UW-Stout/Hydrogen peroxide FA19

Yeast Torture Protocol:

Hydrogen peroxide at a 30% concentration is a strong oxidizing agent and it inflicts corrosive properties. We wanted to see the effects that H2O2 had on different strains of yeast in yeast cultures.

Materials

• H2O2 30% Concentration
• Nitrile Gloves
• P2, P10, P20, P100, P200, P1000 Micro-pipettes
• Yeast Strains (YHR048W, YAL061W, YBL086C, YBL081W, YBR138C, YBR220C, YGL140C, YJR061W, YKR005C, YBR033W)
• Wild Type: BY4736
• 1.5 mL Microcentrifuge tube
• Petri Dishes (33)
• PPE: lab coat, safety glasses and long pants
• Absorption discs

Methods: An initial calibration to determine the volume of hydrogen peroxide to use when plating our cultures was important. Our first two plates consisted of using 1 uL of H2O2 and 5 uL of H2O2 in order to determine which amount of H2O2 would help us get a better zone away from the absorption disc in the center of the petri dish.

Initial Calibration

• Spread 50 uL of a unique yeast strain on two seperate plates with 3-5 glass beads, cover the plates then shake. (technique is important in order to evenly disperse the media)
• Take the cover of the petri dish off for five minutes to dry out. (If performing outside of hood leave top cracked open for 10 minutes.)
• Place dry disc and press down firmly with tweezers to stick in the middle of the petri dish.
• Add 5uL and 1uL of H2O2 to the disc in the center of separate plates.
• Invert the plates and place in a 30℃ incubator for 72 hours.
• After the 72 hours, retrieve plates and measure the radius of the zone of inhibition of both plates with a ruler.

Calibration results:

It was determined that the volume of 5uL would be used to carry on the rest of the experiment as it gave us the largest zone of inhibition.

Yeast strain protocol:

• After determining the most effective volume to use for the zone of inhibition, prepare the yeast strain experiment.
• In order to avoid corrupt data impeding the experiment prepare three plates for every yeast strain that is tested
• Spread 50 uL of a unique yeast strain on three separate plates with 3-5 glass beads, cover the plates then shake. (technique is important in order to evenly disperse the media)
• Repeat step 3. For the remaining yeast strains and wild type
• Take the cover of the petri dish off for five minutes to dry out. (If performing outside of hood leave top cracked open for 10 minutes.)
• Place dry disc and press down firmly with tweezers to stick in the middle of the petri dish.
• Add 5uL of H2O2 to the disc in the center of separate plates.
• Invert the plates and place in a 30 °C incubator for 72 hours.
• After the 72 hours, retrieve the plates and measure the radius of the zone of inhibition of all 33 plates with a ruler.
• The discs that contain the Hydrogen Peroxide might have shifted or fallen off in the process which is why it is imperative to plate three times for each strain. Denote these discs measurements with an “x”