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This experiment aims to compare the effectiveness of salt versus oral mouthwash in inhibiting the growth of oral bacteria such as Treponema denticola, Fusospirochetes, Porphyromonas gingivalis and so on. These are chosen as subjects of experiment as they cause Periodontal Disease or Gingivitis among people. Finding a solution to prevent the growth of these disease-causing bacteria will help solving the problem.
Salt or Sodium Chloride is an ionic compound bonded together by ionic bond between Sodium ion and Chloride ion. Table salt is a processed salt where it is used as a condiment and food preservation. Salt is currently mass-produced by evaporation of seawater. It is also proved to provide medicinal effects. For example, people often heal wounds using salt solution and it is good against infections as it reduces inflammation of wounds. Moreover, researches nowadays have proven salt can bring about an effect in inhibiting the growth of bacteria. This is why people often use a cup of water with two tablespoons of edible salt as mouthwash. Nevertheless, salt solution can be used to treat sore throats. Salt is considered antibacterial because it creates no wet condition for bacteria to grow in. In other words, salt is very good at dehydrating and absorbing water from anything it comes contact with. Salt solution does not kill the bacteria but it inhibits the growth of them. Therefore, it is considered as bacteriostatic.
Mouthwash is a product made for enhancing oral hygiene. Some brands of mouthwash claim that they can help to kill bacteria causing gingivitis and bad breath. Anti-cavity mouth rinse uses fluoride compound to protect against tooth decay. A person has to gargle the mouthwash without the need of brushing and flossing teeth. Besides, mouthwash can also help removing mucus and food particles which are situated down in the throat. This product is bactericidal where it kills bacteria which are found in mouth. Active ingredientsÂ in commercial brands of mouthwash can includeÂ thymol,Â eucalyptol, menthol,Â chlorhexidine gluconate,Â benzalkonium chloride,Â cetylpyridinium chloride,Â methylparaben,Â hydrogen peroxide,Â domiphen bromideÂ and sometimesÂ fluoride,Â enzymes, andÂ calcium. Ingredients also include water, sweeteners such asÂ sorbitol,Â sucralose,Â sodium saccharin, andÂ xylitol. However, there is some evidence which shows that mouthwash contributes to oral cancer. This is shown by a literature review by McCullough and Farah from 2008 published in theÂ Dental Journal of Australia.
Oral hygiene is very important to prevent dental problems and bad breath. One of the common ways to practise oral hygiene is tooth brushing. Some people may use oral mouthwash to kill the disease-causing bacteria situated inside the mouth. One of the common disease regarding oral hygiene is Gingivitis. Gingivitis is a term used to describe non-destructiveÂ periodontal disease. Gingivitis is often caused by types of bacteria stated above. Bacteria activity caused by these microorganisms can lead to severe effects including refractory periodontitis and acute necrotizing gingivitis, resulting in bone resorption and tooth loss. Gargling with oral mouthwash is effective but the price is expensive. Furthermore, it is not always available at any outlets all the time. Practising to gargle with salt water is a convenient method to keep the oral cavity clean. Table salt can be easily obtained from any outlets and the price is far cheaper than any oral mouthwash. One thing good about salt is it does not cause side effects as no artificially made chemicals are contained within it. But how does the effectiveness vary compared to commercial oral mouthwash?
Experimental Hypothesis :
There is a significant difference for the effectiveness of commercial mouthwash in inhibiting oral bacteria compared to salt solution of concentration greater than or equal to 0.4moldm-3.
Null Hypothesis :
There is no significant difference for the effectiveness of commercial mouthwash in inhibiting oral bacteria compared to salt solution of concentration greater than or equal to 0.4moldm-3.
Manipulated variable : Types of antibacterial solution
Responding variable : Number of bacteria colony found on nutrient agar
Fixed variables : Temperature of incubation period, contents of nutrient agar, type of
bacteria cultured, time taken for gargling mouthwash.
