Amoeba Therapy on
Streptococcus Pneumonia
By Sam Casale
Microbial Therapy
• A lesser used form of therapy.
• Involves using microorganisms to fight other microorganisms.
• Phage Therapy is the most commonly used form of microbial therapy
Streptococcus Pneumonia
• Leading cause of pneumonia worldwide
• Contributing cause of meningitis and other streptococcal diseases.
• Every year, approximately 1.6 million people die from streptococcal
pneumonia related illnesses
• More than 80% of these victims are under the age of 5.
• Adept at gaining resistance and immunity to drugs and antibodies and
many new antibiotics are incapable of killing the pathogen
Amoeba Proteus
• Single celled organisms that belong to the Protist group.
• Use a pseudopod to move and capture food by wrapping its body
around its prey and engulfing it.
• Harmless to humans.
• Found in rivers, lakes, streams, and other low current bodies of
waters.
• Phagocytic properties
• Many similarities with white blood cells.
Purpose
Test the effects of Amoeba Proteus in an environment with
Streptococcus Pneumonia to see if Amoeba Proteus is capable of
engulfing, digesting, and killing the pathogen. This would indicate
possible microbial use for therapy.
Hypothesis
The Amoeba Proteus will engulf and digest a
significant amount of streptococcus pneumonia.
Materials
•
•
•
•
•
•
•
•
•
•
•
Streptococcus Pneumonia (AmMS 208)
LB agar plates
Recombinant RFP Protein
Amoeba Proteus
Nikon Eclipse Ti Live Cell S231 (or other cell imaging microscope)
Centrifuge
Centrifugal tubes
Micropipettes+tips
Incubator
NIS- Element viewer (or other program that can view photos in separate color channels)
Adobe Photoshop (or other program that is capable of counting pixels by certain color and
dimensions.)
• Petri dishes
Procedure
1. Streptococcus Pneumonia was grown in an agar plate and labeled
with a Recombinant RFP Protein.
2. With a micropipette, 1ml of amoeba proteus was extracted and
placed in a petri dish.
3. After 24 hours of incubation, varying concentrations of
Streptococcus Pneumonia were extracted from the plate and placed
in centrifugal tubes.
4. After the streptococcus pneumonia was centrifuged, the amount of
streptococcus pneumonia that was placed in the dish with the
amoeba proteus was decided based on the following table:
Concentration of Streptococcus Pneumonia(ML)
0%
0.0001%
0.001%
0.01%
0.1%
Distilled Water
9.9ml
9.899ml
9.89ml
9.8ml
8.9ml
Amoeba Proteus
0.1ml
0.1ml
0.1ml
0.1ml
0.1ml
Strep Pneumonia
0ml
0.001ml
0.01ml
0.1ml
1ml
Total
10ml
10ml
10ml
10ml
10ml
Procedure (continued)
5. As soon as the streptococcus pneumonia was placed with the amoeba proteus,
an amoeba was found under the microscope and photos were taken of the
amoeba every two minutes for ten minutes.
6. This was repeated three times at the same concentration.
7. The same procedure was repeated but with different concentrations for five
trials.
8. A photo viewing program was used to the channel of the photos to TRITC and
the photos were exported.
9. The program that has the ability to count pixels by dimension and color and
have every pixel was used to count every pixel inside the amoeba that was
#d6d6d6 in color and 5X5 pixels in size.
10. The amount of pixels in each photo were counted and compared to see if an
increase in streptococcus pneumonia was found within the amoeba.
Streptococcus Cells in Amoeba (average
amount)
0ml Strep
0.001ml Strep
0.01ml Strep
0.1ml Strep
1ml Strep
2 minutes
0
2
11
23
43
4 minutes
0
6
16
31
59
6 minutes
0
13
20
39
67
8 minutes
0
21
32
45
81
10 minutes
0
29
45
62
107
Total
0
29
45
62
107
Phagocytic Activity of Amoeba on Strep
P Value = 1.16x10ˉ²⁷
Cell Amount Growth
120
107
Amount of Cells
100
81
80
67
60
40
62
59
45
43
39
32
31
20
23
11
0
2
0
2 minutes
45
16
6
0
4 minutes
Control
0.001
20
13
21
0
6 minutes
0
8 minutes
0.01
0.1
1
29
0
10 minutes
Dunnett’s Test
Alpha Value = 0.5
Concentration
0.001ml
0.01ml
0.1ml
1ml
T-Crit
5.32
5.32
5.32
5.312
T> T-Crit: Significant
T< T-Crit: Not Significant
T Value
Result
8.35
Significant
16.38
Significant
36.36
Significant
43.59
Significant
Conclusion
• Every trial showed gain in streptococcus excluding the control.
