A study on the effect of Psidium guajava (guava leaves) and Listerine mouthwash on Streptococcus mutans.

Research Question:
To what extent does increase in concentration (10%, 20%, 30%, 40%, 50% and 60%) of natural leaf extract Psidium
guajava (Guava leaves) and chemical therapeutic mouthwash like Listerine effect the bacterial growth of Streptococcus
mutans suspension measured in terms of pH?
Introduction:
There is an abundance of Pathogens in the world today1. This has led to many people falling ill every now and then. India
is an extremely biodiverse country2 and hence different kinds of medicines are used and preferred by the people living
here. Medicinal practices widely accepted and followed in India consists of Allopathic medicines and Ayurvedic
medicines out of which Ayurvedic medicines still remain the mostly used3. In allopathic medicines different parts of the
plants are used for instance the flower (cloves), leaves (Guava leaves) and roots (ginger)4 to cure problems like
inflammatory gums, dental carriers and common flu. As a result of this in-depth years of research it has come to our
knowledge that leaves and their extracts can inhibit the microbial activity especially that of oral bacteria like
Streptococcus mutans5 (S. mutans). This is an indication that plants that manifest higher level of microbial action can be
utilized as sources that can inhibit the growth of foodborne pathogens.
An online blog “Utilize the Healthy Benefits of the Guava Leaves” published by Nursey live 6 states that people should use
guava leaves in their daily oral hygiene routines. I wondered whether these guava leaves were more effective than
therapeutic mouthwashes like Listerine commonly used by majority of the population. Also there was a keen interest in
finding out how different concentrations of the Psidium guajava (P. guajava) extract and Listerine solution impacted the
growth of S. mutans found in the buccal cavity7. The above study is worthy of investigation as approximately 23.49
million customers use Listerine mouthwash 1 to 5 times a day8. This can be a matter of concern for Listerine contains
many chemicals and alcohol that can lead to organ failure, blindness etc. 9therefore it is important that people are informed
that natural elements like P. guajava might be more or equally effective and are also readily available with no side effects.
Background Information:
Guava Leaves- The guava plant (P. guajava), has been preferred since time in memorial for its therapeutic properties.
After scientists conducted detailed study of the leaf, it was found that P. guajava consists of active components like
flavonoids10. These flavonoids aid in treating gastric diseases like diarrhoea, gastroenteritis and vomiting. Not limited to
this but it also helps in treating oral problems like toothache, inflamed gums, and mouth ulcers11. As the guava plant has
originated in Southern Asian region, researchers have found that people living in rural areas of southern Asian continents
like India have maintained oral hygiene even though they do not have access to modern dental medicines and treatments 12.
Hence because of its medicinal properties since ancient times, P. guajava leaves were chosen for this investigation.
Listerine Solution- Listerine solution is an antiseptic mouthwash which is widely used by different households across the
globe in order to prevent tooth decay, bad breath, plaque formation and to kill bacterial germ present in the buccal
cavity13. The Listerine solution has various active ingredients along with essential oils. It contains-
0.042% of menthol formally known as mint, 0.064% of thymol also known as thyme, 0.06% of methyl salicylate
1
Godfree, Alan. "Pathogens." Pathogens - an Overview | ScienceDirect Topics. 2003. Web. 23 Nov. 2018.
<https://www.sciencedirect.com/topics/engineering/pathogens>.
2
"Megadiverse Countries." Biodiversity A-Z Website. UNEP-WCMC Cambridge, UK, 20 Nov. 2014. Web. 23 Nov. 2018. <http://www.biodiversitya-
z.org/content/megadiverse-countries>.
3 Srinivasan Sandhya. "Allopathy and other Medical Systems." Indian Journal of Medical Ethics [Online], 7. 1 (1999): 5. Web. 24 Jan. 2019
4
Mantz, Annalise. "9 Medicinal Roots For What Ails You." Thrive Market. Thrive Market, 05 Mar. 2019. Web. 23 Nov. 2018.
<https://thrivemarket.com/blog/medicinal-roots>.
5
"Fun Facts About Leaves." Ilona's Garden. 22 Oct. 2018. Web. 23 Nov. 2018.
6
Honey, Sonu. "Utilize The Healthy Benefits Of The Guava Leaves." Blog post. Nurserylive.com, 13 Jan. 2017. Web. 23 Nov. 2018.
<http://blog.nurserylive.com/2017/01/13/utilize-the-healthy-benefits-of-the-guava-leaves-and-gardening-in-india>.
7
Matsui, Robert, and Dennis Cvitkovitch. "Acid Tolerance Mechanisms Utilized by Streptococcus Mutans." Breast Cancer Management : Web. 23
Nov. 2018.
8
"U.S.: Usage Frequency of Listerine Cool Mint (Blue) Mouthwash / Dental Rinse 2018 | Statistic." Statista. Statista, 2018. Web. 23 Nov. 2018.
9
Health, Elements Behavioral. "Drinking Mouthwash Side Effects & Dangers | Hanover, PA." Clarity Way. Web. 23 Nov. 2018.
<https://www.clarityway.com/help-blog/alcoholism-dealing-with-people-who-drink-mouthwash/>.
10
Person. "Psidium Guajava (Guava): A Plant of Multipurpose Medicinal Applications." OMICS International. OMICS International, 28 May 2012.
Web. 23 Nov. 2018.
11
Person. "Psidium Guajava (Guava): A Plant of Multipurpose Medicinal Applications." OMICS International. OMICS International, 28 May 2012.
Web. 23 Nov. 2018
12
Paris, Phillip. "Guava: Origins - Consumption - Nutrition Facts - Health Benefits." Nutritious Fruit. NutritiousFruit.com,Web. Web. 23 Nov. 2018.
13
"LISTERINE® Original Mouthwash." LISTERINE® Antiseptic Mouthwash, Rinse & Oral Care Products. Web. 23 Nov. 2018.