Apparatus : Bottles of inoculum, work table, sterilised petri dishes, micropipette,
1000ml conical flask, Bunsen burner, incubator, 100ml beaker,
electronic balance, 250ml volumetric flask, filter funnel, dropper,
stopper, bio-hazard waste bin.
Materials : Sterilised cotton buds, sterilised distilled water, distilled water,
commercial mouthwash B, disinfectant, tissue papers, Dettol solution,
gloves, teats of micropipette, nutrient agar solution,
aluminium foil, solid Sodium Chloride.
Two trial experiments were conducted in overall. The first trial experiment was carried out to determine which commercial mouthwash is the best in inhibiting the growth of oral bacteria. The commercial mouthwash which has the highest inhibition rate will be compared to salt solution. Three types of commercial mouthwash namely A, B and C were used. Three sterilised petri dishes were obtained. A swab sample was taken before gargling the commercial mouthwash. This was tagged as "before" to indicate the number of bacteria colony found in nutrient agar before commercial mouthwash was used and acted as a control. Three human subjects were volunteered to gargle each type of commercial mouthwash. This was assuming that the number of oral bacteria in every person is the same. After gargling three types of commercial mouthwash for 10 seconds, three swab samples were obtained from every subject. The bacteria were cultured and platted and the number of bacteria colony found on agar was counted. All sterilised petri dishes were then placed in an incubator for a week. The result showed that commercial mouthwash B has the highest bacterial inhibition rate.
Types of commercial mouthwash
Number of bacteria colony found on nutrient agar
Table 1.1 : The number of bacteria colony found on nutrient agar with respective commercial mouthwash used.
The second trial experiment was done to determine the time taken to gargle commercial mouthwash B in order to obtain the maximum inhibition rate. In other words, this trial was carried out to choose a suitable period of gargling mouthwash. Five sterilised petri dishes were obtained. Four types of gargling period had been chosen in this trial which were 5, 10, 15 and 20 seconds. Four human subjects were volunteered to gargle for every period of time. Commercial mouthwash B was used as it gave the highest rate of bacterial inhibition from the first trial conducted. A swab sample was obtained before the trial proceeded. This was labelled as "before" to indicate the number of bacteria colony found in nutrient agar before gargling commercial mouthwash B and acted as a control. Every human subject was ordered to gargle commercial mouthwash B with the respective period of time chosen earlier. After gargling, four swab samples were obtained from each subject. The trial was again assuming that the amount of oral bacteria in every person is the same. The bacteria was cultured and platted and the number of bacteria colony found on nutrient agar was counted. All sterlised petri dishes were then placed in a incubator for a week. The result showed that a period of 10 seconds to gargle commercial mouthwash B has the highest bacterial inhibition rate.
Time taken to gargle commercial mouthwash B / seconds
Number of bacteria colony found on nutrient agar
Table 1.2 : The number of bacteria colony found on nutrient agar with respective period of time to gargle commercial mouthwash B.
Real Experimental Procedures
Preparing salt solution of various concentrations
Mass of an empty 100ml beaker was weighed using an electronic balance.
For 0.1M salt water, 1.4625g of solid Sodium Chloride (NaCl) was weighed.
The weighed solid was then dissolved in the 100ml beaker using distilled water.
The solution was poured into a 250ml volumetric flask using a filter funnel.
The beaker and filter funnel were rinsed with distilled water and added into the volumetric flask.
Distilled water was carefully added into the volumetric flask until the level of solution reached the graduation mark.
Dropper was used instead to prevent any exceed above the graduation mark.
A stopper was fitted on the mouth of volumetric flask and the solution was shaken carefully.
Steps 1 to 8 were repeated by replacing 1.4625g of NaCl with 2.925g for 0.2M, 3.4875g for 0.3M, 5.850g for 0.4M and 7.3125g for 0.5M.
