Effects of Commercial Protein
Powders on Microbial Models
Harry Suver
Grade 11
Central Catholic High School
Gram Staining
• Invented by Hans Christian Gram
• Is a method of differentiating bacterial species into
two groups, Gram-positive and Gram-negative
• Gram-positive bacteria have generally a single
membrane (monoderm) surrounded by a thick
peptidoglycan
• Gram-negative bacteria generally possess a thin layer
of peptidoglycan between two membranes
• When stained, a Gram-positive bacteria results in a
blue/violet color while Gram-negative results in light
red/pink
Gram(-) vs. Gram(+)
Gram-negative


Cell walls contains an extra layer of
lipopolysaccharides for extra
protection.
Outer membrane protects bacteria
from several antibiotics.
Gram-positive
Most pathogenic bacteria in humans
are Gram (+) organisms.
Simple cell wall.
Some antibiotics work against the
formation of the cell wall.
Staphylococcus Epidermidis
• Staph. species consisting of
harmless skin-dwelling
micro-organisms
• Species of Staph. are
commonly used in
microbiological experiments
because of variety of strain
types within each species –
each strain has different
features and toxins
• Gram-positive bacteria
• Most strains are not
pathogenic
Escherichia coli
• Major cause of food-borne
illness
• Consumption of undercooked
meats
• Consumption of contaminated
vegetables or unpasteurized
milk
• Also resides in fecal matter
• Most studied bacteria in the
world and very commonly
used in the lab
• Resides in the digestive tract
of animals
• Not all strains of E.coli can
result in sickness or disease
• Is a Gram-negative bacteria
Whey Protein
• GNC Pro Performance 100%
Whey Protein
• Whey protein is a mixture of
globular proteins isolated
from whey, the liquid material
created as a by-product of
cheese production.
• Whey protein is commonly
marketed and ingested as a
dietary supplement, and
various health claims have
been attributed to it in the
alternative medicine
community.
Soy Protein
• GNC Super Foods 100%
Soy Protein
• protein isolated from
the soybean.
• Made from soybean
meal that had been
dehulled and defatted
Purpose
• The purpose of this experiment was to see if
protein powder had significant effects on the
growth of microbial models
Hypothesis
• Null Hypothesis- The protein powder will not
have a significant effect the growth of the
bacteria
• Alternate Hypothesis- The protein powder will
have a significant effect on the bacteria and
will cause more colonies to grow than the
control groups
Materials
• E.coli
• Staph. E.
• GNC pro performance
Whey Protein
• GNC Soy Protein
• LB media (.5% yeast
extract, 1% tryptone, 1%
sodium chloride)
• Agar plates
• Spreader bars
• Bunsen Burner
• Vortex
•
•
•
•
•
•
•
•
Incubator
Micropipettes
Sterile pipette tips
Sterile dilution fluid
([SDF] (10mM KH2PO4,
10mM K2HPO4, 1mM
MgSO4, .1mM CaCl2,
100mM NaCl)
Sterile test tubes
Test tube rack
Ethanol
Matches
Procedure
1. Bacteria (E.coli and Staph. E.)
were grown overnight in sterile LB
Media.
2. Samples of the overnight cultures
were added to fresh media in a
sterile sidearm flask.
3. The cultures were placed in an
incubator (37°C) until a density of
50 Klett spectrophotometer units
was reached. This represents a cell
density of approximately 10⁸
cells/mL.
4. The cultures were diluted in
sterile dilution fluid to a
concentration of approximately 10⁵
cells/mL.
5. Solutions were made up in sterile
test tubes using sterile fluid and
variable.
6. 0.1mL. Of the cell cultures were
added to the solutions which
brought them to a total volume of
10mL.
7. Solutions were pipetted onto LB
plates using a micro-pipette.
8. Spreader bars were sterilized and
used to spread solutions on the
plates.
9. Plates were put in an incubator set
at 37 degrees Celsius for 24 hours.
10. Colonies were counted, data was
recorded, results were analyzed.
