Cellsens v3

We observed the growth of 26 isolates of S. schenckii on various media (SDA, potato dextrose agar [PDA], and BHIA) and at various temperatures (30, 35, 37, and 40 °C). The growth and transitions to the mycelial and yeast phases, influenced by the culture medium and temperature, were closely monitored under a stereomicroscope (SZ2-ILST, Olympus) for 14 days. The colony diameter was measured by calibrating the ocular micrometer on the imaging software (cellSens V3.2 software, Olympus). The microscopic features of the mold form were evaluated using the slide culture technique on PDA, incubated at 30 °C for 14 days. Morphologies of the yeast-like and yeast forms were obtained from culturing on BHIA at 30 and 35 °C for 14 days. The slides were examined under a microscope (BX43, Olympus), and the cells were measured by a calibrated ocular micrometer on the imaging software (cellSens V3.2, Olympus). The percent growth inhibition (% GI) was calculated using the following formula: [(average colony diameter at low temperature-average colony diameter at high temperature)/average colony diameter at low temperature] × 100. Isolates exhibiting more than 50% GI were classified as having low thermotolerance, whereas those showing less than 50% GI were classified as having high thermotolerance [12 (link)].

Cells were grown to mid-exponential phase at 30 °C in YPD and pelleted by centrifugation for 1 min at 4000 rpm (1500 × g) then resuspended in PBS. 5 μL cell suspension was combined with 1 μL of 0.1 g/L Calcofluor White stain and applied to a glass microscope slide. Alternatively, overnight cultures were prepared for each isolate and wild-type strain in yeast extract peptone dextrose (YPD) at 30 °C, with rotation, and then subcultured to mid-log phase before washing twice in 1× PBS and staining with FM4−64 (BioTracker 640 Red C2(FM4−64) Synaptic Dye, Millipore Sigma) at 10 µM for 10 min. Cells were visualized using an Olympus IX70 Epifluorescent Microscope fitted with a Hamamatsu C11440 camera and taken with Olympus CellSens v. 3.2 software.

Three randomly selected, genetically characterized isolates of lineages A and B, and the single isolate of lineage C, were subjected to morphological analysis. Stool specimens were concentrated using formalin–ethyl acetate sedimentation and wet mounts were examined under differential interference contrast (DIC) and ultraviolet (UV) fluorescence. Length and width of a minimum of 20 oocysts per sample were measured via Olympus cellSens V3.2 software and an Olympus BX51 compound microscope by the same operator. Minimum and maximum dimensions from each lineage were compared using unpaired 2-tailed t-tests. The appearance of unsporulated oocysts from each lineage was also examined in modified acid-fast (Kinyoun) stained smears (1000×) and/or under a UV fluorescence microscope (500×).