1
sometimes referred as wintergreen, 0.092% of eucalyptol also known as eucalyptus, 21.6% of ethanol and 0.02% of
Sodium fluoride (0.01% weight by volume fluoride ion)14.It is proven that all these ingredients combined together have
antiseptic effects. Methyl Salicylate provides an anti-inflammatory effect to the gums15. Most importantly the sodium
fluoride present in the mouthwash solution makes the teeth enamel more resistant to decay and bacteria 16 also these
fluorides make the teeth enamel stronger17. Mouthwashes are popular products that are used to downsize the effects of
these dental caries causing bacteria18, so the popularity of this mouthwash and its usage in majority of the houses make it
an apt chemical to be investigated.
S. mutans - Discovered in 1924 by J Clarke, S. mutans have been found out to be involved in initiation of dental carries19.
This bacteria possess the ability to ferment various kinds of dietary carbohydrates which hence promotes the drop of pH in
the buccal cavity. S. mutans have also known to be highly evolved acid tolerance mechanisms and that is the reason why
they are able to survive after decreasing the buccal cavity’s pH20 . According to a study, it was observed that S. mutans
bacteria produce acid, as a result of this acid production21, the pH in the suspension drops. For the purpose of this
investigation, the S. mutans bacteria can be obtained from the buccal cavity directly22.
pH measurement- pH can be measured either by using a pH strips or pH probe. In order to measure the pH accurately a
Vernier pH probe could be used. If agents like P. guajava leaf extract and Listerine solution are successfully able to inhibit
the growth of bacterial suspension, the acid produced will reduce and the pH in the bacterial suspension would increase
causing a shift of the pH towards being more neutral i.e. pH 7.
Hypothesis:
H1 Alternate hypothesis: Increasing the concentrations (%) of P. guajava leaf extract will increase the pH by inhibiting
the growth of S. mutans bacteria and will be more effective when compared to the different concentrations of Listerine
mouthwash solution.
H0 Null hypothesis: Increasing the concentration (%) of P. guajava leaf extract will have no impact on the pH but the
Listerine mouthwash solution will be more effective in decreasing the growth of S. mutans and increasing the pH when
compared to the P. guajava extract.
Variables:
Table 1: Variables Selected for this Experiment
Units Range
Independent Different Percentage 10%, 20%, 30%,40%, 50%, 60%
Variable concentrations of ( The range was selected by referring to a similar study on effect of
P. guajava extract azadirachtin on S.mutans23)
and Listerine
mouthwash
solution
Dependent Growth of S. Degree of acidity 0 to 14 where 7 is neutral and below 7 is acidic and above 7 is
Variable mutans in terms of and basicity basic.
pH

