Biotwin spirit

For electron microscopy specimen preparation, 80%–90% confluent Vero E6 monolayers in 25 cm² flasks were inoculated with stock virus and monitored for CPE. At the first sign of CPE, culture supernatant was collected, cleared of cellular content by centrifugation (3000× g for 5 min), and subsequently fixed in an equal volume of 2.5% glutaraldehyde in 0.1 M Hepes buffer (pH 6.9) for visualization of virus particles by negative staining. A Beckman Airfuge^® (Beckman Coulter, Brea, CA, USA) was used to concentrate all samples (10 min at 207 kPa), after which droplets of sample were adsorbed to 0.25% formar-coated copper grids for a minimum of 10 min, rinsed twice in deionised, distilled water and stained briefly in 2% phosphotungstic acid (pH 6.9). For ultramicrotomy, the remaining infected monolayers were flooded with the same fixative overnight, then routinely processed (postfixation in 1% buffered osmium tetroxide, graded ethanol dehydration, infiltration with a low viscosity resin (Agar Scientific, Stansted, UK) and overnight polymerisation at 70 °C). Seventy nm sections were cut on a Leica EM-UC6, double stained with saturated uranyl acetate and lead citrate, and viewed at 80 kV on a BioTwin Spirit (FEI Company, Hillsboro, OR, USA). Imaging was done with an Olympus Quemesa CCD camera (Olympus, Tokyo, Japan).

The South African isolates were visualized using a previously described TEM method that was adapted for Gram-negative bacteria by substituting ruthenium red with a 0.5% alcian blue pyridine variant (pH 7.2) [51 (link)]. Ultrathin sections were viewed using a 120 kV BioTwin Spirit transmission electron microscope (FEI Company, Oregon, USA). American Type Culture Collection (ATCC®) isolate M-603, a N. meningitidis serogroup W strain (ATCC-35559™) was used as an encapsulated control. NCDC A7515, a N. lactamica strain (ATCC-23970™) was used as an unencapsulated control.