Procedures For Identification And Quantification Of Spore Traps

Reference data and relevant calculations are as follows:

1. Calibrate the measurements of different magnifications of all objective lenses with a stage micrometer and an ocular micrometer. Attach these measurements to the microscope base for future reference. The magnifications of the microscope should be calibrated at least yearly. The full length of the ocular micrometer under 40 x, 60 x, and 100 x lenses and the diameters of these lenses should be measured also. For instance, the Olympus BH2 microscope has a field of view diameter of 0.39 mm for the 40x objective lens and a 10x ocular lens, and the viewing area is 0.1195 mm2 at the 400x magnification.

2. Measure the adhesive band for the sample deposit for each sample type or obtain this information directly from the manufacturers. For example,

Burkard samplers collect particles on an adhesive band measuring 14 mm by 2 mm (28 mm2), Air-O-Cell, 1.055 mm by 14.4 mm (15.19 mm2), and Allergenco samplers, 14.5 mm by 1.1 mm (16 mm2), respectively.

3. Use the micrometer measurements and spore trace length to calculate the number of passes necessary to cover the whole trace and/or a portion of it (such as 25%), whether counting transverse or longitudinal passes (or traverses). Both the diameter of the lens and the field of view are used to calculate the number of fields needed to cover the whole trace or a portion of it, if using the random field method.

A number of methods are used to examine spore trap samples, such as random fields, transverse traverses (or passes), and longitudinal traverse methods (Fig. 4.2). These methods offer a systematic but random analysis. Traverse methods include two different approaches: using a whole entire field of view or using a micrometer to guide traverses and the counting process. The primary advantage of the micrometer method is the analysis of a narrower, but more manageable analytical area. The microscopist does not have to turn his/her eyes to scan the entire field of view and can more easily focus on the area within the micrometer. This method is less tiring and less stressful for the eyes.40 With a micrometer, it is easier to guide traverses and determine whether a spore on the edge or partially on the micrometer should be counted.

The trace area analyzed ranges from ^25% to 100% depending on the laboratory. There are no governmental, professional, or official requirements on the minimal area that should be analyzed to get a reliable result. For a normal spore load, 25% of the covering area is minimal. If unusually high spore loads (>800 spores/sample) with a relatively even spore distribution are present, the area analyzed might be reduced to less than 25%. Extreme caution should be taken when less than 25% of the trace is analyzed. Some labs examine and then analyze their samples with 100% coverage for large spores and only 25% for the remainder of the trace. This approach, however, is not random, and may indicate a bias toward larger spores.

Fig. 4.2. Fungal sample trace reading methods: (a) transverse traverse; (b) traverse; (c) random fields.

longitudinal

Kapyla and Penttinen statistically compared the three analytical methods (random field, transverse traverses, and longitudinal traverses) to determine the variations of counts and the sample size necessary to yield valid results.22 Their results showed that one or two longitudinal traverses may be unreliable because of uneven distribution of the fungal material on the tape sample of a Burkard sampler. Microscope fields or selected transverse traverses were recommended;22 however, they22 found that systematic traverses provided more effective results than did randomly spaced ones. Sterling et al. found that 12 transverse traverses usually yielded higher counts than did a single longitudinal traverse.24 More empirical studies are needed to improve the quality of the analysis and yield more reproducible results.

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