Amoeba Therapy on Streptococcus Pneumonia By Sam Casale.

Amoeba Therapy on Streptococcus

PneumoniaBy Sam Casale

Microbial Therapy

• A lesser used form of therapy.• Involves using microorganisms to fight other microorganisms.• The therapy used in this experiment has no background or ground

that could be used as a reference.

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 Protists family.• Use a pseudopod to move and capture food by wrapping its body

around its prey and engulfing it.• This type of amoeba is harmless to humans.• Found in rivers, lakes, streams, and other low current bodies of

waters.• Believed to contain phagocytic properties and share 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.)• Pitri dish

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 pitri 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

2 minutes 4 minutes 6 minutes 8 minutes 10 minutes0

20

40

60

80

100

120

0 0 0 0 026

1321

29

1116

20

32

45

2331

3945

62

43

5967

81

107

Population Growth

Control 0.001 0.01 0.1 1

P Value = 1.16x10 ²⁷ˉ

Dunnett’s Test

Concentration T-Crit T Value Result0.001ml 5.32 8.35 Significant0.01ml 5.32 16.38 Significant0.1ml 5.32 36.36 Significant1ml 5.312 43.59 Significant

T> T-Crit: SignificantAlpha Value = 0.5 T< T-Crit: Not 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.

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.

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-amoeba-proteus-kill-salmonella-enteritidis-introduced-by-micro-injection-influence-opsonins-intracellular-killing> • “Streptococcus Pneumonia.” CDC. August 5, 2014.

<http://wwwnc.cdc.gov/travel/diseases-/pneumococcal-disease-streptococcus-pneumoniae>• “Streptococcus Pneumonia.” Wikipedia. December 16, 2014. <

http://en.wikipedia.org/wiki/-Streptococcus_pneumoniae>

Two Factor AnovaVariance 0 1 1 0 4 783.2571

10 minutes

Count 3 3 3 3 3 15Sum 0 87 135 186 321 729Average 0 29 45 62 107 48.6Variance 0 16 1 1 4 1363.686

Total

Count 15 15 15 15 15Sum 0 213 369 600 1071Average 0 14.2 24.6 40 71.4Variance 0 106.3143 162.9714 200.5714 504.8286

ANOVASource of VariationSS df MS F P-value F crit

Sample 9623.28 4 2405.82 366.7409 2.24E-36 2.557179Columns 44891.28 4 11222.82 1710.796 8.43E-53 2.557179Interaction 3694.32 16 230.895 35.19741 1.16E-21 1.850315Within 328 50 6.56

Total 58536.88 74

0.001% AnovaAnova: Single Factor

SUMMARYGroups Count Sum Average Variance

Column 1 5 0 0 0Column 2 5 71 14.2 120.7


Between Groups504.1 1 504.1 8.352941 0.020195 5.317655Within Groups 482.8 8 60.35

Total 986.9 9



Column 1 5 0 0 0Column 2 5 124 24.8 187.7


Between Groups1537.6 1 1537.6 16.38359 0.003696 5.317655Within Groups 750.8 8 93.85

Total 2288.4 9



Column 1 5 0 0 0Column 2 5 200 40 220


Between Groups4000 1 4000 36.36364 0.000313 5.317655Within Groups 880 8 110

Total 4880 9

1% AnovaAnova: Single Factor


Column 1 5 0 0 0Column 2 5 357 71.4 584.8


Between Groups12744.9 1 12744.9 43.58721 0.000169 5.317655Within Groups2339.2 8 292.4

Total 15084.1 9

Amoeba Therapy on Streptococcus Pneumonia By Sam Casale.

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