1. Antimicrobial Effects of Human Saliva Steven Esposito Pittsburgh Central Catholic High School

2. Defense Systems of the Body The immune system is comprised of smaller more specific systems called defense systems. Each defense system has a small line of jobs that it carries out. There are ten main defense systems in the body.

3. Defense Systems Defense System Action Epithelial surface Physical barrier prevents pathogen entry Mucosal surfaceTraps bacteria LysozymeDissolves peptidoglycan Phagocytic cellsIngests and destroys pathogens Ciliated cellsMoves pathogens away from body Fatty acids Prevents bacterial growth Stomach Acid Destruction of ingested pathogens Normal bacterial floraCompetes with pathogens Urine excretionRemoves pathogens from body Complement systemMediates phagocytes function

4. Three Major Salivary Glands Saliva is a key part of the immune system. Without it we would be more prone to infection from harmful microbes. Human saliva is produced by three pairs of major slavery glands NameType Saliva Secreted Parotid Saliva with a watery serous consistency Sublingual and minor slavery glands Viscous mucous fluid Submandibular glandMixture of watery and viscous fluid

5. Human Saliva Is composed mostly of water, about 98-99 percent. Has a very minuscule amount of electrolytes, mucus, antibacterial compounds (thiocyanates, hydrogen peroxide, and secretory immunoglobulin A)

6. Antibacterial Compounds in Saliva Secretory immunoglobulin A Neutralizes bacteria, and enzyme toxins. Serves as an antibody for bacterial antigens, aggregates bacteria, inhibits oral tissues adherence. Thiocyanates Antimicrobial chemical compounds (SCN) Hydrogen Peroxide A weak acid. Hydrogen peroxide is naturally produced as a byproduct of oxygen metabolism.

7. 2 Major Enzymes In Saliva Name Function a-amylase Digests starch and lipid fats in food Lysozyme Acts to cause lysis in bacteria

8. Escherichia coli A prokaryotic, unicellular, bacterial cell which can most frequently be found in the large intestine of endothermic animals. The most studied prokaryote on earth. E. coli are a large and diverse group of bacteria. Some strands can make one very sick, causing diarrhea, while others can cause urinary tract infections.

9. Purpose To assess the effects of various concentrations of human saliva on the survivorship of E. coli.

10. Null Hypothesis and Alternative Hypothesis The saliva will not significantly effect E. coli survivorship. The saliva will significantly effect E. coli survivorship enough to conclude that saliva is antimicrobial.

11. Materials 1.Pipettes 2.DH5 Alpha E-Coli 3. 40 LB Agar Plates 4.Sterile Pipette Tips 5.Spreader Bars 6. Saliva (My Own) 7. 0.22 micron Sterile Filter 8. SDF ( Sterile Dilution Fluid) 9. Test Tubes 10. Incubator 11. Bunsen Burner 12.LB media (0.55 Yeast extract, 1% Tryptone, 1% Sodium Chloride) 13.1.5ml micro-centrifuge.

12. Procedure 1.E. coli was grown overnight in sterile LB media. 2.A Sample of the overnight culture was added to fresh media in a sterile sidearm flask. 3.The culture was placed in an incubator at 37°C until a cell density of 50 Klett spectrophotometer units were reached. This represents cell densities of approximately 10 8 cells/mL. 4. The culture was diluted in sterile dilution fluid to a concentration of approximately 10 5 cells/mL.

13. Procedure 5. Human saliva (my own) was collected and sterile filtered by means of a 0.22 micron syringe filter. 6.The following components were pipetted into sterile 1.5mL micro-centrifuge tubes. 7.The tubes were allowed to incubate for 15 minutes before plating. 8.After exposure to saliva 0.1 mL aliquots of cell suspensions were plated onto nutrient agar. 9.Incubated agar plates for 24 hours. 10.Counted the colonies of bacteria on the plates.

14. Concentration of Saliva Saliva0 mL.01 mL.05 mL0.99 mL SDF0.99 mL0.89 mL0.49 mL0 mL Microbe0.01 mL Final Concentration 0% Saliva 10% Saliva 50% Saliva 99% Saliva

15. P-value 3.65E-11 29.49 26.38 25.23

16. Dunnett's Test Dunnett’s Test purpose- to determine if there is a significant or insignificant variation between the variable groups and the control group.

17. Dunnett's Test 10% Saliva vs. 0% Saliva 29.49Very Significant 50% Saliva vs. 0% Saliva 26.38Very Significant 99% Saliva vs. 0% saliva 25.23Very Significant α=.05 T-critical= 2.76 T-critical

18. Conclusions The data appeared to be significant enough to reject the null hypothesis. The alternative hypothesis was accepted. The saliva will significantly effect E. coli survivorship enough to conclude that saliva is antimicrobial When data was analyzed the 10% concentration of saliva inhibited microbial growth the most, with 50% and 99% coming in second and third respectively. After data was analyzed results showed that the survivorship of the E. coli was reduced by about 75%.

19. Limitations and Extensions Limitations Some lag time when plating could have resulted in extra cell replication prior to Extensions Saliva from different species could be utilized to see if other species saliva also has antimicrobial qualities. Vary the time the saliva samples were exposed to the microbe before plating. Synchronize cell plating. Utilize different microbial models.

20. Sources http://www.newworldencyclopedia.org/entry/Saliva http://digestivesystem.suite101.com/article.cfm/the_ many_purposes_of_saliva http://digestivesystem.suite101.com/article.cfm/the_ many_purposes_of_saliva http://www.thejcdp.com/issue039/de_almeida/de_alm eida.pdf http://www.thejcdp.com/issue039/de_almeida/de_alm eida.pdf