rate.

The clockwork mechanism connected to the impact unit should be wound manually once a week. This is done by turning the nut or key (depending on the model) anti-clockwise as far as it will go, without forcing it. A typical clock sound will be heard when it starts to work. The drum adjustment device is fixed to the clock by a nut. It is essential to place the drum in the position indicated as the start of sampling (start bands), since this marks the sequence of sample collection over the whole sampling period; the length of tape immediately after the start bands corresponds to the first day of sampling.

 

Figure 8 A: Correct positioning of the drum. B: Winding the clock on the impact unit.

Next, the head containing the impact unit is placed inside the metal casing using the guide-rail. It is hermetically sealed to prevent loss of vacuum and suction flow errors. At this point, the vane – previously secured by an anchoring screw – can be released.

Figure 9: Placing the head inside the metal casing of the impact unit.