Pelco easiglow apparatus

Cryo-TEM images were taken on a FEI Tecnai G2 Twin TEM (FEI, Hillsboro OR, USA) at a voltage of 80 kV. Prior to imaging the samples were prepared as follows: Lacey holey carbon grids (Ted Pella, Redding CA, USA) were glow discharged in a PELCO EasiGlow apparatus (Ted Pella, Redding CA, USA) and loaded into a Vitrobot Mark IV vitrification instrument (FEI, Hillsboro OR, USA). Subsequently, 3 μL of sample were carefully deposited onto the grid, blotted for 3 s at a force of −3 and with a drain time of 1 s and then vitrified in liquid ethane. The grids were transferred onto a Gatan 626 cryoholder (Gatan, Pleasanton CA, USA) which was inserted into the TEM instrument.

To identify potential differences in flagellation of P. alvei CCM 2051^T wild-type and the two glycosyltransferase mutants, cells were grown overnight in LB medium. In addition, the capability of purified recombinant, non-glycosylated Hag from E. coli (rHag_{E. coli}) to self-assemble in MQ-water into flagellar filaments was analyzed by transmission electron microscopy (TEM) and compared with the native flagella isolated from P. alvei wild-type cells.
Thirty microliters of these bacterial suspensions were applied to Formvar- and carbon-coated 300-mesh copper grids (Agar Scientific) that were rendered hydrophilic upon glow discharge using a Pelco easiGlow apparatus (Ted Pella). The grids were incubated for 10 min face down on the cell suspensions. Samples were fixed with 2.5% glutaraldehyde, washed three times with MQ-water, and stained with 1% uranyl acetate solution, pH 4.2, for 40 s (Messner et al. 1986 (link)). Samples were investigated using a Tecnai G² 20 Twin TEM (FEI), operating at 120 kV. Pictures were taken with an FEI Eagle 4 K CCD camera (4096 × 4096 pixels).