Antimicrobial Effects of Human Saliva

Steven Esposito

Pittsburgh Central Catholic High School

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.

Defense Systems

Defense System ActionEpithelial surface Physical barrier prevents pathogen entryMucosal surface Traps bacteria Lysozyme Dissolves peptidoglycanPhagocytic cells Ingests and destroys pathogensCiliated cells Moves pathogens away from bodyFatty acids Prevents bacterial growth Stomach Acid Destruction of ingested pathogensNormal bacterial flora Competes with pathogens Urine excretion Removes pathogens from bodyComplement system Mediates phagocytes function

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

Name Type Saliva Secreted

Parotid Saliva with a watery serous consistency

Sublingual and minor slavery glands Viscous mucous fluid

Submandibular gland Mixture of watery and viscous fluid

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)

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.

2 Major Enzymes In Saliva

Name Function

a-amylase Digests starch and lipid fats in food

Lysozyme Acts to cause lysis in bacteria

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.

Purpose

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

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.

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.

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 108 cells/mL.

4. The culture was diluted in sterile dilution fluid to a

concentration of approximately 105 cells/mL.

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.

Concentration of Saliva

Saliva 0 mL .01 mL .05 mL 0.99 mL

SDF 0.99 mL 0.89 mL 0.49 mL 0 mL

Microbe 0.01 mL 0.01 mL 0.01 mL 0.01 mL

Final Concentration

0% Saliva

10% Saliva

50% Saliva

99% Saliva

050

100150200250300350400450

Number of Colonies

0%Saliva

10%Saliva

50%Saliva

99%Saliva

Concentration of Saliva

Average Microbial Survivorship

P-value

3.65E-11

29.49 26.38 25.23

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.

Dunnett's Test

10% Saliva vs. 0% Saliva

29.49 Very Significant

50% Saliva vs. 0% Saliva

26.38 Very Significant

99% Saliva vs. 0% saliva

25.23 Very Significant

α= .05

T-critical= 2.76

T-critical

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%.

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.

Sources

http://www.newworldencyclopedia.org/entry/Saliva

http://digestivesystem.suite101.com/article.cfm/the_many_purposes_of_saliva

http://www.thejcdp.com/issue039/de_almeida/de_almeida.pdf