Antibacterial Property Of Mouthwash Solution

Does the commonly used mouthwash solutions( Colgate Plax, Listerine Spearmint and Wisdom Enamel Protect) have antibacterial property against Eescherichia Coli, determined by disc diffusion method?

I have always wondered why my parents bother me so much about using mouthwash after dinner. When asked repeatedly, the only answer I got was that it helps in destroying harmful bacteria inside our mouth and thus getting rid of foul smell and maintains oral hygiene. Being an inquirer, I wanted to verify this on my own and really wanted to check if these mouthwash which we use have any antibacterial property at all. It is not only the drugs or pills we buy from a pharmaceutical store which shows antimicrobial property; many household or daily life items we use exhibits antimicrobial property. This fact has been claimed repeatedly and proven through a lot of credible researches. After exploring the composition of the mouthwash solutions commonly available in the local market and enough research, it was noted that almost all these oral solutions used contains a chemical as an active ingredient which is claimed to have antimicrobial property. Thus, with a significant personal interests and curiosity, I aimed to explore if these mouthwash solutions exhibits any antibacterial property or not as a topic of my Biology Internal Assessment.

E.coli¹
E.coli is a commonly found bacteria having the shape of a rod. As a structural component, the outer layer consists of lipids made from multiple saccharides unit and a cytoplasmic membrane inside made of peptidoglycan. The type of relationship this bacteria holds with human is mutualistic. It can be both useful as well as threatening to human physiological system. For example- when it survives within human stomach especially within the villi, it uses the nutrients for it’s own survival and helps in the process of absorption by reacting specifically with certain Vitamins like Vitamin-K. Through the process of digestion, often this microorganism makes it’s way to the urinary bladder and inflates it.

Disc-diffusion method²
The susceptibility of a bacteria to a particular antibiotic may be studied using the Kirb-Bauyer disc diffusion method. The bacteria or microorganism to be tested is cultured to a desired concentration in an appropriate broth (usually LR broth is used). The bacteria culture is then passed on to a petri dish filled with nutrient agar medium or MHA medium using a micropipette or a sterile swab. The filter discs impregnated with the antibiotic solution of the right concentration is installed into the agar medium using a forcep or an inoculation loop.

The medium is then incubated at 37^oC for 24 hours. The antibiotic within the filter discs begins to diffuse slowly and as it diffuses and goes away from the disc the concentration of the antibiotic decreases. At a certain radial distance from the filter disc, the concentration of the antibiotic is so low that the growth of the bacteria or the microbes cannot be inhibited. Thus the region along which the growth of the bacteria has been inhibited is marked by a clear circle and is marked as the zone of inhibition. The diameter of the zone of the inhibition is measured from one clear edge to the other using a metric scale.

The aim of the investigation is to detect if the commonly used mouthwash solutions have antibacterial property or not ; to check if the bacteria E.coli is susceptible to mouthwash solutions or not. The mouthwash solutions used in the investigation are –

Colgate Plax

Listerine Spearmint

Wisdom Enamel Protect

All these are commonly used mouthwash solutions and were procured from local supermarket.

The main aim of the investigation is to check if the microorganism E.coli is susceptible to the mouthwash solutions or not and to what extent. The experiment was carried out using disc diffusion method. The diameter of zone of inhibition were measured in each case and compared against a controlled sample (disc impregnated with water).

pH
The activity of a drug or antibiotic against a particular microorganism depends on the pH of the medium. Each drug has an optimum value of pH at which it works best. Certain drugs lose their activity if the pH of the medium is lower than the desired value while certain drugs exhibit more activity if the pH is lower than the optimum value. To control this, the same nutrient medium was used in all petri dishes.

Moisture content
The extent of moisture in the medium does interfere with the mechanism in which the antibiotic or the drug would act against the bacteria. In order to have a check on this, the petri dishes must be treated in a laminar flow before adding the antibiotic solutions to ensure that the plates are free from moisture.

Components of the medium
The chemical composition of the medium has an effect on the zone of inhibition which we record. For example, if the medium used has high concentration of positively charged ions, the diameter of the zone of inhibition might reduce or even do not show up as well even though the bacterium is susceptible to the antibiotic. This is why, choosing the appropriate medium suitable for the microorganism as well as the antibiotic is really important. Although the current investigation uses nutrient agar plate yet MHA (Mueller Hinton Agar) would have been a better choice.

Since the investigation involves studies with pathogens, certain specific safety precautions must be considered in addition to normal safety measures.