Preparing inoculums of bacteria
A sterilised cotton bud was used to obtain the swab sample from mouth.
The cotton bud was then dipped into a bottle of inoculum containing a quarter volume of sterilised distilled water and labelled as 'before'. The cotton bud was then disposed off.
After gargling 50ml salt water with concentration of 0.1M, another swab sample was obtained and dipped into bottle of inoculums containing sterilised distilled water. This was labelled as '0.1M'.
Step 3 was repeated by replacing 0.1M salt water with 0.2M, 0.3M, 0.4M, 0.5M and commercial mouthwash B.
Preparing petri dishes with bacteria
Work table was sprayed with disinfectant to kill all the bacteria present.
Table was wiped with several pieces of tissue papers.
Hands were washed with Dettol solution to ensure no bacteria were found.
Gloves were worn on both hands.
Three sterilised petri dishes were obtained.
A bottle of inoculum (before) was taken.
A micropipette was used and calibrated to 200 microlitres.
Teat of micropipette was placed at the edge of micropipette. Hands were forbidden to touch the teat so that it would not be contaminated with bacteria.
The bottle of inoculum was opened using a hand and the end of micropipette was pressed gently to suck the content of inoculums.
The cap of bottle was closed and the content of inoculum was placed in the first petri dish. The lid was opened slightly till the teat can be placed in the petri dish.
The end of micropipette was pressed fully to release the sucked content of inoculums.
The teat was then expelled into a bio-hazard waste bin.
Same procedures were repeated for second and third petri dishes.
Steps 6 to 13 were repeated by replacing bottle of inoculum (before) with bottle labelled 0.1M, 0.2M, 0.3M, 0.4M, 0.5M and commercial mouthwash B.
Preparing bacterial lawn
Nutrient agar solution was poured into a 1000ml conical flask.
The solution was left to cool down for several minutes.
After several minutes, the mouth of conical flask was heated with a Bunsen burner. This was done so that the aluminium foil could be capped on the mouth of conical flask for sterilising purpose.
The mouth of conical flask was placed gently at a petri dish. The culture solution was poured into the petri dish until it was one-third full. This process is known as platting.
Aluminium foil was capped back on the mouth of conical flask.
The petri dish containing bacteria and agar solution was swirled gently by pressing and moving the petri dish on the table.
The petri dish was left on the table for 10 minutes. This was done to ensure that the culture solution is solidified.
Steps 1 to 7 were repeated for every petri dishes. All petri dishes were inverted and stored in an incubator for a week.
After one-week incubation period, all petri dishes were observed for bacterial activity.
A complete aseptic procedure was used throughout the experiment. The work table was sprayed using a disinfectant to kill all the foreign bacteria present. Hands were washed with Dettol solution and gloves were worn so that I would not be infected by the bacteria when having a meal. Sterilised petri dishes were used because it contained only the oral bacteria when experiment was carried out. This was important to maintain the validity of results obtained. Sterilised distilled water was also used to make sure only oral bacteria were cultured. Teats of the micropipette were disposed off into a bio-hazard waste bin because they were contaminated with bacteria and could possibly infect other people. Disposing off the teats will solve the problem. The lid of petri dish was opened slightly when introducing bacteria into the petri dish. This was done to prevent any foreign bacteria in the lab to grow and reproduce in the petri dish containing agar solution. Distilled water was used to prepare various concentrations of salt solution to make sure no other impurities would affect the concentration or molarity of the solution. The electronic balance was tarred to reset the reading value before weighing a specific mass of solid Sodium Chloride. Dropper was used to prevent any exceed above the graduation mark of volumetric flask when dilution was carried out. Mouth of conical flask was heated with Bunsen burner to ensure no contamination from other bacteria. Aluminium foil was capped back on the mouth of conical flask to prevent any foreign bacteria from entering the conical flask. At the end of the experiment, all petri dishes were sent for autoclaving for disposal purpose.
