Mcolorphast ph indicator strips

To study the effect of pH on cryptococcal growth, the yeast cells were grown in minimal medium (15 mM dextrose, 10 mM MgSO₄, 29.4 mM KH₂PO₄, 13 mM glycine, 3 μM thiamine-HCl) buffered with 100 mM citrate buffer (Sodium citrate and citric acid) at various pH ranging from 4.2 to 5.4 at 0.2-pH unit increments. The pH of the medium was measured using an Accumet Basic AB15 pH meter (Thermo fisher Scientific, Waltham, MA). The pH was further verified by use of MColorpHast pH-indicator strips (EMD Millipore, Jaffrey, NH) before and after the growth assay to ensure the pH keep constant throughout the assay.
To study the utilization of urea in C. neoformans, cells were grown in minimal medium at pH 5.5 with substitution of 7.5 mM urea or ammonium sulfate for glycine as sole nitrogen source and substitution of 7.5 mM urea for dextrose as sole carbon source. Cryptococcal strains were also grown in Sabourand broth to determine their growth and doubling time.
Growth studies were done using a Bioscreen C plate reader (Growth Curves USA) starting the cultures with 10⁵ yeast cells per well in honeycomb plate in different conditions mentioned above at 30 °C and measuring cell density for 72 h.

Seed cultures were grown under 30 μmol photons m⁻² s⁻¹ at 30 °C in BG11 with appropriate antibiotic(s) in 100 mL Erlenmeyer flasks (VWR) until OD₇₅₀ = 1.5–2.0. The seed cultures were then used to inoculate 25 mL experimental cultures to OD₇₅₀ = 0.1 in BioLite 25 cm² plug-sealed tissue culture flasks (Thermo Fisher Scientific). The medium used for experimental cultures was BG11 with addition of 50 mM NaHCO₃ (Sigma-Aldrich) and appropriate antibiotic(s) (final concentrations: chloramphenicol, 10 μg mL⁻¹; spectinomycin, 25 μg mL⁻¹; erythromycin, 25 μg mL⁻¹; and kanamycin, 25 μg mL⁻¹). All experimental cultures were prepared in quadruplicates. The flasks were shaken horizontally at 120 rpm, under 50 μmol photons m⁻² s⁻¹ at 30 °C. Two milliliters of culture were sampled from each flask every second day for measurements and 2 mL of fresh BG11 medium with addition of 500 mM NaHCO₃ (Sigma-Aldrich) and appropriate antibiotic(s) were added back. The pH of experimental cultures was measured with MColorpHast™ pH-indicator strips (pH 6.5–10) (Merck) and the cultures pH were adjusted to the range between 7–8 using 37% HCl (Sigma-Aldrich).