14
"LISTERINE® Mouthwash Active Ingredients." LISTERINE®. J&JCI, 2016. Web. 23 Nov. 2018. <https://www.listerine.com/active-ingredients>.
15
"Methyl Salicylate-Menthol Topical : Uses, Side Effects, Interactions, Pictures, Warnings & Dosing." WebMD. WebMD, Web. 23 Nov. 2018.
16
"Fluoride (Sodium) Oral : Uses, Side Effects, Interactions, Pictures, Warnings & Dosing." WebMD. WebMD, 2018. Web. 23 Nov. 2018.
17
"Does Mouthwash Work?" National Dental Care. NDC Australia Pty Ltd., 23 June 2018. Web. 23 Nov. 2018.
<https://www.nationaldentalcare.com.au/does-mouthwash-work/>.
18
"Does Mouthwash Work?" National Dental Care. NDC Australia Pty Ltd., 23 June 2018. Web. 23 Nov. 2018.
<https://www.nationaldentalcare.com.au/does-mouthwash-work/>.
19
Matsui, Robert, and Dennis Cvitkovitch. "Acid Tolerance Mechanisms Utilized ByStreptococcus Mutans." Future Microbiology 5.3 (2010): 403-
17. US National Library of Medicine. 2010. Web. 23 Nov. 2018. <https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2937171/>.
20
Matsui, Robert, and Dennis Cvitkovitch. "Acid Tolerance Mechanisms Utilized ByStreptococcus Mutans." Future Microbiology 5.3 (2010): 403-
17. US National Library of Medicine. 2010. Web. 23 Nov. 2018. <https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2937171/>.
21
Forssten, Sofia D., Marika Björklund, and Arthur C. Ouwehand. "Streptococcus Mutans, Caries and Simulation Models." Nutrients 2.3 (2010):
290-98. National Center for Biotechnology Information, U.S. National Library of Medicine. Multidisciplinary Digital Publishing Institute (MDPI),
Mar. 2010. Web. 23 Nov. 2018. <https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3257652/>.
22
Forssten, Sofia D., Marika Björklund, and Arthur C. Ouwehand. "Streptococcus Mutans, Caries and Simulation Models." Nutrients 2.3 (2010):
290-98. National Center for Biotechnology Information, U.S. National Library of Medicine. Multidisciplinary Digital Publishing Institute (MDPI),
Mar. 2010. Web. 23 Nov. 2018. <https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3257652/>.
23
Kankariya, Amitr, Alokr Patel, and Sankets Kunte. "The Effect of Different Concentrations of Water Soluble Azadirachtin (neem Metabolite) on
Streptococcus Mutans Compared with Chlorhexidine." Journal of Indian Society of Pedodontics and Preventive Dentistry 34.2 (2016): 105. National
Center for Biotechnology Information, U.S. National Library of Medicine. Apr. 2016. Web. 23 Nov. 2018.
<https://www.ncbi.nlm.nih.gov/pubmed/27080957>.
2
 Control Variables Units Need of control How to control?
Amount of bacteria ml If the amount of the S. mutans suspension is not To avoid this, the bacterial
suspension (S. mutans) fixed, the amount or number of bacteria will vary, suspension should be
then the guava leaf extract and Listerine might not measured using a measuring
be able to inhibit the growth and change the pH cylinder (25ml ±0.5) and a
within the same time frame, leading to the dropper.
inaccuracy in the results24.
Amount of the P. guajava ml If the amount of the leaf extract solution was not To avoid this, 10 ml of guava
extract solution fixed then there would be different amounts of leaf extract was added to the
flavonoids in beakers hence the reaction would vary beakers using a measuring
in the same time period and hamper the results25. cylinder (25ml ±0.5) for each
trial.
Amount of Listerine ml If the amount of Listerine Solution is not fixed then To avoid this, 10 ml of
Mouthwash solution there would be different amounts of Sodium Listerine solution was added
Fluoride and Alcohol in the beaker hence the to the beakers using a
reaction time would vary in the same time period measuring cylinder for each
and hamper the results. 26 trial.
Usage time of the PH Seconds If the duration during which the probe was recording To avoid this, the reading time
sensor (Vernier) (±0.2) the data varies then the pH levels would increase if was fixed in the Lab Pro
kept for a longer time period. (I.e. greater than Interphase used to collect the
60secs/1 minute)27. data readings.
Temperature of the ºC If the temperatures of the Listerine solutions and the To avoid this, all the solutions
solutions P.guajava leaf extract solution vary, the activity along with the bacterial
ethanol in Listerine solution would decrease as the suspension were kept inside
ethanol highly volatile and volatile nature increases the incubator at 37oC32.
with increase in temperature28 and Sodium fluoride
(components of Listerine) at higher temperature
would start becoming alkaline29. Similarly the
activity of flavonoids (found in guava leaves) would
decrease30 present would not be the same. Hence if
the temperature was optimum the enzymes would be
the most efficient Resulting in a decreased pH vice-
versa31.

Time of exposure of S. Hours If the time of exposure of S. mutans to the Listerine To avoid this, the exposure

