Transcript The Effects of Sweeteners on Prokaryotes

The Effects of
Sweeteners on
Prokaryotes
By Michael Gleason
Grade 9
Pittsburgh Central Catholic High School
Why study sweeteners?
Artificial sweeteners may cause glucose intolerance
Can cause type 2 diabetes
Change composition and function of micro bacteria
Artificial sweeteners may lead to obesity and metabolic
changes
Tested on mice and humans
Microbial Flora
Comprised of all the microorganisms that reside in or
on the human body (examples: skin, saliva, gut)
Most species are none pathogenic microorganisms
that either have no effect on the host or offer some
benefit
Almost 1000 species of bacteria live in the gut of an
average human body
Escherichia Coli
Major cause of food illness
70,000 cases a year in U.S.
Consumption of undercooked meats and
contaminated vegetables
Also in fecal matter
Most studied bacteria
Can survive outside of host organism
Stevia
Sugar substitute
Extracted from Stevia Rebaudiana leaves
Up to 150 times the sweetness of sugar
Taste has longer duration than sugar
Sucralose
Majority is not broken down in body
About 320-1000 times sweeter than sugar
Common brand names are Splenda and Zerocal
Sugar
Carbohydrates composed of carbon, hydrogen, and
oxygen
Found in sugarcane and sugar beet
About 168 million tons of sugar in 2011
Experiment
The objective is to explore the effect of
sweeteners on E. coli survivorship
Hypothesis
Null Hypothesis: The sweeteners will not have
significant effect on the survivorship of E. coli
Alternative- The sweeteners will significantly reduce
the survivorship of the E. coli.
Materials
E. coli
Micropipettes
Micro tubes
stevia, sucralose, cane sugar (10% solutions)
Sterile Dilution Fluid (SDF) (per 1 liter) (100mM KH2PO4,
100mM K2HPO4, 10mM MgSO4, 1mM NaCl)
 42 LB agar plates
LB media (Per Liter:1% Tryptone, 0.5% Yeast Extract, and 1%
NaCl)
2 syringe Sterile Filters
Bunsen Burners
Spread Bar
Incubator
Ethanol
Matches
Vortex
Procedure
Labeled each plate with correct concentration, sugar and time
Bacteria (E.coli ) was grown overnight in sterile LB media.
A sample of the overnight culture was added to fresh media in a
sterile sidearm flask.
The cultures were placed in incubators at 37°C until a density of
50 Klett spectrophotometer units were reached.
The cultures were diluted in sterile dilution fluid to a
concentration of approximately 105 cells/mL.
Created variable concentrations (slide following)
Vortexed each tube and allow to sit at room temperature for 15
min
Procedure continued
0.1 ml of each concentration was plated on
appropriate plate
Using a sterilized spreader bar, they were spread on
the plate
The plates were capped
E. Coli plates were incubated for 24 hours at 37
degrees Celsius
Resulting colonies were counted
Concentration 1% (measurements in ml)
SDF
E. Coli
Sweeteners
Concentrations
9.9
.1
0
0%
9.89
.1
.01
.1%
9.8
.1
.1
1%
Average survivorship 10 minutes
2500
2000
P 0.106923
Number of Colonies
P 0.01423
1500
Control
P 0.000104
Stevia
Cane Sugar
1000
Sucralose
P 0.0000121
500
0
0% control
.1% solutions
1% solutions
E. Coli 10 minute Dunnett’s Test
T-crit = 4.4
Stevia .1
T=1.361
not significant
Stevia 1
T=9.561
significant
Cane Sugar.1 T=9.660
significant
Cane Sugar 1 T=12.145
significant
Sucralose .1
T=1.776
not significant
Sucralose 1
T=15.294
significant
Average survivorship 30 minutes
2500
2000
P 0.088064
P 0.008254
Number of Colonies
1500
P 0.00024
Control
Stevia
Cane Sugar
Sucralose
1000
P 0.00000341
500
0
0% Control
0.1% Solutions
1% Solutions
E. Coli 30 minute Dunnett’s Test
T-crit = 4.4
Stevia .1
T=1.764
not significant
Stevia 1
T=12.143
significant
Cane Sugar.1 T=15.088
significant
Cane Sugar 1 T=17.451
significant
Sucralose .1
T=3.795
not significant
Sucralose 1
T=22.062
significant
Conclusion
The Null hypothesis is rejected due to Stevia .1 10 and
30 minutes, Sucralose 10 and 30 minutes
Sweeteners did have significant effect on E. Coli
survivorship.
Time exposed did not change the effects of
sweeteners
Extensions
Could have used more sweeteners: aspartame for
example
More plates
Limitations
Only three concentrations used
Plating slightly unsynchronized