Préparation d'échantillon de bactéries pour TEM

Fix for minimum 4h at 4C with 2.5% glutaraldehyde in 0.1M cacodylate at pH 7.2
Wash 3x using cacodylate buffer and centrifuge at 2000g 5 min
Remove supernatant and immobilize with same amount low melting point agarose (3% final)
Cut in stick of 0.5 by 0.5 by 5 mm and wash 2x in cacodylate buffer
Post-fix in 1% OsO4 for 1.5h at RT
Wash 3x in cacodylate buffer
Dehydrate with grading ethanol (30%, 50%, 70%, 95%, 100%, 15 min each)
Wash 3x 15 min with 100% ethanol
Transfer and wash 2x in 100% propylene oxide
Transfer to EPON-812¹⁾ (without hardner) 50% in propylene oxide and incubate 16h in a rotary mixer
Remove caps and evaporate propylene oxide for 4h
Replace with EPON-812 and incubate for 16h in a rotary mixer
Transfer to freshly mixed EPON and hardner (BDMA or DMP-30) and incubate for 2.5h
Include in silicone mold and incubate 1 day at 37C and 3 days at 60C
Cut sections with Reichert-Jung Ultracut E Ultramicrotome using a 35° Diatome diamond blade
Sections are stained 5 min in 2% uranyle acetate and 3 min in 3% lead citrate using NaOH pellets for scrubbing CO2
Grids are imaged on a JEOL JEM-2100plus

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