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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 CCD camera (software EMMENU 4; TVIPS GmbH, Gauting, Germany)

For more details see:
Klingl A, Moissl-Eichinger C, Wanner G, Zweck J, Huber H, Thomm M, Rachel R. Analysis of the surface proteins of Acidithiobacillus ferrooxidans strain SP5/1 and the new, pyrite-oxi­dizing Acidithiobacillus isolate HV2/2, and their possible involvement in pyrite oxidation. Arch Microbiol. 2011 Dec;193(12):867-82. doi: 10.1007/s00203-011-0720-y

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Fig. 1: Ultrathin sections of Epon-embedded cells, grown on pyrite crystallites. Freeze substitution was performed with ethanol containing glutaraldehyde, formaldehyde and uranyl acetate. (A) Acidithiobacillus sp. HV2/2. (B) Higher magnification of the selected area in (A). Py: pyrite , C: cytoplasm; CM: cytoplasmic membrane; PP: periplasm with peptidoglycan; OM: outer membrane; SL: S-layer; arrow heads: pili. .Bars: 200 nm (A) and 100 nm (B).