Transmission Electron Microscopy of M. bovis

Approximately, 1 × 10⁸ CFUs of M. bovis in 100 μl of PBS was transferred to a non-tissue culture treated flat-bottom 96-well plate and incubated with 30 μM of NK-lysin peptides or PBS at 37°C in a humidified atmosphere of 5% CO₂ for 60 min. The bacterial suspension was mixed with an equal volume of 3% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4 and processed for transmission electron microscopy as described previously [17 (link), 28 (link)]. Briefly, after fixation, bacterial pellets were rinsed in cacodylate buffer, post-fixed in 1% osmium tetroxide, dehydrated in alcohols, and embedded in epoxy resin. Ultrathin sections of the bacterial pellets were cut and stained with uranyl acetate and lead citrate. Sections were examined with a FEI Tecnai G2 Biotwin (FEI Co., Hillboro, OR) transmission electron microscope and images were taken with Advanced Microscopy Technologies (AMT Inc., Danvers, MN) imaging camera.