The Effect of Potassium Nitrate on Microbes By Liam O'Malley 9th Grade Central Catholic High School

Runoff ●Flow of water that occours when excess water runs over the earth's surface ●Often pick up pollutants on the way ●Pollutants could include fertelizer containing potassium nitrate ●Could potassium nitrate runoff be damaging microbial flora?

Purpose To determine the effect of potassium nitrate (KNO3) on microbial survivorship.

Hypotheses Null Hypothesis: Potassium nitrate will not have a significant reduction on microbial survivorship. Alternate Hypothesis: Potassium nitrate will significantly reduce microbial survivorship.

Potassium Nitrate ●Chemical compound KNO3 ●Ionic salt of Potassium K+ and Nitrate NO- ●Used in fertilizer, gunpowder, food preservatives, and stump remover ●Been used for 100s of years

Yeast ●Saccharomyces cerevisiae ●Eukaryotic microorganisms ●Fungi Kingdom ●1% of microorganisms ●1500 species ●Most common cell model today

Escherichia Coli ●Large and diverse group of gram (-) bacteria ●Free living, symbionts, or pathogens ●Live in the intestinal tract of many mammals ●Most strains are not pathogenic ●Serves as a common prokaryotic cell model

Materials ●Yeast ●YEPD plates (0.5% Yeast extract, 2% peptone, 2% glucose) ●E. coli ●LB plates (0.5% yeast extract, 1% tryptone, 1% sodium chloride) ●Alcohol ●Matches ●Pipettes ●Spreader bar ●Test tubes ●Test tube rack ●Vortex mixer ●Sterile Tips ●Beakers ●Sterile Dilution Fluid [SDF] (100mM KH2PO4, 100mM K2HPO4, 10mM MgSO4, 1mM NaCl) ●10% stock solution KNO3 (Mixed from Stump remover)

Pre-Procedure ( Same for LP and Infusion ) 1.E. coli and S. cerevisiae were grown overnight in sterile LB and YEPD media. 2.Samples of the cultures were added to media in sterile sidearm flasks. 3.Cultures were placed in an incubator (37°C and 30°C) until a density of 50 Klett spectrophotometer units was reached. This represents a cell density of approximately 10^8 and 10^7 cells/mL. 4.The cultures were diluted in sterile dilution fluid to a concentration of approximately 10^4 cells/mL

Procedure (Liquid Pulse) 1.Sterile LB and YEPD agar plates were labeled. 10 ml solutions using sterile water were made using the following concentrations. Two sets of each concentration were made. (Next Slide) 2.The solutions were vortexed to prepare for pipetting mL of the 10 mL solutions were pipetted onto the LB and YEPD agar plates: (36 plates total)

Potassium Nitrate Concentrations 0%.1%1% SDF9.9 mL9.89 mL9.8 mL 10% KNO3 Stock0 mL0.01 mL0.1 mL Microbe0.1 mL Total Volume10 mL

Procedure (continued) (Liquid Pulse) 1.The plates were incubated at 37°C and 30°C for 24 and 48 hours. 2.The resulting colonies were counted. Each colony is assumed to have arisen from one cell.

Procedure (Infusion) ●The following volumes of 10% KNO3 stock were spread onto LB and YEPD agar plates to create the following approximate concentrations. 200μL stock (10%), 20μL stock -180 ul water (1%) and 2μL stock -198 μL water (.1%). ●The plates were incubated at 37°C for one hour. ●100μL aliquots of bacterial suspensions from the CONTROL tube were spread onto the infused plates. ●The resulting colonies were counted visually. Each colony was assumed to have arisen from one cell.

P-Value: P-Value:

KNO3 LP Effects on E. coli Dunnett's Test T-ValueT-CritSignificant Variation or Not 0.1% Not 1% Not

P-Value: P-Value: 3E-06

KNO3 Infusion Effects on Yeast Dunnett's Test T-ValueT-CritSignificant Variation or Not 10% Not Sig 1% Sig 0.1% Not Sig

KNO3 Infusion Effects on E. coli Dunnett's Test T-ValueT-CritSignificant Variation or Not 10% Sig 1% Not 0.1% Sig

Conclusions The Null Hypothesis was accepted for all cases except for the 1% Yeast Infusion, the 0.1% and the 10% E. coli Infusion. Overall, the Potassium Nitrate had no significant effect on microbial survivorship.

Limitations Extensions ●Slight variations in plating times ●Limited different concentrations ●Only one exposure time ●Possibly too much variation within groups ●Do a test with a Gram (+) bacteria ●More concentrations ●Test growth ●Test synergistic effects with KNO3 and other variables

KNO3 LP Effects on Yeast ANOVA

KNO3 LP Effects on E. coli ANOVA

KNO3 Infusion Effects on Yeast ANOVA

KNO3 Infusion Effects on E. coli ANOVA