My study researched the inhibitory properties of resveratrol on the bacterial growth of Streptococcus Mutans. If you have any questions about my research please feel free to contact me via email at julia.barquin@icloud.com. All Rights Reserved.

1. The Inhibitory Effects of
Resveratrol on Planktonic Growth
and Biofilm Formation of Caries
Causing Streptococcus Mutans
Julia Barquin
American Heritage School,Boca/Delray
Cell Biology/Biochemistry
Grade 11

2. Background Information
What is Streptococcus Mutans?
• This bacteria is very important to study because
virtually everyone in the world carries it. It is one of
the bacteria predominantly found in the human
mouth.
• S. mutans is a gram positive, anaerobic bacterium
which forms a biofilm and secretes lactic acid on the
surface of the teeth, degrading their calcium
phosphorus matrices.
• Cavities caused by the bacteria are the reason for half
of all dental visits in the U.S.
• Though not fatal, tooth decay is one of the most
common infectious diseases in humans.

3. Problem
Certain active ingredients used in name brand toothpastes and
mouthwashes to prevent bacterial growth are toxic!
• FDA recently banned in soaps but not toothpastes, Triclosan is a
hazardous active ingredient presently used in certain oral hygiene
products, including Colgate Total®.
• Studies have shown that triclosan can cause health problems such as,
- endocrine disruption, leading to organ dysfunction
-altered testosterone and thyroid serum levels
- compromised affected immune system and fertility
- heart disease and heart failure

4. Problem
Triclosan and other compounds used as antibacterial agents
present a threat to the health of the general community
• A need exists for an novel effective, non-toxic,
antibacterial active ingredient to be used in oral hygiene
products

5. Objective
To examine the inhibitory capabilities of a novel, naturally
occurring compound that will be able to efficiently disrupt
the growth of Streptococcus Mutans.
• With an equally effective safer alternative to dangerous
antiseptic ingredients, improvements can not only be made to
oral hygiene products, but also various other common
antibacterial products that utilize the same active ingredients.

6. Research
A Natural Antibacterial Compound
• Resveratrol
- Resveratrol is a plant derived
polyphenol compound found in the skin
of grapes and berries and several plant
species.
- This compound is produced by plants
to assist when under attack by pathogens
such as bacteria
- It is often taken as a supplement in pill
form due the compounds list of health
benefits and antioxidant properties
- Best known for its presence in red wine

7. Experimental Design
In order to determine the inhibitory capabilities of
resveratrol on the bacterial growth of S. Mutans, I carried
out different experiments to observe different aspects of
bacterial growth.
A. Purple Broth Growth Test - Planktonic Growth
B. Crystal Violet Biofilm Assay - Biofilm Formation
C. ESEM Imaging - Extracellular Matrix and Biofilm
Formation
D. Human Participant Trials - Plaque Accumulation

8. Hypotheses
• If resveratrol is added to the S.mutans living media, than
resveratrol will inhibit the growth.
• If resveratrol is added to the S. mutans biofilm, than resveratrol
will inhibit and disrupt the formation of the biofilm.
• If a test group brushes their teeth with toothpaste containing
resveratrol, then this group will have the smallest amount of
plaque on their teeth compared to the control group.
• If a test group rinses their mouths with mouthwash containing
resveratrol, then this group will have the smallest amount of
plaque on their teeth compared to the control group.

9. The results of the test will
signify if the bacterial planktonic
growth was disrupted by the
resveratrol
Swabs from test tubes will also
be taken and smeared on
different agar plates and they
will be incubated at the same
temperature in order to observe
growth post treatment
The tubes are shaken at 37
degrees celsius for 24 hours
Procedure Part A: Purple Broth Growth
Test (Planktonic Inhibition)
A 10% sucrose solution of
purple broth growth was
prepared.
10 mg of resveratrol/DMSO
stock solution will be added to
each of the 10 tubes for the
test.
Different concentrations of
resveratrol will be added to 40
different test tubes containing
the broth, sugar, and bacteria.

10. Results
Since S. Mutans is a facultative anaerobe, the bacteria secrete lactic acid as
a byproduct of biological functions. The lactic acid changes the color of the
purple broth to yellow. A positive test is yellow, negative remains purple.

