2.0 Materials and method

Transcription

1 2.0 Materials and method 2.1 Materials Mouthwash Six types of mouthwash were used in this method- Corsodyl, Dentyl Active, Listerine, Oral B, Oraldene and RetarDex (table 2.1). All of the mouthwashes are easily found in supermarkets in the United Kingdom. They were chosen depending on their active ingredients. Corsodyl, Oraldene and RetarDex were chosen because of their unique active ingredients. Dentyl Active, Listerine and Oral B mouthwashes contain sodium fluoride. Dentyl Active and Oral B are both alcohol free and contain 0.05% sodium fluoride. Dentyl active also contains Cetylpyridinium chloride, an antibacterial agent. All six brands of mouthwash advertise that they are antibacterial. CloSYS II, the active ingredient in RetarDex, is a brand of stabilised chlorine dioxide. The mouthwashes were stored in sterile Sterilin tubes at room temperature. Table 2.1 Table showing active ingredients of the Mouthwash brands chosen. Mouthwash Corsodyl Dentyl Active (alcohol free) Listerine Total Care Oral B (alcohol free) Microorganism and cultures Oraldene Active Ingredient Chlorhexidine digluconate (0.2% w/v) Sodium fluoride (0.05%), Cetylpyridinium chloride Sodium fluoride (0.022%) Sodium fluoride (0.05%) Hexetidine (0.1g/100ml) RetarDex (alcohol free) CloSYS II The primary cause of dental plaque and tooth decay is Streptococci mutans. The standard strain of S. mutans was used. The bacteria were regularly sub-cultured on nutrient agar reinforced with glucose and sucrose. The S. mutans were also regularly sub-cultured in nutrient broth, also reinforced with sugars. The cultures were routinely inspected to ensure that the culture was not contaminated. This was done through daily inoculation and gram staining, whereby a gram positive result indicated that no contamination had occurred. 18

2 2.1.3 Growth of Streptococci mutans The Streptococcus mutans was first grown on nutrient agar however, after 24 hours at 37 C there was no growth on the agar. Emilson and Bratthall's (1976) study on S. mutans growth on various media showed that the optimum growth medium of S. mutans is mitis-salivarius (MS) agar. In spite of this, it is not economical for this experiment. In the oral cavity S. mutans utilises sugars, sucrose and glucose in particular, to initiate dental plaque. Sucrose is also found in MS agar which may contribute to the growth on that medium. Therefore a nutrient agar reinforced with 0.2g of sucrose and 0.2g of glucose was used to culture the bacteria, which resulted in sufficient growth, providing a suitable and economical alternative to the MS agar. The bacteria had also failed to grow in nutrient broth; however when the broth was reinforced with 0.2g of sucrose and glucose growth of the bacteria had occurred. To guarantee optimum growth of the S. mutans in the medium a range of sugar concentrations were used (table 2.2). Table 2.2 Table showing the percentage sugar concentrations and amount of glucose and sucrose added to 100ml of nutrient agar Sugar concentration % Glucose (g) Sucrose (g) The nutrient agar was prepared and different amounts of sucrose and glucose were added. 0.2ml of broth containing the S. mutans was placed in 6 labelled petri dishes. The agar was added to each dish and the dishes were turned to ensure that the agar and bacteria were evenly mixed. The plates were left for 24 hours at 37 C. The plates were divided into sixteenths and the colonies were then counted to produce quantitative results (as shown in fig 2.1). 19

3 Fig. 2.1 Images showing Streptococci mutans growth on nutrient agar containing different concentrations of sugar (shown as a percentage) % 1% 2% 3% 4% 5% The procedure was repeated for more reliable results. Fig 2.2 shows that the sugar concentration for optimum growth of the Streptococci mutans is 0.4%. Fig 2.2 Table and graph showing the number of colonies of S. mutans counted in one sixteenth of the agar plates containing nutrient agar with different sugar concentrations. Sugar concentration %Number of colonies:average Attempt 1Attempt

