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
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Saccharomyces cerevisiae
Eukaryotic microorganisms
Fungi Kingdom
1% of microorganisms
1500 species
Most common cell model today
Escherichia Coli
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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
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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.
3. 0.1 mL of the 10 mL solutions were pipetted onto the LB
and YEPD agar plates: (36 plates total)
Potassium Nitrate Concentrations
0%
.1%
1%
SDF
9.9 mL
9.89 mL
9.8 mL
10% KNO3 Stock
0 mL
0.01 mL
0.1 mL
Microbe
0.1 mL
0.1 mL
0.1 mL
Total Volume
10 mL
10 mL
10 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: 0.023786
P-Value: 0.31943
KNO3 LP Effects on E. coli Dunnett's
Test
T-Value
T-Crit
Significant
Variation or Not
0.1%
2.8655780848
3.68232
Not
1%
0.3785352277
3.68232
Not
P-Value: 0.006035
P-Value: 3E-06
KNO3 Infusion Effects on Yeast Dunnett's Test
T-Value
T-Crit
Significant
Variation or Not
10%
2.51156160906 3.238872
Not Sig
1%
7.57874731154 3.238872
Sig
0.1%
0.24234366403 3.238872
Not Sig
KNO3 Infusion Effects on E. coli Dunnett's Test
T-Value
T-Crit
Significant
Variation or Not
10%
3.8572568850
3.238872
Sig
1%
1.5406426425
3.238872
Not
0.1%
3.7743861316
3.238872
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
● Slight variations in
plating times
● Limited different
concentrations
● Only one exposure time
● Possibly too much
variation within groups
Extensions
● 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