Types of antibacterial solution
Number of bacteria colony found on nutrient agar
0.1M Salt Water
0.2M Salt Water
0.3M Salt Water
0.4M Salt Water
0.5M Salt Water
Commercial Mouthwash B
Table 1.3 : Number of bacteria colony found on nutrient agar with respective types of antibacterial solution used.
1st reading was obtained from first petri dish.
2nd reading was obtained from second petri dish.
3rd reading was obtained from third petri dish.
Graph 1.1 : Bar chart of mean number of bacteria colony found on nutrient agar against types of antibacterial solution.
There is no significant difference for the effectiveness of commercial mouthwash in inhibiting oral bacteria compared to salt solution of concentration greater than or equal to 0.4moldm-3. The calculated U-values are more than the Ucrit value which is zero at 5% significance level. The null hypothesis is not rejected as the U-values are not lower than Ucrit value. Therefore, null hypothesis is accepted and the experimental hypothesis is rejected. There is insufficient evidence to state that there is a significant difference for the effectiveness of commercial mouthwash in inhibiting oral bacteria compared to salt solution of concentration greater than or equal to 0.4moldm-3.
0.4M salt solution
Commercial mouthwash B
âˆ‘Rank sample 1
âˆ‘Rank sample 2
Table 1.4 : Calculations for Mann-Whitney Test.
Formulae for calculating U-values :
U1 = n1n2 + n1(n1+1) - âˆ‘Rank sample 1
U2 = n1n2 + n1(n1+1) - âˆ‘Rank sample 2
n1 = size of the sample 1 (0.4M salt solution)
n2 = size of the sample 2 (Commercial mouthwash B)
âˆ‘Rank sample 1 = total rank of sample 1
âˆ‘Rank sample 2 = total rank of sample 2
Calculations of U-values for both samples
U1 = (3)(3) + (3)(3+1) - 14.5 = 0.5
U2 = (3)(3) + (3)(3+1) - 6.5 = 8.5
Significance level = 5%
Value of Ucrit according to table = 0
From the experiment conducted, it is shown that the mean number of bacteria colony found in nutrient agar due to commercial mouthwash B is lower than other salt solutions with various concentrations. When the concentration of salt solution is less than 0.4moldm-3, there is a large difference for the mean number of bacteria colony found between commercial mouthwash B and salt solution. The result changes when concentration of salt solution increases to 0.4moldm-3 where there is only a small difference for the mean number of bacteria colony found in nutrient agar.
Commercial mouthwash B and salt solution are proven for inhibiting growth of oral bacteria. The number of oral bacteria found in mouth will drop significantly after gargling because antibacterial solutions kill or prevent the bacteria from growing. The most suitable method to find out the effect of antibacterial solution on the growth of oral bacteria is to count the number of bacteria colony found on nutrient agar after gargling the solutions. The results obtained are compared with the number of bacteria colony found before gargling the solutions which acts as a control. Temperature of incubation is set constant at 36.9oC where it represents the exact body temperature of a human body.
Commercial mouthwash B contains an active ingredient namely Chlorhexidine gluconate. It has both bactericidal and bacteriostatic mechanisms of action. It is a type of cell membrane agent. It disrupts the structure of cell membrane, causing the rigidity of the cell membrane to be broken down. This active chemical binds onto lipopolysaccharides, situated at outer membrane of Gram-negative bacteria, disrupting the structure lipid bilayer consisting of phospholipids. When the fluid lipid bilayer is broken down, cell organelles and metabolites no longer bordered by cell membrane. Loss of metabolites results in death of a bacterium.