• Hands must be cleaned with disinfectant or soap before starting the work and after finishing it.
• Hand gloves must be used throughout.
• Workstation must be disinfected using ethanol.
• No cultures must be brought outside the laboratory.
• The forceps used must be flame sterilised before using it.
• All caps of the reagents, bacteria culture should be replaced.
• Always a teat pipette or a bulb pipette must be used so that pipetting is not done using mouth.

As the investigation is a bacteriological investigation, each and every waste is a biohazard and disposing them in the correct procedure is really important to ensure that the environment is not harmed in any way during the investigation. All solid waste must be collected in a biohazard bag, treated in an autoclave and then thrown to the garbage bin. All solutions used must be diluted before disposing them into the waste chemical bin, nothing should be disposed off directly into the sink.

Bacteria culture and studying antibiotic susceptibility is a standard practice and does not involve any major ethical issues.

• A sterile nutrient agar plate was taken for the lawn of growth.
• The bacteria culture was added to streak the plate using a micro pipette.
• The plates were allowed to dry for 10 minutes.
• The filter discs impregnated with the mouthwash (using a micropipette) solutions were inserted using a flame sterilised forceps. Three discs of the same type were inserted in each of the plate to collect the data in triplicate.
• The discs were gently pressed on the agar plates using the flame sterilised forceps.
• The lid of the petri discs were closed and turned upside down, marking was done on the cover of the petri dishes to separate the filter discs.
• The petri dishes were then incubated for 24 hours at 37⁰C.
• The dishes were taken out and the diameter of the zone of inhibition was measured using a metric ruler.

The same process was followed for all the mouthwash solution as well as for water as a control.

The mouthwash solutions impregnated into the filter discs were observed to diffuse in case of mouthwash Listerine Spearmint and Wisdom Enamel protect and no diffusion was noted in case of the mouthwash Colgate Plax and control (water). The zone of inhibition was clearly circular in case of Wisdom Enamel while in case of Listerine Spearmint, colony was observed.

The diameter of the zone of inhibition was measured from edge to edge of the clear area.

The data plotted above depicts that control (water) and Colgate Plax has no action against the bacteria. It means that the bacteria E.coli is resistant to it while Listerine Spearmint and Wisdom Enamel has displayed zone of inhibition and the diameter measured are 19 mm ± 0.5 and 21 mm ± 0.5 respectively. It shows that these two mouthwash solutions have action against the bacteria and the bacteria E.coli is not resistant to it.

The zone of inhibition obtained for Wisdom Enamel is longer than that obtained for Listerine Spearmint as indicated by the values of the diameter.

As we have only one factor here diameter of zone of inhibition with three independent levels- Colgate Plax, Listerine Spearmint and Wisdom Enamel, one way ANOVA was used. Moreover, one way ANOVA has been chosen as the data set chosen is quantitative data instead of beinb categorical and the basic aim of the statistical test is to determine if the diameter of zone of inhibition depends on the type of mouthwash or not.

Significance level ( α ) = 0.05 (50%)

Null: There is no correlation between type of mouthwash used and the zone of inhibition

Alternate: There is a correlation between type of mouthwash used and the zone of inhibition

N ( total number of values) = 3 + 3 + 3 = 9

n (number of values in each category) = 3

df_between = n − 1 = 3 − 1 = 2

df_{wit hin} = N − n = 9 − 3 = 6

df_total = N − 1 = 9 − 1 = 8

[ df_total = df_between + df_{wit hin} ; it shows that our calculation to determine degrees of freedom is correct ]

http://users.sussex.ac.uk/~grahamh/RM1web/F-ratio%20table%202005.pdf

Critical value = 5.14

SS_between = \(\frac{39^2+40^2+41^2}{30}-\frac{(39+40+41)^2}{9}\)= 0.67

SS_{wit hin} = (0² + 18² + 21² + 0² + 19² + 21² + 0² + 19² + 22² − \(\frac{39^2+40^2+41^2}{3}\)= 811.33

SS_total = (0² + 18² + 21² + 0² + 19² + 21² + 0² + 19² + 22² − \(\frac{(39+40+41^2)}{3}\) = 812.00

F = mod \(\bigg(\frac{SS_{between }}{df_{between}}-\frac{SS_{wit\ hin}}{dfwit\ hin}\bigg)\)= mod\((\frac{0.67}{2}-\frac{811.33}{6})\) = 134.887

Since the value of F (134.887) is higher than critical value (5.14), the null hypotheses is rejected and the alternate hypotheses is accepted.

Thus it is statistically proved that there is a correlation between the type of mouthwash solutions used and their antibacterial property against E.coli.

