UW-Stout/Calcofluor White SP21

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Calibration Protocol

The calibration protocol for calcofluor white involved diluting the calcofluor white into various concentrations, from 1000 uM to 10 uM. The calibration is designed to stress, but not kill the yeast, as calcofluor white can poison yeast by binding to the cell wall. Some stress could also be attributed to the potassium hydroxide the yeast was treated with before the calcofluor white. However, calcofluor white is hazardous to human life as well; the vapors from it are irritants for the eyes and lungs.

Materials

Method

  1. Dilute calcofluor white to desired concentrations by placing 25 uL calcofluor white in a microcentrifuge tube with the desired amount of demineralized water. The dilutions are as follows:
    1. 1000 uM, 1.03 uL
    2. 500 uM, 27.05 uL
    3. 200 uM, 130.13 uL
    4. 100 uM, 260.25 uL
    5. 50 uM, 520.50 uL
    6. 20 uM, 1301.25 uL
    7. 10 uM, 2602.50 uL
    8. 0 uM, only water for a control strain
  2. Place 50 uL of wild yeast in a column on the plate, followed by 25 uL of potassium hydroxide solution. Mix the yeast solution with the pipette tip and wait 1 minute.
  3. Place 25 uL of the calcofluor white solution into the corresponding well and mix the well with the pipette tip once again.
  4. Place well plate into plate reader with the absorbance at 600 nm. Let run for the entire cycle and record observations.

Observations

Looking at the graph, the most consistent line of fit was the 200 uM solution. It had the highest correlation factor, which is important for getting consistent results with not that much variation as the other part of the calcofluor white was Evan’s Blue Dye, which absorbs all but blue light. The orange light (coming from the plate reader) was being absorbed more by the higher concentrations of the blue dye, so the 200 uM solution was the best choice as it could stress the yeast and have more comprehensible data (For context, the A7 dataset is the control with 25 uL of potassium hydroxide and 25 uL of water).

Calcofluor Graph

Yeast Stress Protocol

After establishing that a 200 uM concentration would be the best choice, the next step is to test them against the different yeast strains. The order of these are as follows:

  1. A: YOR387C
  2. B: YKL121W
  3. C: YBR225W
  4. D: YNL018C
  5. E: YLR426W
  6. F: YGL138C
  7. G: Wild Strain