4 2.2 Method The method used for the investigation is an adaptation of the Antibiotic resistance testing technique and involves impregnating filter paper discs (5mm diameter) with each mouthwash and placing on a bacterial lawn. All equipment, including pipette tips, filter paper discs and glass containers, nutrient agar and nutrient broths were autoclaved for 15 minutes at 121 C, 15psi above atmospheric pressure to prevent contamination from other bacteria. The experiment was performed in a fume cupboard and all equipment was sterile to prevent bacteria in the environment from affecting the results Preliminary experiment The mouthwash diffuses into the agar, and therefore amount of mouthwash infused into the filter paper discs will determine the size of the diameter and zone of potential bacterial inhibition. In order to gain the maximum zone of inhibition the filter discs must be fully saturated. To determine how much mouthwash should be inserted into the filter discs Amaranth Red dye was used. Four discs were each impregnated with μl, 1μl, 2μl or 3μl of the dye. The discs were placed on a nutrient agar plate and left for 24 hours. The diameters of the spread of the dye were measured (fig 2.3) and the discs impregnated with 2μl and 3 μl of dye had the same diameter of spread, therefore the filter paper disc was saturated with 2μl of dye. Therefore in the experiment 2 μl of mouthwash was used. Fig 2.3 A) Image showing the spread of diffusion of Amaranth Red dye inserted into filter paper discs in different amounts B) Table showing the difference in diameter of the spread of the dye A) B) Amount of dye inserted into disc (μl) Diameter of dye spread(mm)

5 The figure (fig 2.3 A) also shows that a diffusion gradient is produced. This will help in interpreting the results, and determining whether higher concentrations of mouthwash are needed to have an effect on the S. mutans Producing a Bacterial Lawn In order to produce a bacterial lawn the bacteria was grown in nutrient broth supplemented with sugar (0.4%). 100 μl of the broth containing bacteria is added to a petri dish. 5ml of nutrient agar is poured into the dish. The amount of agar used must be measured as the depth of the agar in the plate will affect the distribution of the mouthwash through the agar. The liquids are swirled clockwise 5 times, there the disc is rotated 90 and again swirled clockwise 5 times. The disc is again rotated by 90 and the liquids are swirled 5 times anti-clockwise twice. This ensures that the bacteria are distributed evenly throughout the agar, allowing for constant growth and therefore more reliable results. After 24 hours at 37 C the growth of the bacteria was even, however deficient, therefore 200μl of broth was used instead of 100 μl, which increased the quantity of the bacterial growth within 24 hours Method used to determine the bactericidal properties of mouthwash Once the nutrient agar for the bacterial lawn had set, the filter paper discs were impregnated with 2μl of a mouthwash. Each petri dish can hold four filter discs, whilst retaining enough space to allow for diffusion of the mouthwash in the agar, therefore each mouthwash was infused in four filter paper discs. The four discs containing the same mouthwash were placed in each agar plate. The discs were placed to allow a 25mm diameter of spread (the maximum for the dye was 23mm). The petri dishes were sealed with tape and kept at 37 C for 24 hours to allow the mouthwash to diffuse and act on the bacteria. After 24 hours the plates were viewed. The zone of inhibition (area of no bacterial growth around the filter paper disc) was measured with a ruler and the area was 22

6 calculated. Once the results were noted, the filter paper discs were removed to prevent any residual mouthwash from diffusing into the agar. The plates were again left at 37 C. The plates were checked every 24 hours to monitor growth of the bacteria in the previously inhibited areas. The plates that showed growth of the bacteria in the inhibited zones were noted, along with the day number.the experiment was repeated for reliable results and to negate any anomalies. The filter paper discs were placed before the bacteria was able to establish a bacterial lawn as this would show the inhibition and killing properties of the mouthwash against Streptococci mutans. If the lawn was left to grow first the results would not show the preventative properties of the mouthwash Control In order to ensure that the inhibition of the bacterial growth was a result of the mouthwash, controls were designed. In one control standard distilled water was used to impregnate the filter paper discs instead of mouthwash to ensure that the inhibition was not due to the presence of liquid but because of the active ingredients found in the mouthwash. Another control was designed to ensure the growth of the bacterial lawn from the culture. It was also used to ensure that the growth was even throughout the lawn. This control was repeated for each individual plate. During the mixture of the agar and the bacteria, the control and the test were mixed simultaneously. The controls were then used as a comparison between the plates to ensure equal growth between the plates and also compared to the relative plate in the case of anomalies in the results. The controls also ensured that there was continual bacterial growth throughout the experiment and that the growth as not inhibited by other factors, such as lack of nutrients. 23

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