Salt solution has a different mechanism in inhibiting growth of oral bacteria. Every microorganism needs an aqueous environment to thrive in. In low concentration of salt solution, the surrounding environment is hypotonic. The solute concentration remains higher than the surrounding solution. Oral bacteria have the ability to pump in ions with the energy comes from ATP by respiratory enzyme found in mesosomes. This ion pump moves ions from surrounding solution into the body of oral bacteria. There is water potential from surrounding solution to the cytoplasm of oral bacteria. Water moves into the cell by osmosis and this gives an aqueous environment which is favourable for oral bacteria to grow and reproduce. At high concentration of salt solution, the solute concentration in the surrounding solution is greater than the cytoplasm of oral bacteria. This is because the ion pump cannot keep up to pump in more ions efficiently. There is water potential from cytoplasm of bacteria to surrounding solution. Water moves out from cell by osmosis. Oral bacteria are dehydrated and eventually die within a minute. 
However, there is a difference in mean number of bacteria colony found when different antibacterial solutions are used. The difference is mainly because active ingredient in commercial mouthwash B kills the bacteria and they can no longer reproduce again. When salt solution is used, bacteria may move away from the solution which has high solute concentration. Bacteria are not killed and may have the chances to reproduce again.
75% lake water
50% lake water
25% lake water
10% lake water
Table 1.4 : number of bacteria found in respective water sample.
From the above data, it was an experiment conducted Dr.Claude E. Zobell and D. Quentin Anderson to investigate the number of bacteria found in different water sample. It can be seen that the number of bacteria colony decreased tremendously when the bacteria were cultured in a sample of sea water. Sea water has a high concentration of salt which will provide a high solute concentration compared to cytoplasm of oral bacteria. The difference from my data and theirs will be discussed in the evaluation section.
There are a few limitations found in this experiment. Contamination may occur when introducing bacteria into the petri dish. Foreign bacteria found in the air may enter the petri dish and reproduce in the nutrient agar. This was inevitable because lamina floor was broken down and could not be used for this experiment. A known species of a bacterium cannot be used as it is not available in the lab. Therefore, swab samples were obtained and these contained a mixture of bacteria as stated above. Experiment cannot be conducted only on a type of bacterium. Besides, nutrient agar solution may not be sterilised completely as the autoclave machine in the lab was broken down and spare parts were not available by that time. Therefore, nutrient agar solution was sterilised using a pressure cooker. This caused other foreign bacteria to survive and reproduce in the nutrient agar. The period of incubation chosen is a week because the bacteria cultured from swab samples are low in number. This requires a longer period for bacteria to grow and reproduce. The human subjects involved may not gargle the antibacterial solutions in a correct way. This will affect the validity of data obtained during observation. Lastly, the genetic makeups of bacteria found in swab samples are not known. This could not be prevented as a specific strain of bacterium was not recognised. As a result, some of the bacteria may have mutated and probably resistant to the antimicrobial solutions. The results obtained will be affected. These are the reasons why the findings are different from this experiment and experiment conducted by Professor above.
The experiment can be modified by using a known strain of bacterium. This will increase the validity of data significantly. Moreover, different type of nutrient agar which is favourable for oral bacteria to grow can be used. This will cause the bacteria to grow and reproduce faster than expected. Thus, results can be obtained earlier. Furthermore, nutrient agar solution can be sterilised with an autoclave machine so any foreign bacteria present will be killed.
There is no significant difference for the effectiveness of commercial mouthwash in inhibiting oral bacteria compared to salt solution of concentration greater than or equal to 0.4moldm-3. Null hypothesis is accepted.
Source 1, 2 and 3 are published books thus the information are reliable. Sources 4 to 8 are journals. They are written by scientists all around the world and these are obtained from Science Direct webpage. Therefore, it is trustable. All the information should be factual and accurate. Source 9 and 16 are websites containing journals as well. Its journals are well-known and most of them are written by famous scientists. Sources 10,11,12,13 and 15 are websites of Wikipedia. Most of the data and information obtained here have citations and they are partially reliable and valid. Source 14 is the official website of Australian Dental Association. The information contained here is mainly about the effect of chlorhexidine. It is very reliable. Therefore, it should not contain any biased points based on arguments made.