The basic aim of the investigation was to answer the research question-

Does the commonly used mouthwash solutions( Colgate Plax, Listerine Spearmint and Wisdom Enamel Protect) have antibacterial property against Eescherichia Coli, determined by disc diffusion method?

As clearly indicated by the statistical test, there is a correlation between the type of mouthwash solution used and its antibacterial property against E.coli.

To be more precise, we could see that among the three mouthwash chosen – Colgate Plax, Listerine Spearmint and Wisdom Enamel, any clear zone of inhibition was not seen in case of Colgate Plax which suggests that bacteria are resistant to this solution or in simple words Colgate Plax does not have any action against the bacteria E.coli while the bacteria Listerine Spearmint and Wisdom Enamel displays antibacterial action as zone of inhibition was observed in those cases. The diameter of zone of inhibition in case of Listerine Spearmint was 19 mm ± 0.5 while for Wisdom Enamel was 21 mm ± 0.5; it indicates that E.coli is more susceptible to Wisdom Enamel in comparison to Listerine Spearmint.

Although the value of diameter of zone of inhibition is longer in case of Wisdom Enamel, we cannot claim that it has a better antibacterial property than the other two mouthwash samples used because the zone of inhibition depends on a lot of factors which have not been controlled here like rate of diffusion, viscosity of the medium, concentration of the mouthwash solution used and so on. Moreover, a pharmaceutical drug with a longer diameter of zone of inhibition may not be a suitable choice for therapeutic use as it might have some strong adverse effects.

The active ingredient of Colgate Plax⁴, Listerine spearmint⁵ and Wisdom enamel⁶ are cetylpyridinium chloride, eucalyptol, cocamidopropyl betaine.

Eucalyptol or extracts of Eucalyptus plants are found to show antimicrobial property and in some cases they are more effective than commonly used antibiotics like Gentamicin and refampin⁷ . Although any evidence of study against E.coli was not obtained but the antibacterial property of Eucalyptus oil has been already evidenced.

Qauternary ammonium salts exhibits antimicrobial property. Cocamidopropyl betaine is a quaternary ammonium salt and thus it has antibacterial property as already evidenced⁸ . Infact, some quaternary ammonium salt like benzalalkonium chloride are used as an active ingredient for various disinfectant solutions.

The diameter of zone of inhibition depends on the amount of organism in the culture medium. Ideally, it is supposed to be standardised using a standard McFarland solution by matching the turbidity visually after adding saline solutions or by using a spectrophotometer to ensure that the absorbance of the culture medium is 1 at a wavelength of 600 nm before it is inserted into the agar plate. None of these was possible due to the unavailability of the required solution or a spectrophotometer in the laboratory.

The type of culture medium used has a large effect on the action of a antibiotic or a pharmaceutical drug in a commercial available oral medicine on a bacteria. Ideally, MHA is a better choice of medium than nutrient agar for E.coli as nutrient agar may have high concentration of thymidine compounds which might inhibit the action of antibiotics against the bacteria.

Although it is confirmed through the statistical tests that there is a correlation between the type of mouthwash solutions and their antimicrobial properties but it is difficult to conclude which one is best due to the lack of certain specific quantitative and categorical data like rate of diffusion, adverse effects if any and so on.

The zone of inhibition was clear for Wisdom enamel while for Listerine Spearmint colony was observed which indicates that there are some bacteria within the area of zone of inhibition having resistance to the active drug component of these solutions.

• The investigation was conducted with utmost care of safety precautions especially keeping in mind the fact that it is a bacteriological experiment and the investigation deal with a pathogen.
• Appropriate methodology relevant to the aim of the research has been used.
• Statistical test has been used to arrive at a valid conclusion to the research question.
• The conclusion arrived has been supported by scientific facts as evidenced from some reliable researches already done.
• All the secondary sources used were credible, reliable and authentic and does display the awareness of information literacy.
• Acknowledgement of all sources referred to has been done throughout the report to avoid any academic misconduct or malpractice.

If given a chance and the appropriate infrastructural support, I would like to extend the investigation to determine the minimum dosage of the solutions – Wisdom enamel and Listerine Spearmint to treat this bacteria E.coli. I would use 6 wells in each agar plate for each sample of the mouthwash. The culture medium injected into these wells will be varied in their concentration through serial dilution method. The minimum dosage required will thus be recorded after incubating the plates for requisite time interval (24 hours) at 37oC. This method of assay is known as MIC (Minimum Inhibitory Concentration) method.

I would also like to apply the same investigation for other pathogens to get an idea about the spectrum of the antibiotic solutions used.

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