Fei tecnai g2 biotwin transmission electron microscope

The three females sampled in the late season were used for nsEM analyses. The extracts were prepared and treated using the method commonly applied for honey bees⁶¹ . Each individual was placed in 2.4 ml 0.001 M potassium buffer (PB), pH 6.7, containing 0,2% sodium diethyldithiocarbamate (DIECA) to prevent melanization and then mechanically homogenized (Ultraturrax – Ika Werk, Staufen, Germany). The extracts were emulsified with 0.3 ml chloroform and 0.3 ml diethyl ether, and then cleared by low-speed centrifugation at 4500 g for 30 min. The supernatants were separated and again centrifuged at 9500 g for 30 min. Next, 100 μl of each supernatant was ultracentrifuged with an Airfuge (Beckman, Indianapolis, USA), operating at 21 psi 82000 g for 15 min, and fitted with an A100 rotor holding six 175 μl test tubes equipped with specific plastic adapters which permit to directly pelleting virions onto 3 mm carbon-coated Formvar copper grids. The grids were negatively stained with 2% sodium phosphotungstate (NaPT) at pH 6.8 for 90 seconds and observed with a FEI Tecnai G2 Biotwin transmission electron microscope (FEI Company, Hillsboro, USA) operating at 85 kV at 16500–43000 magnifications.