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Pollen Counting Methods
The pollen is collected with a rotorod contact sampler, located on the roof of Peter Engle science center. The sampler collects the pollen on two 1.52x1.52x32 mm pre-greased polystyrene rods. A thin coat of Silicone Grease captures the pollen and mold spores on the leading edge of the rod. When counting, a rod is placed in one of the deep grooves of a stage adapter. A few drops of Calberla's stain are applied and then the rod is covered with a standard 22x22 mm coverslip, with the edge of the coverslip about one mm from the distal end of the rod. Only the area of the rod under the coverslip is counted. When the rod has been counted, it is removed from the stage adapter with a pair of forcepts, dabbed dry with a kimwipe, and moved to permanent storage. The Stage adapter is rinsed off and dabbed dry with a kimwipe. All pollen counting is done with a light microscope under 400x magnification.
A counting reticle that has been calibrated with a stage micrometer is used when determining the number of pollen grains on the slide. The counting reticle is a glass disk that contains a grid and is inserted into the eyepiece of the microscope. The reticle is used to help measure pollen and spore size, to keep track of which pollen or spores have been counted and, in cases of high pollen concentration, to determine how much of the rod has been counted.
Statistical tests have determined that 400 pollen grains is a sufficient amount to attain an accurate pollen count. If there are less than 400 pollen grains on the rod, then the whole rod is counted. If there are more than 400 pollen grains on the rod, then only a percentage of the rod is counted. In this case, while counting the pollen, the number of "swipes" made are also recorded. A swipe is one full length of the rod, either long (horizontally) or short (vertically). For example, a short swipe would start at the top (or bottom) of the slide and go straight down (or up), counting all pollen that is either completely inside the reticle or touching the top or left edge of the reticle. A swipe is completed when the reticle reaches the bottom (or top) of the rod. Using the calibrated reticle size, the number of swipes, and the known total area of the rod, the percentage of the rod that was counted can be determined (Equation 1). If 100% of the rod was counted, 157.143 short swipes or 10.857 long swipes is entered into the database. This is done in order to give an exact value of 100% for the percentage of the rod counted.
After the raw number of pollen grains has been recorded, it is inserted into an equation that determines the amount of pollen grains per mm3. This equation also takes into account the duration of the sample, the volume of air sampled, the percentage of rod sampled and the duty cycle during the sample (Equation 2).
Equation 1:
| # of swipes x swipe area (mm2) 33.44 mm2 | x 100 | ||||||
| Swipe area - long = 3.08 mm2 = 0.14 mm (height of reticle) x 22 mm (width of coverslip) short = 0.2128 mm2 = 0.14 mm (width of reticle) x 1.52 mm (height of rod) | |||||||
| 33.44 mm2 = total area of rod = 22 mm (width of coverslip) x 1.52 mm (height of rod) | |||||||
| Equation 2: | |||||||
| # of grains counted 3.12 m3 | x | 1440 minutes Sample duration (minutes) | x | 10 % %Duty Cycle actually used | x | 100 % % of rod actually counted |
3.12 m3 = the calculated volume of air Rotorod Sampler measures in standard conditions (1440 minutes, 10% Duty Cycle and 100% of rod counted)