1. Amoeba Therapy on Streptococcus Pneumonia By Sam Casale

2. 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

3. 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

4. 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.

5. 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.

6. Hypothesis The Amoeba Proteus will engulf and digest a significant amount of streptococcus pneumonia.

7. 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

8. 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:

9. Concentration of Streptococcus Pneumonia(ML) 0%0.0001%0.001%0.01%0.1% Distilled Water9.9ml9.899ml9.89ml9.8ml8.9ml Amoeba Proteus 0.1ml Strep Pneumonia 0ml0.001ml0.01ml0.1ml1ml Total10ml

10. 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.

11. Streptococcus Cells in Amoeba (average amount) 0ml Strep0.001ml Strep0.01ml Strep0.1ml Strep1ml Strep 2 minutes02112343 4 minutes06163159 6 minutes013203967 8 minutes021324581 10 minutes0294562107 Total0294562107

12. Phagocytic Activity of Amoeba on Strep P Value = 1.16x10ˉ²⁷

13. Dunnett’s Test ConcentrationT-CritT ValueResult 0.001ml5.328.35Significant 0.01ml5.3216.38Significant 0.1ml5.3236.36Significant 1ml5.31243.59Significant T> T-Crit: Significant Alpha Value = 0.5T< T-Crit: Not Significant

14. 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.

15. 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.

16. 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.

17. References “Amoeba Proteus.” Wikipedia. December 19, 2014 “Amoeba Proteus: Structure and Functions.” David Wang Blog. Savanat, T.; Favillard “Amoeba Proteus On Salmonella.” Ebscohost. “Streptococcus Pneumonia.” CDC. August 5, 2014. “Streptococcus Pneumonia.” Wikipedia. December 16, 2014.

18. Two Factor Anova

19. 0.001% Anova

20. 0.01% Anova

21. 0.1% Anova

22. 1% Anova