24
"GCSE Bitesize: Temperature, PH and Enzymes." BBC. BBC, 17 Dec. 2007. Web. 23 Nov. 2018.
25
Yartsev, Alex. "Control Variables: Volume and Pressure | Deranged Physiology." Deranged Physiology. Derangedphysiology.com, 16 June 2015.
Web. 23 Nov. 2018. <https://derangedphysiology.com/main/cicm-primary-exam/required-reading/respiratory-system/Chapter%20534/control-
variables-volume-and>.
26
Yartsev, Alex. "Control Variables: Volume and Pressure | Deranged Physiology." Deranged Physiology. Derangedphysiology.com, 16 June 2015.
Web. 23 Nov. 2018. <https://derangedphysiology.com/main/cicm-primary-exam/required-reading/respiratory-system/Chapter%20534/control-
variables-volume-and>.
27
He, Zhongqi, Dr. "Using a PH Meter | Protocol." JoVE Science Education Database. Www.jove.com, Web. 23 Nov. 2018.
28
"Volatility of a Liquid." Duke University- The Alcohol Pharmacology Education Partnership. Wordpress, Web. 23 Nov. 2018
<https://sites.duke.edu/apep/module-4-alcohol-and-the-breathalyzer-test/biology-and-chemistry-connections/volatility-of-a-liquid/>.
29
"Sodium Fluoride." National Center for Biotechnology Information. PubChem Compound Database. U.S. National Library of Medicine, 25 Mar.
2015. Web. 23 Nov. 2018.
30
Sharma, Kavita, Eun Young Ko, Awraris D. Assefa, Soyoung Ha, Shivraj H. Nile, Eul Tai Lee, and Se Won Park. "Temperature-dependent Studies
on the Total Phenolics, Flavonoids, Antioxidant Activities, and Sugar Content in Six Onion Varieties." Journal of Food and Drug Analysis 23.2
(2015): 243-52. ScienceDirect. June 2015. Web. 23 Nov. 2018. <https://www.sciencedirect.com/science/article/pii/S1021949814001379>.
31 Gera, Kanika, and Kevin S. McIver. "Laboratory Growth and Maintenance of Streptococcus Pyogenes (The Group A Streptococcus,
GAS)." Current Protocols in Microbiology. John Wiley & Sons, Ltd, 16 Feb. 2018. Web. 23 Nov. 2018.
<https://currentprotocols.onlinelibrary.wiley.com/doi/10.1002/9780471729259.mc09d02s30>.
32
Gera, Kanika, and Kevin S. McIver. "Laboratory Growth and Maintenance of Streptococcus Pyogenes (The Group A Streptococcus,
GAS)." Current Protocols in Microbiology. John Wiley & Sons, Ltd, 16 Feb. 2018. Web. 23 Nov. 2018.
<https://currentprotocols.onlinelibrary.wiley.com/doi/10.1002/9780471729259.mc09d02s30>.
3
mutans bacteria to the mouthwash is not fixed, then the time given for time was fixed to 3 hours
Listerine Mouthwash Flavonoids (found in guava leaves) and Sodium using a timer.
Solution and P.guajava fluoride (component of Listerine) to inhibit the
leaf extract solution growth of S. mutans would vary. Hence the greater
the exposure time is, the more effective the
P.guajava leaf extract and the Listerine solution
would be.
Equipment: 11. Funnel
3
1. 70 beakers (250 cm each) 12. pH probe (Vernier) (± 0.2)
2. 10 guava leaves (P. guajava) 13. Marker (to label the samples)
3. Listerine mouthwash solution 14. Weighing Machine (Wensar) (± 50 mg)
4. 2 measuring cylinders 15. Incubator (30º C)
5. Tap water 16. Sucrose (34.23 gm)
6. Purified water 17. Cellophane wrap ( to cover the beakers)
7. Blender 18. Petri Dish
8. Dropper 19. Cotton balls
9. Stirrer 20. Timer (Stop watch)
10. Filter paper
Safety, ethical or environmental issues:
1. Use gloves in order to protect your hands from any cuts or exposure of chemical and bacterial infection.
2. Be careful while handling the bacterial solution as it might be a possible pathogen.
3. All equipment and apparatus should be sterilized.
4. Disinfectant should be used before and after use of the bacterial suspension.
5. Label all beakers carefully.
6. No food should be kept where microbes are being stored.
7. Use pH probe and lab pro software with caution and make sure all the data is saved.
8. Glass apparatus like conical flasks and test tubes were handled with care.
9. In case of a spill or a broken apparatus, the area should be covered with 10% bleach solution or 70% ethanol and
paper towels.
10. A mask should be worn at all times when working with the bacteria in order to avoid any infection.
Methods for Solution preparation:
A) Preparation of S. mutans suspension:
1. The buccal cavity was rinsed with purified water before preparing the bacterial solution.
2. 30 ml of water was taken in the mouth and was gargled thoroughly inside the buccal cavity for 2 minutes in order to
obtain the bacteria found in mouth. According to a study by Amitr Kankariya et.al 33, it was seen that the S. mutans
bacteria will be present in the suspension when the above method of extraction was used.
3. The bacteria solution obtained from mouth was poured into a 25 ml beaker.
4. The beaker was labelled as “Bacteria Suspension”.
5. 200 ml of tap water was added into the bacteria suspension to increase its volume.
6. 1M sucrose was added in the bacteria suspension to promote S. mutans growth as sugar is a nutrient for bacteria34.
7. To prepare 1M sucrose, 34.23 grams of sucrose was added to 65.77ml of water.
8. The Bacteria suspension was placed in the incubator at 37ºC for 48 hours35.
9. The beaker was taken out from the incubator and then pH level was measured using a PH probe.
B) Preparation of the P. guajava leaf extract and Listerine solution :
1. 10 fresh P. guajava leaves were taken approximately of the same size, age and from the same branch36.
33
Kankariya, Amitr, Alokr Patel, and Sankets Kunte. "The Effect of Different Concentrations of Water Soluble Azadirachtin (neem Metabolite) on
Streptococcus Mutans Compared with Chlorhexidine." Journal of Indian Society of Pedodontics and Preventive Dentistry 34.2 (2016): 105. National
Center for Biotechnology Information, U.S. National Library of Medicine. Apr. 2016. Web. 23 Nov. 2018.
34
Harrison, D. M., A. J. Daley, K. Rautenbacher, P. M. Loughnan, E. Manias, and L. J. Johnston. "Bacterial Contamination of Oral Sucrose
Solutions." Archives of Disease in Childhood - Fetal and Neonatal Edition 92.2 (2007): US National Library of Medicine National Institutes of
Health. BMJ Publishing Group, Mar. 2017. Web. 23 Nov. 2018. <https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2675471/>.
35
Gera, Kanika, and Kevin S. McIver. "Laboratory Growth and Maintenance of Streptococcus Pyogenes (The Group A Streptococcus,
GAS)." Current Protocols in Microbiology. John Wiley & Sons, Ltd, 16 Feb. 2018. Web. 23 Nov. 2018.
<https://currentprotocols.onlinelibrary.wiley.com/doi/10.1002/9780471729259.mc09d02s30>.
36
Azwanida, NN. "A Review on the Extraction Methods Use in Medicinal Plants, Principle, Strength and Limitation." Medicinal & Aromatic
Plants 04.03 (2015): Print.

