Transmission Electron Microscopy of Ehrlichia chaffeensis

Purified E. chaffeensis DCs and RCs were resuspended in PBS and used in transmission electron microscopy analysis by following the methods described previously^{12 (link)}. Briefly, all TEM preparation steps were followed by repelleting samples by centrifugation at 4 °C for 5 min at 200 × g, unless otherwise specified. Pelleted DCs and RCs (in 10 μl volume) were fixed with 0.5 ml of Karnovsky’s fixative containing 2% paraformaldehyde, 2.5% gluteraldehyde in 0.1 M cacodylate buffer (pH7.4) overnight at 4 °C. The cell-free E. chaffeensis organisms were then washed three times with 1 ml of 0.1 M cacodylate buffer, post-fixed in 2% osmium tetroxide in the same cacodylate buffer, washed, enblock stained in 1% aqueous uranyl acetate for 1 h, washed, dehydrated in a 50–100% ascending series of acetone, infiltrated and embedded in Spurr’s resin. Ultrathin sections (<95 nm thick silver to gold) were cut using ultramicrotome, examined with a CM 100 TEM (FEI Company, Hillsboro, OR, USA now Thermo Fisher Scientific), and images captured with a Hamamatsu C8484 digital camera using a AMT digital image capture system (Chazy, NY).