Z1 counter

To assess proliferation, cells were counted daily using a hemocytometer or Z1 Beckman Coulter counter before and after rapamycin induction. For the clonogenic survival assay, cells were induced for 72 h with rapamycin or DMSO, counted and diluted to 1.6 parasites mL⁻¹ in HOMEM with 20% heat-inactivated FCS and 100 nM rapamycin or DMSO. The cells were plated out in two to four 96-well flat bottom plates by adding 200 µL cells per well. Plates were sealed and incubated at 25 °C for 3–4 weeks. Surviving clones were counted by visual inspection of each well using a light microscope; any well containing live parasites was counted as a surviving clone. The percentage of surviving clones of the total cells plated is shown in graphs.

To test for ether resistance, cells were sporulated as described above, and sonicated briefly to break the clumps into separate cells. For each sample, 100 cells were counted for sporulation efficiency. Cell concentration was measured with a Z1 Beckman-Coulter Counter, and with this information, each culture was diluted so that the final concentration of asci was 5x10⁶ asci/ml, and 5-fold serial dilutions were spotted identically on two YPD plates. One plate served as a no-ether control, while the other was inverted over ether soaked filter paper (Whatman #3, 1003–090) for 1 hour. Plates were incubated at 30°C for 2–3 days and scanned.

T. thermophila strains relevant to this work are listed in Table 1. Unless otherwise stated, cells were cultured in SPP [2% proteose peptone, 0.2% dextrose, 0.1% yeast extract, 0.003% sequestrene (ferric ethylenediaminetetraacetic acid)] and starved in 10 mM Tris buffer, pH 7.4, both at 30° while shaking at 99 rpm. PP medium (2% proteose peptone, 0.003% sequestrene), for cells grown in 96-well plates, also included 250 μg/ml penicillin G, 250 μg/ml streptomycin sulfate, and 0.25 μg/ml amphotericin B fungizone (Gibco). For most uses, cells were grown overnight to medium density (1.5–3 × 10⁵ cells/ml) in a volume of SPP equal to one-fifth of the nominal culture flask volume. Cell densities were determined using a Z1 Beckman Coulter Counter. Cells in 96-well or drop plates, including cells under drug selection, were grown for 3 d at 30° in moisture chambers. Scoring and screening of cells was done on an inverted microscope at 100× magnification.

Cells were seeded in 25 cm² flasks at a density of 4 × 10⁵ cells/flask 1 day before the experiment and then treated for 15, 24 or 48 h. At the end of treatment, an aliquot of each sample was collected to count the number of cells through a Z1 Counter (Beckman Coulter). The remaining cell suspension was centrifuged, incubated in a 3:1 distilled water/medium mixture for 5 min and fixed in a 3:1 methanol/acetic acid mixture. Finally, cells were dropped on slides and stained with the conventional Giemsa method. For each experimental point, 1000 cells were analysed to count the number of mitoses. At least 200 mitoses were analysed to identify the different mitotic figures.