4
2. The P. guajava leaves were washed thoroughly and pat dried using a filter paper.
3. 100 ml of tap water was added to a beaker and the 10 leaves were added to it.
4. A blender was then used to crush the leaves.
5. The crushed leaves formed a solution which was then filtered using a sieve to obtain pure P. guajava leaf extract.
6. Tap water was added to the pure leaf extract in order to make the volume up to 100 ml of extract.
7. To prepare 10 ml of solution at different concentrations of P. guajava leaf extract and Listerine Solution, the following
measurements were used.
Concentrations of P. guajava leaf Volume of pure P. guajava leaf Volume of tap water (ml)
extract and Listerine Solution extract and Listerine solution (ml)
10% 1 ml 9 ml
20% 2 ml 8 ml
30% 3 ml 7 ml
40% 4 ml 6 ml
50% 5 ml 5 ml
60% 6 ml 4 ml
Methodology (for the main lab):
1. 60 beakers were taken and 30 each were divided respectively for Psidium guajava leaf extract and Listerine solution.
2. The time was so managed that pH of beakers taken out after 3 hours from the incubator was calculated using a pH
probe while the other set of breakers with different concentrations were prepared and placed for another round in
incubator.
3. In total 5 trials were run for each concentration, where in first row 5 beakers were labelled as 10% P. guajava extract
and the second row was labelled as 10% Listerine Solution.
4. 10 ml of each 10% P. guajava solution and 10% Listerine solution was added to the respective beakers for all the 5
trials.
5. 5 ml of the bacterial suspension was added to the all the 10 beakers.
6. The beakers were placed in an incubator for 3 hours at 37ºC37.
7. After 3 hours, the pH in each beaker was measured using a pH probe and the device was connected to the Lab Pro
Interphase which was then connected to a laptop using a Lab Pro Software as shown in image 2 below.
8. For each trial, the pH probe was inserted inside the respective beakers and the pH data was recorded at an interval of
one minute.
9. The same process was repeated for the remaining beakers (20%, 30%, 40%, 50%, and 60%) for Listerine as well as
Psidium guajava leaf extract.
10. The data collected was recorded in the table prepared (table 3 and 4).
11. The mean pH for Psidium guajava solution results and for Listerine solution results was calculated (appendix 1).
12. Percentage difference between the mean pH before and after the exposure of P. guajava extract and Listerine solution
was calculated.
13. Graph was also plotted to see the percentage difference between the mean pH before and after the exposure of P.
guajava extract and Listerine solution to observe the changes in trend.
14. Average pH of bacterial suspension without any P. guajava leaf extract and Listerine solution was calculated
using pH probe and its value was 5.751.

37
Gera, Kanika, and Kevin S. McIver. "Laboratory Growth and Maintenance of Streptococcus Pyogenes (The Group A Streptococcus,
GAS)." Current Protocols in Microbiology. John Wiley & Sons, Ltd, 16 Feb. 2018. Web. 23 Nov. 2018.
<https://currentprotocols.onlinelibrary.wiley.com/doi/10.1002/9780471729259.mc09d02s30>.

5
Image 1: Trial 1- 10% concentration of the two solutions Image 2: pH probe and Logger Pro software

Analysis
Qualitative data- It was observed from table 3 and table 4 that the pH of S. mutans bacteria suspension was showing
values closer to neutral when added in the P. guajava solution in comparison to the Listerine solution. The colour of the
solutions (between 10% and 60%) were different because of the varying concentration of the above extracts, therefore as
the concentrations increased the solution’s colour became a darker green for Psidium guajava leaf extract and darker blue
for Listerine solution. Moreover, there were no changes observed in the smell of the Psidium guajava leaf extract and
Listerine solution after the addition of the bacterial suspension.
Quantitative data- Five trials were made up for all the different concentrations (10%, 20%, 30%, 40%, 50% and 60%)
(The range was selected by referring to a similar study on effect of azadirachtin on S.mutans38). All the solutions were
kept at a constant temperature of 37oC in the incubator. The raw data of pH values can be seen in the table 1 and 2 below
along with the mean pH at each concentration and its standard deviation. The pH of S. mutans bacteria suspension before
addition of the extract and solution was 5.751.

Table 1: Effect of the increasing concentration of P. guajava extract on the pH of S.mutans bacterial solution39.
Different Mean pH Standard
concentrations of P. Deviation
of P. guajava pH of P. guajava extract at different concentrations guajava
extract Error (+/- 0.2) extract

1st trial 2nd trial 3rd trial 4th trial 5th trial Error
(+/- 0.2)

10% 6.223 6.225 6.298 6.482 6.130 6.272 0.132

20% 6.823 6.725 6.798 6.982 6.851 6.836 0.094
30% 7.443 7.368 7.374 7.130 7.632 7.389 0.180
40% 7.902 7.923 7.817 7.951 7.117 7.742 0.353
50% 7.854 7.813 7.884 7.907 7.521 7.796 0.158
60% 7.990 7.614 7.908 7.963 7.928 7.881 0.152

38
Kankariya, Amitr, Alokr Patel, and Sankets Kunte. "The Effect of Different Concentrations of Water Soluble Azadirachtin (neem Metabolite) on
Streptococcus Mutans Compared with Chlorhexidine." Journal of Indian Society of Pedodontics and Preventive Dentistry 34.2 (2016): 105. National
Center for Biotechnology Information, U.S. National Library of Medicine. Apr. 2016. Web. 23 Nov. 2018.
<https://www.ncbi.nlm.nih.gov/pubmed/27080957>.

