Glutaraldehyde solution grade 1

Cryptococci were washed three times in PBS (pH 7.3 ± 0.1) and fixed in 2.5% glutaraldehyde solution grade I (Electron Microscopy Sciences) in sodium cacodylate buffer 0.1 M (pH 7.2 ± 0.1) for 45 min at room temperature. Next, the cells were washed three times in 0.1 M sodium cacodylate buffer (pH 7.2 ± 0.1) containing 0.2 M sucrose and 2 mM MgCl₂ (Merck Millipore) and adhered to 12-mm diameter round glass coverslips (Paul Marienfeld GmbH and Co.) previously coated with 0.01% poly l-lysine (Sigma) for 20 min. Adhered cells were then gradually dehydrated in an ethanol growing series of 30%, 50%, and 70% for 5 min and 95% and 100% twice for 10 min (Merck Millipore). The coverslips were then critical-point-dried using an EM DPC 300 critical point drier (Leica) and mounted on specimen stubs using a conductive carbon adhesive (Pelco Tabs). Next, the samples were coated with a thin layer of gold or gold-palladium (10 to 15 nm) using the sputter method (Balzers Union). Finally, samples were visualized on a scanning electron microscope (Carl Zeiss Evo LS 10) operating at 10 kV with an average working distance of 10 mm and images were collected with their respective software packages.

In brief, C. neoformans cells grown for 7 days at 50 μg/mL were washed three times in PBS pH 7.4 and fixed in 2.5% glutaraldehyde solution grade I (Electron Microscopy Sciences, Hatfield, PA, USA) in sodium cacodylate buffer 0.1 M pH 7.2 for 1 h at room temperature. Then, the cells were washed three times in 0.1 M sodium cacodylate buffer pH 7.2 containing 0.2 M sucrose and 2 mM MgCl₂ (Merck Millipore Darmstadt, Germany), and adhered to 12 mm diameter round glass coverslips (Paul Marienfeld GmbH Co. KG, Germany) previously coated with 0.01% poly-L-lysine (Sigma-Aldrich, Darmstadt, Germany) for 20 min. Adhered cells were then gradually dehydrated in an ethanol (Merck Millipore, Darmstadt, Germany) series (30, 50, and, 70% for 5 min and 95 and 100% twice for 10 min). The coverslips were then critical-point-dried using an EM DPC 300 critical point drier (Leica, Germany) and mounted on specimen stubs using a conductive carbon adhesive (Pelco Tabs™, Stansted, Essex, UK). Next, the samples were coated with a thin layer of gold-palladium (10-15 nm) using the sputter method (Balzers Union FL−9496, Balzers, FL) (Carl Zeiss Evo LS or FEI Quanta 250), operating at 10–20 kV.