12. Data & Graphs
anova t-test et

13. Procedure Part B: Biofilm Formation Test
All data was recorded and pictures were taken
The optical density of the solution was then
measured at 575 nm.
The bound dye was then extracted using
200µL of ethanol/acetone mix.
Then the bacteria were immersed in water
twice to rinse the dye.
The remaining bacteria then were stained with
50µL of 0.1% crystal violet for 15 minutes at
room temperature
The plates were then blotted with paper
towels and air dried.
500 µL of pre ordered samples of S. mutans
will be transferred to BHI medium and grown
at 37°C to an optical density of 0.5 at 600 nm.
5.5 ml of BHI/bacterial media will be added to
all 4 tubes
Control group will receive 5 ml of BHI media
and instead of bacteria it got 100µL of DMSO.
A different concentration of resveratrol was
administered to each group of 8 micro wells
Plates were incubated at 37°C for 48 hours
The plates were slowly immersed in water and
dumped out to remove planktonic cells

14. Results
The more crystal violet stain absorbed, the more biofilm was intact after resveratrol treatment

15. Data & Graphs
As resveratrol concentrations increased, percent biofilm formation decreased.

16. As resveratrol
concentrations increased,
absorbency decreased. The
p value is less than .01,
indicating it is viable data.

17. Procedure Part C: ESEM Data
Swabs from the 250µL culture and the control culture were put on glass slides
and observed under and Environmental Scanning Electron Microscope
DMSO- Clear biofilm and continuous
matrix is seen. Continuous bacteria.
Matrix formation evident. Biofilm
formation.
Resveratrol- S. mutans in scattered
patches, no biofilm formed. Little to no
evidence of extracellular matrix.

18. The second with 20 random subjects aged 15-18
(with signed consent forms).
Both groups used mouthwash and toothpaste
twice daily, the experimental group getting
products with a .8% concentration of resveratrol
instead of regular.
Before and after each trial individuals chewed
plaque indicating tablets and had a picture
taken of their teeth.
A grid was laid over each photo, and the total
teeth area divided by the total plaque area
provided the percentage growth.
Paired T-Tests were used to evaluate
significance of the data collected
These experiments were devised to
test the inhibitory properties of
resveratrol outside of a laboratory
setting, in vivo.
Two different human participant
trials were carried out.
The first with 10 random adults divided
into a control and an experimental
group.
They used normal toothpaste or
resveratrol infused toothpaste for twice
daily for two weeks.
The resveratrol was at a 3%
concentration in the experimental group
Procedure Part D & E: Human Participant
Trials

19. Results: Part 1
Experimental Group Before and After Photos

20. Data & Graphs:
Part 2

21. Results: Part 2
Control Group Before and After Photos:

22. Results: Part 2
Experimental Group Before and After Photos:

23. Data & Graphs:
Part 2

24. Data & Graphs: Part 2

25. Conclusion
The purpose of this research was to determine if
resveratrol could exhibit inhibitory effects on the caries
causing bacteria, Streptococcus Mutans.
The results of this study indicate that resveratrol can
• disrupt planktonic growth of S. Mutans
• disrupt biofilm/ECM formation of S. Mutans
• significantly lower plaque formation in the human
mouth

26. Applications & Future Research
• This research is important because resveratrol could potentially replace
toxic antiseptic chemicals in not only oral hygiene products, but also a
large array of products containing the same harmful compounds.
• In the future I would like to research other similar powerful polyphenols
and their inhibitory properties on bacterial growth. I also hypothesize that
resveratrol may be an effective inhibitor of the growth of other bacteria in
the streptococcus family, including S. Pyogenes and S. Pneumoniae and
would like to research that as well.
• Resveratrol could also potentially be used in topical analgesics for minor
cuts and burns to prevent bacterial infection, analgesic throat sprays to
target strep throat, and a combination of polyphenols could be used as a
novel antibiotic

27. Acknowledgements
A very special thanks to…
• Dr. Brian P. Ramos, Scientific Research Advisor
• American Heritage Plantation Laboratory, BSL 2
• My Parents, for supporting my scientific endeavors!