6
Table 2: Effect of the increasing concentration of Listerine solution on the pH of bacterial suspension.
Different Mean pH Standard
concentrations of Deviation
of Listerine pH of Listerine mouthwash at different concentrations Listerine
solution Error (+/- 0.2) solution

1st trial 2nd trial 3rd trial 4th trial 5th trial Error (+/-
0.2)

10% 6.457 6.223 6.954 6.383 6.238 6.451 0.298

20% 6.934 6.701 6.891 6.934 6.812 6.854 0.099
30% 6.792 6.824 6.941 6.934 6.872 6.873 0.066
40% 7.180 7.130 7.194 7.118 7.133 7.151 0.034
50% 7.317 7.318 7.398 7.428 7.364 7.365 0.049
60% 7.816 7.723 7.930 7.772 7.721 7.792 0.086
Mean pH and Percentage difference: The data collected after the 5 trials of both the independent variables was first
arranged as illustrated in table 1 and table 2. Then the mean value of the 5 trials for all the concentrations (10%,
20%, 30%, 40%, 50% and 60%) were calculated using the following formula:

Mean pH=
Following these mean values, the data was also processed by calculating the percentage difference (%) between the mean
pH of the bacterial suspension before addition of P. guajava and Listerine Solution and the mean pH of bacterial
suspension after addition of P. guajava and Listerine Solution.
The percentage difference was calculated using the formula written below:

Mean pH after experiment at a particular concentration − Mean pH before experiment at that concentration
× 100
Mean pH before experiment at that concentration
The processed data obtained from the formula can be seen in Table 3 and Table 4.

Table 3: Percentage difference in pH of P. guajava extract post the addition of bacteria suspension.
Different Mean pH of S. mutans Mean pH of S. Difference Percentage
concentrations of P. bacteria suspension mutans bacteria difference
guajava extract before addition of P. suspension after P.
added to the guajava guajava extract is
bacterial suspension added
10% 5.751 6.272 0.521 9%
20% 5.751 6.836 1.085 19%
30% 5.751 7.389 1.638 28%
40% 5.751 7.742 1.991 35%
50% 5.751 7.796 2.045 36%
60% 5.751 7.881 2.130 37%

7
Table 4: Percentage difference in pH of Listerine solution post the addition of bacterial suspension.
Different Mean pH of S. mutans Mean pH of S. Difference Percentage
concentrations of bacteria solution before mutans bacteria Difference
Listerine addition of Listerine suspension after
mouthwash added Solution Listerine solution is
to the bacterial added
suspension
10% 5.751 6.451 0.700 12%
20% 5.751 6.854 1.103 19%
30% 5.751 6.873 1.122 20%
40% 5.751 7.151 1.400 24%
50% 5.751 7.365 1.614 28%
60% 5.751 7.792 2.041 35%

Table 5: Comparison of percentage change in P. guajava extract and Listerine solution.

Concentration of Percentage difference Percentage difference
solutions in Psidium guajava in Listerine solution
extract
10% 9% 12%
20% 19% 19%
30% 28% 20%
40% 35% 24%
50% 36% 28%
60% 37% 35%

Graph 1: Comparison between percentage difference in pH of bacterial suspension before and after addition of P.
guajava and Listerine Solution.

45%

40% 37%
35% 36% 35%
35%
Percenate Difference

30% 28% 28%

24%
25%
19% 19% 20%
20%

15% 12%
9%
10%

5%

0%
10% 20% 30% 40% 50% 60%
Percentage differnce in Psidium guajava leaf extractConcentrations
Percenatge difference in Listerine solution y = 0.0417x + 0.084
Linear (Percentage differnce in Psidium guajava leaf extract )
y = 0.0566x + 0.0753

8
 Pearson Correlation Coefficient40
The Pearson Correlation Coefficient is a statistcal test used to find the degree and strength of correlation between
increasing concentrations of P. guajava leaf extract and Listerine Solution and the pH increase of the S. mutans bacteria
suspension. The following formula is hence used to calculate the Pearson Correlation Coefficient:

∑ (𝑥 − 𝑥̅ )(𝑦 − 𝑦)
𝑟=
∑ (𝑥 − 𝑥̅ ) ∑ (𝑦 − 𝑦)

