Cell Envelope Structure of a Gram-negative Thermotolerant y-Proteobacterium Acidithiobacillus sp., Strain HV2/2 and its Interaction with Pyrite
Application Note for Leica EM AFS2 - Life Science Research
Sample Preparation:Cells of the the moderately thermophilic Acidithiobacillus sp.strain HV2/2, were centrifuged at 20,0009xg and then cryo-immobilized by high-pressure freezing on gold carriers (Leica EM PACT2, Leica Microsystems GmbH, Vienna, Austria). Freeze substitution of the samples was performed in the automatic freeze substitution unit Leica EM AFS2 (Leica Microsystems GmbH, Vienna, Austria) according to standard protocols (9 h at -90°C; 8 h at -60°C; 8 h at -30°C). Two substitution solutions were used: 94.6% ethanol plus 5.4% H2O, containing 0.5% glutardialdehyde, 1% formaldehyde, and 0.5% uranyl acetate, or acetone with 5% H2O, containing 2% OsO4 and 0.25% uranyl acetate. Finally, samples were washed twice in pure ice-cold acetone at 0°C, gradually embedded in Epon at room temperature and cured at 60°C for 72 h. Sections with a nominal thickness of about 50–70 nm were cut using an ultramicrotome (Leica EM UC6, Leica Microsystems GmbH, Vienna, Austria) with a diamond knife.
They were collected on uncoated copper grids (400 square mesh), and contrasted with 2% uranyl acetate and lead citrate. Transmission electron microscopy (TEM) was performed on a Philips CM12 (FEI Co., Eindhoven, NL) with a LaB6 cathode at 120 keV. The micrographs were recorded with a slow-scan