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Periodontal disease note
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Unbranded Mouthwash
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Recommended: Periodontal disease note
How branded and unbranded mouthwash effect cell numbers?
Research and Rationale:
In 2001 it was reported by BBC news that serious dental diseases could be one cause of diabetes. Gingivitis is the periodontal disease that is talked about in the article, this is a disease that causes inflammation to the gums and if left untreated could lead to periodontitis which eventually leads to the loss of teeth. There is no hard evidence for gum disease leading to diabetes however a spokeswomen for the British Dental Association (BDA) said “Studies are currently underway to explore further the relationship between periodontal disease and diabetes.” She also said that there was growing evidence linking periodontal diseases with other medical conditions as well as
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diabetes. Periodontal diseases are gum diseases that are caused by bacteria and plaque that builds up when the teeth are not taken care of. If periodontal diseases are linked to other medical conditions then it is vital to carry out this investigation as to see what mouthwash can help the prevention of bacteria building up and causing periodontal diseases. http://news.bbc.co.uk/1/hi/health/1283644.stm http://www.medicalnewstoday.com/articles/241721.php http://www.medicinenet.com/gum_disease/article.htm There are many different forms of bacteria found in the mouth such as: Streptococcus mutans- This bacteria feeds of sugars and starches that humans eat, and produces plaque and acids that breaks down the tooth enamel and causes cavities Porphyromonas gingivalis- this is a bacteria that leads to periodontitis which is a disease that effects the tissue and the bone that support the teeth which could lead to tooth-loss Escherichia coli- Usually found in the intestines but some can be fund in the mouth, fresh foods usually contain E.Coli and while some are not dangerous, E,Coli found in the environment such as food or animal faeces can be. http://www.colgate.com/app/CP/US/EN/OC/Information/Articles/ColgateNewandNow/Personal/2013/May/article/SW-281474979087677.cvsp http://bioweb.uwlax.edu/bio203/s2007/allmann_ambe/ http://www.livestrong.com/article/161300-what-are-the-different-types-of-bacteria-found-in-the-human-mouth/ Bacteria come into two categories, Gram-Positive and Gram negative.
The scientist named Hans Christian Gram devised a method in 1884 to differentiate between the two different forms of bacteria relating to structural differences in their cell wall.
http://micro.digitalproteus.com/morphology2.php
On the left is the gram positive bacteria which has a thicker peptidoglycan layer. This is the ayer that retains a crystal violet dye when added to it whereas gram-negative has a thinner layer of this and so unable to retain the dye and is coloured red/pink.
Gram-positive bacteria has a single membrane surrounded by a thick peptidoglycan layer whereas Gram-negative bacteria has a thin peptidoglycan layer in between two membranes and because of this, gram-negative bacteria is more resistant against antibodies due to their impenetrable cell wall.
http://www.diffen.com/difference/Gram-negative_Bacteria_vs_Gram-positive_Bacteria
Mouthwash for many people is something that is used everyday, gram-negative bacteria are very strong bacteria and are hard to kill with such structural properties and from investigating this I am able to see what form of mouthwash is better for the human mouth on killing such bacteria.
Hypothesis: My hypothesis is that branded mouthwash would decrease cell numbers better than unbranded mouthwash. The two mouthwashes I shall be using is listerine and a medicinal one that is not branded. The reason for this hypothesis is because listerine contains substances such as eucalyptol, methyl salicylate and menthol as the active ingredients in them. Listerine also contains an antiseptic substance called thymol with strong antimicrobial properties to fight against tooth decay and infection. The other mouthwash that I will be using is corsodyl that is alcohol free. Alcohol free mouthwashes use a substance in them called cetylpyridinium chloride (CPC) which is an antiseptic that destroys and cleanses bacteria by disturbing the membrane of them eventually killing them. This substance kills gram-positive bacteria, and because it does it would kill a lot of the gram-negative bacteria and so wouldn't be unbranded mouthwash wouldn't be as efficient as branded mouthwash. http://www.dentistryiq.com/articles/2011/09/cpc.html Plan Hypothesis: Branded mouthwash will kill more cell numbers than unbranded mouthwash Independent variable: the mouthwash used in the experiment Dependant Variable: the size of the inhibition/clear zone Pilot study: Aim of the pilot For the investigation there are several different methods that could be used. I shall use three different methods to gather enough information to see which bring the best results. I shall use the pour plate, spread plate and liquid culture method. The aim of the pilot study is to deduce with method would give me better results and the quantities of each substance I may need for this investigation to be successful. Equipment 2 spread plates 2 pour plates 3 liquid cultures(containing nutrients needed for growth of bacteria) 2 bottles of agar Branded mouthwash Unbranded mouthwash Cork borer Sterile pipettes Alcohol(control) Calorimeter 3 cuvettes Disinfectant Bio-hazard tape Regular tape Bunsen burner Paper discs Spread Plate First you warm up the 2 bottles of agar until they have fully melted but cool enough to still touch and work with. Open the bottles up fully and working under the flame of the bunsen burner add 5 drops of the gram positive bacteria. After adding the bacteria still under the flame of the bunsen pour the agar bottle onto the spread plate. Make sure to do these last two stages quickly as the agar starts to go back into the solid form again. Pick up one plastic disc and using a pipette add the branded mouthwash covering the whole disc.