Bx51 camera

Intracellular parasite loads were analyzed as previously described [19 (link)]. Briefly, cells were infected and treated with nucleotides and were fixed onto slides, stained using a panoptic stain (Laborclin®, PR, Brazil), and counted using a Primo Star light microscope (Zeiss, Germany), with a 40x objective (100x for representative pictures). Images were acquired using a Bx51 camera (Olympus, Tokyo Japan) operated using the Cell^F software. We calculated the “infection index,” representing the overall infection load, based on the count of about 100 cells in a total of five fields to obtain the number of infected macrophages and the average number of parasites per macrophage. Individual amastigotes were clearly visible in the cytoplasm of infected macrophages. The results were expressed as the infection index II = (%infected macrophages) × (amastigotes/infected macrophage)/100.

Trichophyton tonsurans strain RCPFF 214/898, isolated from a human patient with onychomycosis, was obtained from the Russian Collection of Pathogenic Fungi, St. Petersburg (Russia). Identity was confirmed by sequencing the rDNA ITS locus. The strain was grown on solid CzA at 28ºС and investigated after 5, 10, 20 and 30 days of cultivation. Colonies were photographed with an Olympus BX 51 camera. For scanning electron microscopy (SEM), small parts of fungal colonies were fixed in 3% glutaraldehyde (in cacodylate buffer, pH 7.2) for 3 h, post-fixed overnight in 1% osmium tetroxide in the same buffer, dehydrated by ethanol series (30%→50%→70%), critical-point dried (HCP-2) for 15 min, coated with gold and observed in a JSM 35 scanning electron microscope (Jeol, Tokyo, Japan).
For transmission electron microscopy (TEM), blocks of nutrient medium with parts of fungal colonies were fixed during the 3 h in 3% glutaraldehyde and post-fixed for 10 h in 1% osmium tetroxide. Subsequently, samples were dehydrated through an ethanol and acetone series and embedded in epon-araldite epoxy resin. Ultrathin sections were cut with an Ultratome 2088 (LKB, Bromma, Sweden), stained with uranyl acetate and lead citrate and were investigated under a TEM Jem 100 SX (Jeol).

The intracellular parasite load was analyzed 24 h after nucleotide treatment, by light microscopy. Cells were plated onto 24-well glass coverslips and treated as described above. After 24 h of incubation with nucleotides, cell supernatants were discarded and samples were fixed with 4% paraformaldehyde for 10 min and then stained with panoptic stain (Laborclin^®, PR, Brazil), according to the manufacturer’s instructions. Slides were examined in a Primo Star light microscope (Zeiss, Germany), using a 100× oil-immersion objective. Images were acquired using a Bx51 camera (Olympus, Tokyo Japan) operated by the Cell^F software and were used to estimate the “infection index,” which represents the overall infection load. The “infection index” was calculated using following formula:
$\%\text{ Infection = }\frac{\text{(total number of infected cells }\times \text{ 100)}}{\text{Total number of cells}}$