Aluminum pin stubs

Two hundred microliters from each aliquot were filtrated onto a 25 mm polycarbonate filter (pore size: 0.45 μm, Merck KGaA, Darmstadt, Germany). Subsequently, the filters were air-dried under sterile conditions and then mounted onto 25 mm diameter aluminum pin stubs (Agar Scientific Ltd., Stansted Essex, United Kingdom) using double-sided carbon adhesive discs. The filter samples were coated with 5-6 nm platinum in a Cressington 200HR Sputter Coater (Cressington Scientific Instruments Ltd., Watford, UK). Spores of bacterial and fungal origin were identified based on morphological characteristics. Spores were counted in 100 randomly selected imaged fields at ×3,000 magnification and reported as the number of spores m⁻³ air. The number of spores was estimated by extrapolating the counts in the selected fields to the whole filter using the following formula: $Sporesper{m}^3=\frac{n\times \text{filtration}\text{filter}\text{area}\left(\mu m^2\right)\times \text{Dilution}\text{factor}}{\text{k}\times \text{FESEM}\text{image}\text{field}\text{area}\left(\mu m^2\right)\times \text{sampled}\text{air}\text{volume}\left(m^3\right)}$ where
n = number of particles counted on the filter; k = 100; filtration filter area of 25 mm filter = 227 × 10⁶ μm²; FESEM Image field area at 3,000 = 1064 μm². Dilution factor: 35 [14 (link)]. The lowest detectable number of particles (LOD) with FESEM was 3.73 × 10⁴ m⁻³ at a total air sampled volume of 1 m³.