The ‘r’ value of the Pearson Correlation Coefficient of increasing concentrations of Guava leaf extract is 0.978 and for
Listerine Solution is 0.940. This implies that both these solutions are cases of strong positive correlation. However,
because the ‘r’ value of increasing P. guajava leaf extract is closer to 1, this means that it has a stronger positive
correlation with increasing pH when compared to Listerine Mouthwash.
Results:
The results showed that with the increase in the concentrations (10%, 20%, 30%, 40%, 50% and 60%) of both Psidium
guajava extract and Listerne solution, the pH of the S. mutans bateria suspension kept on increasing i.e. becoming neutral.
Before the addition of the extract and Listerine the pH of the S. mutans bacteria suspension was 5.751 which indicates an
acidic nature and hence is harmful for the teeth enamel. However when 10 ml of Psidium guajava extract was added to
the suspension, the pH started increasing from 6.272 at 10% to 7.881 at 60% concentartion which clearly depicts that with
increasing concetrations of guava leaf extract the bacteria is unable to survive and produce acid leading to gradual
increase in pH. Hence this shifted the acidic pH towards neutral pH. When the same was done for Listerine solution, the
increase in pH was from 6.451 at 10% to 7.792 at 60%. Yet again showing a similar trend. Hence it was proved that both
the solutions were effective in downsizing the effect of S. mutans bacteria.
But the research question aimed at which one of two solutions was more effective. For this, first, the mean pH before and
after addition of Psidium guajava and Listerine Solution was recorded and calculated in for the 5 trials in Table 1. The
positive correlation was observed with the gradually increasing concentrations of P. guajava and mean pH. Though the
pH values at 20% and 30% i.e 6.836 and 7.389 were the closest to the neutral pH of 7 and signify that it is best suited for
the human buccal cavity. Although at further increasinng concentrations till 60%, the mean pH value kept on increasing
and was signifying the basic nature. A similar increasing pH value trend was observed in Listerine Solution as shown in
table 2. The mean PH increased from 6.451 to 7.792 from 10% to 60% and the best suited concentration was found to be
at 30% and 40% (6.873 and 7.151) as the pH values obtaines were closest to the neutral pH. The standard deviation of the
data collected and recorded both in Table 1 and Table 2. The maximum standard deviation was 0.353 at 40% and the
minimum was 0.094 at 20% for Psidium guajava solution. Whereas the maximum standard deviation for Listerine
solution was 0.298 at 10% and the minimum was 0.034 at 40%.
When observed in Table 3, a comparison between the mean pH of S. mutans bacteria suspension before and after addition
of P. guajava extract was noted. Then the percentage difference was calculated as well. The minimum percentage
difference was 9% observed in 10% concentration of P. guajava extract however the maximum percentage difference of
37% was observed in 60% concentration P. guajava extract. Similarly in Table 4, the minimum percentage difference of
12% was observed at 10% concentration of Listerine solution and the maximum difference of 35% was obtained at 60%
concentration of Listerine solution. Hence as the concentrations of both the solutions increased, the percentage difference
also increased, but as seen in Graph 1, the percentage difference of P. guajava extract and S. mutans bacteria suspension
was greater than the percentage change between Listerine solution and S. mutans bacteria suspension. Though both the
solutions showed a similar trend which was a strong positive correlation calculated using the Pearson Correlation test.
Even the equation displayed on graph 1 for P. guajava leaf extract i.e. 𝑦 = 0.0417𝑥 + 0.084 and for Listerine solution
𝑦 = 0.0566𝑥 + 0.0753 was able to support that P. guajava leaf extract was more effective. The gradient for P. guajava
leaf extract was 0.0417 which is less than 0.0566 of the Listerine Solution. As a trend line will always be steeper for a line
whose gradient value is closer to 0. Therefore the trend line for P. guajava leaf extract is steeper and supports that P.
guajava leaf extract was more effective.
To further study error bars were also plotted in Graph 1. At 20% concentration the error bars were overlapping which
indiates that the difference between the values is not that significant. However at 60% concentration the error bars were
partially overlapping. This indicates that the difference between the extract and the solution is probably not that
statistically significant. But at conectrations 10%, 30% 40% and 50% the error bars do not overlap which it indiactes that
the difference is staistically different. Similarly for the P. guajava leaf extract at 40%, 50% and 60% concentration, error
40
"Pearson Product-Moment Correlation." Pearson Product-Moment Correlation. 25 July 2010. Web. 23 Nov. 2018
https://statistics.laerd.com/statistical-guides/pearson-correlation-coefficient-statistical-guide.php

9
bars overlapped which signify that no significant difference occured. As for Listerine solution, at 20% and 30%
concentration, the error bars overlapp which yet again signifies that no significant difference has taken place. Hence it can
be inferred that 40% concentration for P. guajava leaf extract was equally effcetive as 60% concentration of P. guajava
leaf extract. Likewise 20% and 30% Listerine solution have exactly same effects on inhhibiting the growth of S.mutans
bacteria.
Discussion and Evaluation:
P. guajava leaf extract and Listerine Solution, both have had a positive effect on inhibiting the growth of S. mutans
bacteria measured in terms of pH, as the pH of the S. mutans bacteria suspension became neutral from acidic. Moreover,
through this investigation, it was also confirmed by the data that P. guajava leaf extract was more effective when
compared to Listerine solution in terms of increasing the pH at different concentrations of each solution. Although it was
best at 20% and 30% for P. guajava extract and 30% and 40% for Listerine solution discussed in the results above.

Graph 2
"PH." PH | Listerine Global HCP. Web. 23 Nov. 2018.

