Uranyl acetate and lead citrate

Transmission electron microscopy was used to study the ultrastructural damage to M. tuberculosis caused by treatment with WBCATH. Briefly, bacilli (Mtb, H37Rv) were cultured in Middlebrook 7H9 broth (Difco Laboratories) supplemented with Middlebrook OADC enrichment media (BBL; BD, Franklin Lakes, NJ, USA) until the logarithmic phase was achieved. Then, viable bacilli (1 × 10⁷) were placed in the wells of 96-well plates and were exposed to WBCATH for 18 h at the MICs determined previously. Subsequently, fixation was performed with 1% glutaraldehyde dissolved in 0.1 M cacodylate buffer (pH 7); postfixed in 2% osmium tetraoxide for 4 h, and the fixed bacilli suspension was washed three times with cacodylate buffer, dehydrated with increasing concentrations of ethanol and gradually infiltrated with Epon resin (Pelco, Hawthorne, CA, USA). Thin sections were contrasted with uranyl acetate and lead citrate (Electron Microscopy Sciences, Fort Washington, PA, USA), and examined with an FEI Tecnai transmission electron microscope (Hillsboro, OR, USA).

Cells were fixed overnight in a 1:1 mixture of 5% glutaraldehyde (#16120, Electron Microscopy Sciences, Hatfield, PA) and PBS at 4°C. Following three washes with PBS, cells were treated with a 1:1 mixture of 2% osmium tetroxide (#19170, Electron Microscopy Sciences) and PBS for 2 h at 4°C. Samples were rinsed three times in deionized water then stored overnight at 4°C in uranyl acetate (#22400, Electron Microscopy Sciences) en-bloc stain. Following two washes with water, cells were dehydrated in a graded ethanol series, then infiltrated with Spurrs resin (#14300, Electron Microscopy Sciences) using acetone as the transitional solvent. Infiltrated samples were embedded in fresh resin which was then polymerized overnight at 70°C. Approximately 90 nm sections were cut with a diamond knife, collected on mesh grids, and post-stained with uranyl acetate and lead citrate (#17800, Electron Microscopy Sciences). Cells were photographed using a Hitachi H-7000 transmission electron microscope (Tokyo, Japan) operating at 75KeV. Images were digitized at 800 dpi and analyzed using Magnification Version 2 (Orbicule, Inc., Leuven, Belgium). The percentage of the cell cytoplasm occupied by organelles of the lysosomal system (primary lysosomes, secondary lysosomes, and lipid-laden membrane whorls) was determined for 11 cells of each treatment.

The scutellum of 2 pharate adults, 4 just emerged adults (2 females and 2 males), 4 three day old adults (2 females and 2 males) and 8 ten-day-old adults (4 females and 4 males) of B. oleae were dissected from anaesthetized insects and fixed for 3 h in 2.5% glutaraldehyde in cacodylate buffer (Electron Microscopy Sciences, Hatfield, England), pH 7.2. The fixed scutelli were repeatedly rinsed in sodium cacodylate buffer and post-fixed for 1 h at 4 °C in 1% osmium tetroxide in sodium cacodylate buffer (Electron Microscopy Sciences). The samples were then repeatedly washed in the same buffer, dehydrated in ascending ethanol concentrations and finally embedded in an Epon-Araldite resin mixture (Sigma-Aldrich). Afterwards, ultra-thin sections were cut using a Leica EM UC6 ultramicrotome (Leica Microsystem GmbH, Wetzlar, Germany), collected on Parlodion (Spi-Chem, West Chester, PA, USA) coated copper grids, stained with uranyl acetate and lead citrate (Electron Microscopy Sciences, Hatfield, England) and examined using a Technai G2 Spirit BioTwin Transmission Electron Microscope (FEI company, Hillsboro, OR, USA) equipped with a CCD camera Veleta (EMSIS) 2048 × 2040.