Concentrations in Tubes
CONTROL
5%
10%
STERILE
FLUID
VARIABLE
9.9 mL
7.9 mL
7.9 mL
0.0 mL
2.0 mL
(of 25% stock)
2.0 mL
(of 50% stock)
BACTERIA
0.1 mL
0.1 mL
0.1 mL
TOTAL
VOLUME
10 mL
10 mL
10 mL
5 replicates for each group
Stat Analysis
ANOVA
• Shows if there is any
variations between the
mean of 2 or more variable
groups
Alpha=.05
Dunnetts Test
• Shows the variation
between a control group
and an experimental groups
T crit= 3.03
Staph with Soy Protein
170
165
P-value = 0.037219 = Significant
Number of Colonies
160
155
150
145
140
135
130
125
Control
5%
10%
Dunnett's Test(Staph w/ Soy)
Concentration T-value
Significance
5%
1.207
Not Significant
10%
3.34
Significant
180
160
Staph with Whey Protein
P-value = .116984 = Not Significant
140
Number of Colonies
120
100
80
60
40
20
0
Control
5%
10%
Dunnett’s Test (Staph w/Whey)
Concentration T-value
Significance
5%
-0.99281
Not Significant
10%
1.2264
Not Significant
E.Coli with Soy Protein
450
400
P-value = 0.003653 = Significant
Number of Colonies
350
300
250
200
150
100
50
0
Control
5%
10%
Dunnetts Test (E.coli w/ Soy)
Concentration
T-value
Significance
5%
3.2442
Significant
10%
4.4968
Significant
E.Coli with Whey Protein
350
340
P-value = 0.016782 = Significant
Number of Colonies
330
320
310
300
290
280
270
Control
5%
10%
Dunnetts Test (E.coli w/ Whey)
Concentration T-value
Significance
5%
-0.5841
Not Significant
10%
3.8646
Significant
Conclusion
• The Null Hypothesis can be partially rejected, and
the Alternate Hypothesis can be partially
accepted.
• Soy protein had significant effects on both
bacterial models at all concentrations and caused
more colonies to grow.
• Whey protein did not have significant effects on
either bacterial model at any concentration.
• While on some occasions the 5% groups had less
colonies than the control groups, they had
colonies that were larger in size
Limitations
• Did not have access to
any other model
organisms.
• Lag time between
counting of colonies
Continuations
• Experiment could be
expanded to test
protein powder on stem
cells or algae.
• Could compare protein
powder’s effectiveness
to growth hormones
and natural animal
proteins.
ANOVA (Staph w/ soy)
SUMMARY
Groups
Column 1
Column 2
Column 3
ANOVA
Source of
Variation
Between
Groups
Within
Groups
Total
Count
5
5
5
SS
376.1333
Sum
Average Variance
700
140
62.5
725
145
62.5
761
152.2
3.7
df
MS
F
P-value
F crit
2 188.0667 4.383838 0.037219 3.885294
514.8
12
890.9333
14
42.9
ANOVA (Staph w/ whey)
SUMMARY
Groups
Column 1
Column 2
Column 3
ANOVA
Source of
Variation
Between
Groups
Within
Groups
Total
Count
Sum
5
5
5
SS
3782.8
8798.8
12581.6
Average Variance
700
140
20.5
615
123
1678
809
161.8
501.2
df
MS
2
1891.4
12 733.2333
14
F
2.579534
P-value
0.116984
F crit
3.885294
ANOVA (E.coli w/ soy)
SUMMARY
Groups
Column 1
Column 2
Column 3
ANOVA
Source of
Variation
Between
Groups
Within
Groups
Total
Count
5
5
5
SS
15257.2
9855.2
25112.4
Sum
Average Variance
1530
306
348.5
1685
337
698
1918
383.6
1417.3
df
MS
2
P-value
F crit
7628.6 9.288822 0.003653 3.885294
12 821.2667
14
F
ANOVA (E.coli w/ whey)
SUMMARY
Groups
Column 1
Column 2
Column 3
ANOVA
Source of
Variation
Between
Groups
Within
Groups
Total
Count
5
5
5
SS
4813.333
4930
9743.333
Sum
Average Variance
1530
306
305
1480
296
247
1690
338
680.5
df
MS
F
P-value
F crit
2 2406.667 5.858012 0.016782 3.885294
12 410.8333
14
References
• CentralCatholichs.com/biology
• http://www.gnc.com/home/index.jsp