EFFECT OF STERILANTS ON THE SURVIVAL OF YEAST Pearse Walsh Grade 9 Central Catholic High School

Anti-Microbials Natural : ●Lemon Juice ●Cinnamon Oil ●Grapefruit Seed Extract Artificial : ●Hydrogen Peroxide ●Chlorhexidine Gluconate ●Alcohol

YEAST, SACCHAROMYCES CEREVISIAE ● Eukaryotic organism, meaning it is a cell with a nucleus, membrane, and cytoplasm ● Fungus ● Normal flora, meaning it naturally occurs in the body, of the gastrointestinal tract and skin ● Can cause infection of blood (fungemia), or infection of muscosa, an area of the body that is exposed to air and secretes mucus

CHLORHEXIDINE GLUCONATE ● Antifungal and antibacterial ● Commonly paired with alcohol for use in sterilizing the skin before an injection or incision ● Active ingredient in many mouthwash products ● Corrosive at high concentrations ● Combination of a chlorhexidine salt and a gluconate acid ● Attack cells via binding to and rupturing the cell wall/membrane

HYDROGEN PEROXIDE ● Used in non-chlorine bleach (often used to bleach hair) ● Antibacterial ● In a previous study, supported growth in two yeasts, C. albicans and C. neoformans ● Destroys cells via oxidation, a process in which electrons from one atom are taken by another ● Also corrosive

INGREDIENTS ● 3% Hydrogen Peroxide ○ Sterilized Water ○ Hydrogen Peroxide ●4% Chlorhexidine Gluconate ○Cocamide DEA, FragrancePOFL 147, Glucono Delta Lactone, Hydroxyethylcellulose, Isopropyl Alcohol, Lauramine Oxide, PEG 75 Lanolin, Purified Water, Tridecyl 12Alcohol ○Chlorhexidine Gluconate

SYNERGY Hydrogen Peroxide and Chlorhexidine Gluconate have shown to have pro-synergistic effects against the survival of bacteria The variables acted together via chlorhexidine gluconate breaking the cell wall which allowed for more hydrogen peroxide to enter the cell

PURPOSE ●To compare the effectiveness of one solution against another in eradicating yeast, and see if there is a synergistic effect between Chlorhexidine Gluconate and Hydrogen Peroxide.

HYPOTHESIS Null: The sterilants will not significantly affect the survival of S. cerevisiae. Alternative: The sterilants will significantly affect the survival of S. cerevisiae.

MATERIALS ● Saccharomyces cerevisiae (yeast) ● 3% hydrogen peroxide solution ● 4% chlorhexidine gluconate solution ● 20 test tubes filled with 9.9 ml of sterile dilution fluid ● 60 observation plates

PROCEDURE 1.Created 1% concentrations of the hydrogen peroxide and chlorhexidine solutions 2.Created a 5ml tube with 2.5ml of each 1% variable solution to produce the third variable, to create a 1% concentration of 0.5% of each previous variable. 3.Poured tubes and replicant tubes with their assigned amounts (next slide) 4.Added 0.1 ml of the yeast to each tube 5.Left the tubes for 5 minutes (time for the yeast to exposed to the variables) 6.Plated and spread 1 ml aliquots from each tube, three replicant plates per tube 7.Incubated for 24 hours 8.Recorded visible yeast colonies

TUBE AMOUNTS CONTROL0.001%0.010%0.100% Sterilized Dilution Fluid9.9ml9.89ml9.8ml8.9ml Yeast0.1ml Variable0ml0.01ml0.1ml1ml Total10ml

KEY QUESTIONS ● Which of the variables was most effective at killing yeast? ● Did the varying concentrations produce varying effects? ● Was the hypothesis supported? ● Did the variables effect vary significantly from the control?

P value = 4.59E-13

P value = E-17

P value = E-16

ANOVA (0.001%) ANOVA is a statistical test that is used to identify if there was significant variation between results.

DUNNETT'S TEST  Confidence interval of 95% VariablesT valueConclusion Hydrogen Peroxide.001%-8.8Significant Chlorhexidine Gluconate.001%-80.7Significant Mixture.001%-85.7Significant Hydrogen Peroxide.01%-33.8Significant Chlorhexidine Gluconate.01%-145Significant Mixture.01%-146.5Significant Hydrogen Peroxide.1%-98.5Significant Chlorhexidine Gluconate.1%-146.5Significant Mixture.1%-146.5Significant

T-CRIT VALUES

CONCLUSION The null was rejected at all levels based on the evidence. The chlorhexidine gluconate and hydrogen peroxide solution was the most effective yeast sterilant. All of the tested sterilants were significantly effective at killing yeast.

Limitations: Only one species of yeast was tested. Limited number of replicants. Extensions: Alcohol can be added as a variant, because of its wide use as a sterilant alongside chlorhexidine gluconate. Bacteria or virus can be used as subjects in future testing. Errors in testing: Due to limited man power yeast was not added to each tube simultaneously.

SOURCES

RAW DATA CG0.00%0.01%0.10%CONTROL: Hydrogen Peroxide0.00%0.01%0.10% SYN0.00%0.01%0.10% w/

ANOVA (0.001%)

ANOVA (0.01%)

ANOVA (0.1%)