(Repeat this for branded and control as well) Still under the bunsen burner flame add the three discs into the spread plate where the culture is contained and seal up with regular tape ensuring the tape is stuck on horizontally on the plate at three areas Also add on the plates bio-hazard tape Repeat this experiment for the gram negative bacteria Pour plate First under the flame of the bunsen burner open the pour plate and add 5 drops of the gram positive bacteria and using the spreading knife, spread the contents equally across the plate. Still under the flame, open the pour plate and using a cork borer, dig in 3 different wells for were your branded, unbranded and control should be. Using a sterile pipette add 3-4 drops of each of the substances into each well ensuring it is only in the well and not anywhere else. Carefully seal the pour plate up using the same technique as the spread plate Also add on the plates bio-hazard tape When being put away ensure the pour plate stays up right so that the contents in the wells does not fall out Repeat tis experiment for gram negative bacteria Liquid culture With the liquid culture in the flasks there is already nutrients and agar and the bacteria itself for the bacteria to grow. There are three flasks each one containing branded, unbranded and the control each one containing gram negative The bacteria had been left for a few days for it to grow before adding anything else using a cuvette we first use water to check if the calorimeter works well. We then take a sample of each culture put it in the cuvette Place the cuvette in the calorimeter ad take down the readings this could be then repeated the gram positive. Risk Assessment The risks that are involved in this experiment are: Chemicals may get into the eyes e.g. the bacteria which could potentially be harmful Bacteria may get onto the hands which could be passed around if come into contact with someone else. The bunsen burner flame could potentially be harmful and burn something. Hot agar could be spilled and be harmful when in contact with the skin To overcome these risks that are involved when doing this investigation is: Wear goggles and gloves when carrying this out Ensure to wash your hands after the experiment is over Make sure do not put your hands in your mouth or rub your eyes when doing experiment Be aware of the bunsen burner flame ensure if not in use to put it on safety flame or turn off Be very carful when working with the hot agar, before using make sure it is coool enough for you work with. Results These results show the area of the clear zones for each of the different plates. Spread plates after 48 hours Mean area in cm² (+) Mean area in cm² (-) Branded 7.02 26.89 Unbranded 60.82 223.65 Control --------------------------- 0.44 Repeat Mean area in cm² (+) Mean area in cm² (-) Branded 14.52 32.17 Unbranded 29.22 45.69 Control --------------------------- 0.06 Pour plates after 48 hours Mean area in cm² (+) Mean area in cm² (-) Branded --------------------------- 3.3 Unbranded 13.53 15.9 Control 0.39 0.05 Repeat Mean area in cm² (+) Mean area in cm² (-) Branded 0.04 8.55 Unbranded 32.67 0.87 Control --------------------------- 0.28 Liquid culture(-) Branded Unbranded Control Arbitrary units 87 88 79 Outcome of pilot From doing the pilot I can see the best method to use is the spread plate method as it brought out reliable results as the original and repeat had similar results The liquid culture method was not good enough as the results for it are far too vague and I wouldn't be able to form a conclusion from it. Also during the experiment both the broths generally looked the same so the results were quite close still making me unable to draw a conclusion. I wouldn't use the pour plate method as the results don't show a difference and are not reliable as the one experiment had no clear zone for the branded, gram positive bacteria and the other experiment shows that the branded bacteria showed a bigger clear zone than the unbranded bacteria for the gram negative experiment making the results in conclusive. This may have been due to the pour plates having wells with the substances in them which could have spilled out leaving the contents across the whole plate mixed up. The results of the spread plate show me a trend with branded and unbranded mouthwash and with gram-negative and positive bacteria. The spread plate was also easier to do as the contents wouldn't move out of place once the agar was set. Once the agar set as well the gram-positive and gram- negative bacteria was spread out evenly because it was put in when the agar had melted whereas with the pour plate, it may have not fully been spread evenly. List of apparatus Alternative Reason for choice Paper discs Wells Use of paper discs: easier to place absorbs all the substance better clear zones formed no need to worry about spilling out Sterile pipettes Normal pipettes Use of sterile pipettes: needs to be done under sterile condition prevention of contamination Alcohol (control) Water Use of alcohol: alcohol is a good control to compare to alcohol in mouthwash and may kill bacteria rather than other substances in mouthwash Bunsen Burner Stove Use of bunsen burner: Burning flame, kills external bacteria more available where experiment is done. Key Variables From the pilot, with the spread plate experiment I used 5 drops of the gram-negative and gram-positive when I added it to the agar, the results showed that 5 drops of it was enough so that is going to be kept constant. If I change the volume of bacteria for each different spread plate this would change the size of the clear zones, so if the volume of bacteria increased then the clear zones would be smaller as there is more bacteria so it would be harder for the contents in the mouthwash to kill them. Another variable that should be kept the same is the incubation time, for my experiment I incubated it at room temperature (23°C) which under the circumstances worked well and had good clear zones. If I kept fluctuating the temperature for different spread plates then the number of bacteria would multiply as bacteria work better in warmer conditions so this will again affect the size of the clear zones. The time of incubation I left my spread plates for were for a maximum of 48 hours for each of them and results were gathered then. All the spread plates should be kept incubated for the same time and results should be taken all at the same time as the longer the spread plates are incubated for the greater the size of the clear zones and I would not be able to draw valid results from it.