Now, the question which arises is that, why does the pH of a solution increase when the bacteria is killed? The reason
behind this is that some bacteria like S. mutans produce acid as they grow41 as they are acidophiles. The acid release hence
decreases the pH of the surrounding environment. The pH of Listerine solution is 6.0242. But it was supported that after
rinsing the mouth with Listerine, the mean salivary pH goes to over 7.31 as illustrated in the graph 2. Therefore the
tendency of listerine to maintain the pH by buffering ensures that pH returns to the neutral level a point 43.The pH of the P.
guajava leaf is also almost neutral, close to basic. Therefore as the number of acid producing bacteria reduces, the pH of
the solution returns back to normal. As a result, the investigation can hence infer that the mean pH values collected post
exposure were accurate. This investigation was conducted to see how effective Listerine mouthwash is in real life when
compared to other naturally available elements like P. guajava . After the experiment it was found that the natural
flavonoids like Quercetin found in guava leaves were more effective44 than sodium fluoride found in Listerine
mouthwash. Previously conducted study by George Sam, 2015, compared the effect of neem (Azadirachta indica) with
toothpaste on plaque and gingivitis, and also supports the finding of this research45.
The reason why flavonoids such as quercetin present in the P. guajava leaf is so effective is because they project an
antibacterial effect. According to a study, flavonoids clearly illustrate the in-vitro inhibitory effect on the bacteria isolated
from the buccal cavity that is the oral streptococci and bacteria in saliva 46. This means that the action of flavonoids have
controlled dental caries. This is so because greater the concentration of the solutions the more amount of flavonoids and
sodium fluoride will be present and will be able to effectively kill more S. mutans within the fixed time frame. As
mentioned earlier, the sodium fluoride is of the major components of Listerine mouthwash. The sodium fluoride protects
the teeth in 3 ways. It aids in remineralization of the teeth, helps by hardening the enamel and lastly fluoride also contains
antibacterial properties47. After delving deeper into the antibacterial properties of fluoride, it came to my knowledge that
41
Blamire, John. "Effect of PH on Growth Rate." BIOdotEDU. Science at a Distance, 2000. Web. 23 Nov. 2018
42
"Acidity Index of Common Mouthwashes." Blog post. Http://www.wellnessdentistrynetwork.com. Http://www.wellnessdentistrynetwork.com,
2015. Web. 23 Nov. 2018.
43
"PH." PH | Listerine Global HCP. Web. 23 Nov. 2018.
44
Sankari, S. Leena, N. Aravindha Babu, Vidya Rani, C. Priyadharsini, and K. M. K. Masthan. "Flavonoids – Clinical Effects and Applications in
Dentistry: A Review." J Pharm Bioallied Sci.., July 2014. Web. 25 Jan. 2019. <https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4157275/>.
45
Abhishek, KN, and S. Supreetha. "Effect of Neem Containing Toothpaste on Plaque and Gingivitis." Effect of Neem Containing Toothpaste on
Plaque and Gingivitis (2015): Print.
46
Sankari, Sleena, Naravindha Babu, Vidya Rani, C. Priyadharsini, and K. M K Masthan. "Flavonoids - Clinical Effects and Applications in
Dentistry: A Review." Journal of Pharmacy and Bioallied Sciences 6.5 (2014): Print.
47
"How Does Fluoride Prevent Cavities? -." The Best Toothpastes for a High Decay Rate- OTC vs RX, Brands. 2018. Web. 23 Nov. 2018.

10
fluoride have the ability to constrain the bacterial enzymes that are necessary for the uptake of sugars (i.e. a source of
nutrition for bacteria)48. As a result, as the bacteria fails to provide itself with nutrition, its activities decrease and
ultimately results in a decrease in their number. Graph 1 therefore supports this theory because as the concentration of
Listerine solution increases from 10% to 60%, the pH also increases because the more amount of sodium fluoride that is
present will inhibit more bacterial activity. However if the Listerine solution would be old or expired, then it would not
have been that effective. To avoid this it was made sure that the Listerine solution for all the trials was taken from the
same bottle and the mouthwash used was within 6 months of manufacturing date. This is so, as after Listerine expires all
the active ingredients would not be effective.
Conclusion:
In the investigation above, the effect of increasing concentrations of P. guajava leaf and Listerine solution were seen on
the growth of S. mutans, a bacteria found in the buccal cavity using a pH probe, thus, the research question framed was as
follows:
To what extent does increase in concentration (10%, 20%, 30%, 40%, 50% and 60%) of natural leaf extract P.
guajava (guava leaves) and chemical solutions like Listerine mouthwash solution effect the bacterial growth of
Streptococcus mutans solution measured in terms of pH?
It was concluded that both P. guajava leaf extract and Listerine solution were able to inhibit the growth of S. mutans had a
positive effect on the pH of the S. mutans bacteria suspension. However, it was also observed that P. guajava leaf extract
was slightly more effective in increasing the pH of the streptococcus bacteria when compared to the Listerine solution.
Hence the alternative hypothesis was confirmed by the data.
Because of the naturally present flavonoids in P. guajava leaf and Sodium fluoride present in Listerine Solution, the S.
mutans bacteria is destroyed and hence an increase in the pH is observed which is again confirmed by both the greater
positive regression line in Graph 1 of guava leaf extract and the Pearson correlation proves the alternative hypothesis.
Limitations and Improvements:
There are still some limitations to this study. For making the P. guajava leaf extract 100 ml of water was used and thus the
extract obtained was not in its purest form as it did contain water beforehand. The same applies for Listerine mouthwash
as the solution contained 40% water. Therefore the solution obtained were not in its purest form. Secondly while
collecting the P. guajava leaf, there is a high probability that some of the leaves collected might be old and some might be
new. In this case the new leaves would be more effective as they have a higher content of active flavonoids. Therefore in
order to avoid this, all the leaves were collected from the same branch as that would ensure that leaves have received the
same amount of nutrition and would also ensure that the leaves grew roughly around the same time. Method of extraction
of S.mutans cannot ensure that whether enough amount of bacteria is obtained or not. Cultured S.mutans strains could
overcome this problem. Growth of bacteria is monitored by change in pH. Probably a spectrophotometer could be used to
measure the growth of the bacteria by measuring the transmittance and absorbance of light of a particular wavelength.
Further Study
Other medicinal plants like neem, tulsi (ocimum) and spices like clove, turmeric, and garlic could also be experimented to
find their impact on the bacteria.
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Appendices: Appendix 2- Percentage difference in Table 3 and Table 4
Appendix 1- Mean values in Table 1 and 2
For 10% concentration of Guava leaf extract
For 10% concentration of Guava leaf extract
. . . . . . .
= = 6.272 = × 100 =9%
.

12