• Every trial was above the f-crit value and displayed significant results
that support the idea that Amoeba Proteus are capable of engulfing
and digesting streptococcus pneumonia.
• The data supports the initial hypothesis.
Possible Errors/Limitations
• No major complications or problems were found in the experimental
process or data collection in the experiment that could have impacted
the outcome of the experiment.
• The exact amount of streptococcus pneumonia might differ from the
amount that was collected based off the pixel counting program’s
collection.
Extensions
• Other bacteria would have been tested
• More trials to find an average would have been conducted
• Other types of amoeba would have been tested.
References
• “Amoeba Proteus.” Wikipedia. December 19, 2014
<http://en.wikipedia.org/wiki/Amoeba_pro-teus>
• “Amoeba Proteus: Structure and Functions.” David Wang Blog.
<https://davidwangblog.wordpr-ess.com/structure-and-functions/>
• Savanat, T.; Favillard “Amoeba Proteus On Salmonella.” Ebscohost. <
http://connection.ebscoh-st.com/c/articles/15832563/ability-amoebaproteus-kill-salmonella-enteritidis-introduced-by-micro-injection-influenceopsonins-intracellular-killing>
• “Streptococcus Pneumonia.” CDC. August 5, 2014.
<http://wwwnc.cdc.gov/travel/diseases-/pneumococcal-diseasestreptococcus-pneumoniae>
• “Streptococcus Pneumonia.” Wikipedia. December 16, 2014. <
http://en.wikipedia.org/wiki/-Streptococcus_pneumoniae>
Variance
Two Factor Anova
0
1
1
0
4 783.2571
3
0
0
0
3
87
29
16
3
135
45
1
3
186
62
1
3
15
321
729
107
48.6
4 1363.686
10 minutes
Count
Sum
Average
Variance
Total
Count
Sum
Average
Variance
15
15
15
15
15
0
213
369
600
1071
0
14.2
24.6
40
71.4
0 106.3143 162.9714 200.5714 504.8286
ANOVA
Source of VariationSS
Sample
9623.28
Columns 44891.28
Interaction 3694.32
Within
328
Total
58536.88
df
4
4
16
50
74
MS
F
P-value
F crit
2405.82 366.7409 2.24E-36 2.557179
11222.82 1710.796 8.43E-53 2.557179
230.895 35.19741 1.16E-21 1.850315
6.56
0.001% Anova
Anova: Single Factor
SUMMARY
Groups
Count
Column 1
5
Column 2
5
ANOVA
Source of VariationSS
Between Groups
504.1
Within Groups 482.8
Total
986.9
Sum
Average Variance
0
0
0
71
14.2
120.7
df
1
8
9
MS
F
P-value
F crit
504.1 8.352941 0.020195 5.317655
60.35
0.01% Anova
Anova: Single Factor
SUMMARY
Groups
Count
Column 1
5
Column 2
5
ANOVA
Source of VariationSS
Between Groups
1537.6
Within Groups 750.8
Total
2288.4
Sum
Average Variance
0
0
0
124
24.8
187.7
df
1
8
9
MS
F
P-value
F crit
1537.6 16.38359 0.003696 5.317655
93.85
0.1% Anova
Anova: Single Factor
SUMMARY
Groups
Count
Column 1
5
Column 2
5
ANOVA
Source of VariationSS
Between Groups
4000
Within Groups 880
Total
4880
Sum
Average Variance
0
0
0
200
40
220
df
1
8
9
MS
F
P-value
F crit
4000 36.36364 0.000313 5.317655
110
1% Anova
Anova: Single Factor
SUMMARY
Groups
Count
Column 1
5
Column 2
5
ANOVA
Source of VariationSS
Between Groups
12744.9
Within Groups2339.2
Total
15084.1
Sum
Average Variance
0
0
0
357
71.4
584.8
df
1
8
9
MS
F
P-value
F crit
12744.9 43.58721 0.000169 5.317655
292.4