Reynold s lead citrate

Mycobacteria from log phase were harvested and washed with 0.1 M phosphate buffer (PBS). Cells were subsequently fixed using 2.5% glutaraldehyde. Post fixation was carried out in 1% osmic acid. Following several rinses with ddH₂O, samples were dehydrated in a series of different concentrations of ethanol and 100% acetone. For transition solvent, resin: acetone (2:1) were used overnight. Epoxy resin-812 was used for 1 h for embedding. 90 nm sections were cut and stained with uranyl acetate and Reynold’s lead citrate (Ted Pella, Inc). After drying, transmission electron microscopic (TEM) images of the sections were taken using TEM-1400plus. The cell wall thickness was measured for each strain as follows: 100 mycobacteria in the visual field were randomly selected, the largest distance between the outer membrane and the inner membrane of each cell was measured and the data were statistically analyzed using One Way ANOVA with Bonferroni correction. For scanning electron microscopy (SEM), ethanol dehydrated samples were dried in freeze-drier and coated with 10 nm gold film using ion sputter. Scanning electron microscopic images were taken using HITACHI SU8010.

HeLa cells grown in T-25 mL flasks were infected with C. trachomatis L2 (Ct; MOI of 5) and treated with 5 μM iAkt from 2 h pi until its fixation at 24 h pi. Infected cell monolayers were fixed with 2% glutaraldehyde/PBS for 1 h at 37°C. Then, cells were removed with 1% gelatin/PBS, gently centrifuged (15 min at 1200 rpm) and washed three times with PBS. After that, cells were incubated with Osmium tetroxide/Potassium ferricyanide/PBS (1:1:1) for 90 min. Samples were dehydrated using increasing acetone series and embedded in Spurr’s resin (Ted Pella Inc., United States). Thin sections were cut with an ultramicrotome (Leica ultracut R, Austria) and stained with 1% uranyl acetate and Reynold’s lead citrate (Ted Pella Inc., United States) before they were observed with a Zeiss 900 electron microscope (Zeiss, Germany). Images were processed using Adobe Photoshop CS5 (Adobe Systems, Inc., San Jose, CA, United States).