Evaluation of Yeasts Exposed to Heavy Metals see ref

3.3.1. Culture Maintenance and Growth

1. Maintain stock cultures of Candida albicans or Candida maltosa (R-42) on SAB slants by transferring to fresh slants every 3-4 wk.

2. Grow working cultures in DYNB with agitation at 22°C for 18 h.

3.3.2. Preparation of Yeast Inoculum

1. Harvest cells from DYNB with centrifugation.

2. Wash cells twice with ddH2O via centrifugation and suspend pellet in MES buffer to an optical density at 600 nm of 0.6 in a 1.0-cm light path (equivalent to 2.33 x 106 cells/mL of C. maltosa or 1.20 x 107 cells/mL of C. albicans).

3.3.3. Exposure of Cells to Heavy Metals

1. Add 20 ^L of various concentrations of heavy metals (Ag[I] or Hg[II]) to 0.98 mL of cell suspension in microcentrifuge tubes and incubate tubes at 22°C for 1 h.

2. After exposure to heavy metals, harvest cells with centrifugation at 10,000g for 2 min.

3. Suspend cell pellet in 1.0 mL MES buffer for staining procedure.

3.3.4. Fluorescent Probe Staining Procedure

1. Add 20 ^L of PI stock solution and incubate for 5 min at 22°C.

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