Research II Paper

‘Using an in vitro Closed Chamber System as a Model, Can Selected Sugar

Alcohols, found in Sugar-free Chewing gum be an effective method of reducing

the concentration of Streptococcus mutans in Dental Plaque?”

By

Katie Dill

Submitted in partial fulfillment of the requirements for

BIOL 4801

Rogers State University

Fall 2015

Approved by Faculty Mentor: ____________________________________

Date of submission: ____________________________________________

Katie Dill Effects of Selective Sugar alcohols in Dental Plaque

ABSTRACT

Effects of Selective Sugar Alcohols on Streptococcus mutans in Dental Plaque

December 9, 2015

Department of Biology

Rogers State University

Abstract: Streptococcus mutans is the most abundant, pathogenic bacterium that grows in the

human oral cavity. When this bacterium attaches to dental plaque on our teeth, it can erode away

hard enamel creating dental decay. While currently methods of reducing this bacteria exist,

dental caries is still one of the world’s most common diseases that is treatable but can be

expensive to fix, painful and in extreme cases lead to cardiovascular disease, which is why

research into developing new methods of removing this bacteria from dental plaque should be a

priority (Allman, 2007, Burks, 2014). This research contributes to this development by testing

the effectiveness of sugar alcohols present in sugar free chewing gum, on S.mutans within human

dental plaque, using an in vitro closed chamber system to eliminate variability found with in vivo

testing and to isolate the sugar alcohols as the sole cause of the reduction of this bacteria and not

the mechanics of chewing gum itself removing plaque from teeth.

Data was collected from the samples, one ceramic disc per sample, from eight

experimental trials each tested a selected sugar alcohol or control (sucrose) at a five percent

concentration against the S.mutans present in the biofilm on ceramic discs grown in the

laboratory using a diluted saliva solution composed of saliva, sucrose broth and sterile water.

The biofilm was removed from each disc through sonication and from this biofilm a pellet was

obtained using centrifugation. The pellet underwent a 1:2 dilution set that was then plated onto

sucrose agar using the spotting technique and incubated overnight before clear S.mutans colonies

were counted and quantitatively analyzed by calculating concentration factor in colony forming

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units per milliliter (CFU/mL) for each sample. These values were compared between each sugar

type at the end of the experiment. Among the sugar alcohols only 16% of the samples taken

exhibited concentration of S.mutans lower than the control. However, it cannot be said as to

whether or not the sugar alcohols, xylitol, sorbitol, or erythritol are effective in lowering the S.

mutans concentration within dental plaque, given that the sample size obtained from each xylitol

and sorbitol trials was too small and because half of the erythritol samples showed to be effective

at lowering the S.mutans concentration, while the other half contradicts this finding. Although,

since all mannitol samples obtained are higher is concentration of S.mutans than sucrose it can be

stated that mannitol is not effective at lowering the concentration of S.mutans.

Approved by Faculty Mentor: ____________________________________

Date of submission: ____________________________________________

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Introduction

In our mouths we have over 700 bacteria taxa that are free-floating or attached to the

structures within our mouths such as the teeth, gums, cheeks and tongue (Forssten, Bjorklund, &

Ouwehand, 2010). The bacteria that attach to the surface of our teeth can lead to the production

of a biofilm called dental plaque. Dental plaque is composed of microorganisms that can cause

tooth decay by releasing various acids through fermentation of carbohydrates. The acids erode

teeth enamel causing periodontal disease, therefore chemical or mechanical methods are

generally used to remove or prevent dental plaque from building up. This research reviews the

formation of dental plaque on teeth, it’s relation to tooth decay and discusses the possibility of

the effectiveness of sugar alcohols found in sugar-free chewing gum in the reduction of the most

common decay causing bacterium found in the flora of the human mouth, Streptococcus mutans.

Free floating bacteria attach to the surface of teeth, and form dental plaque in three

distinct phases 1) attachment, 2.) formation of microcolonies and 3.) formation of sublingual

plaque biofilm. Dental plaque is a biofilm of microorganisms that are protected by structural

features which include a slime layer to defend against host defense mechanisms or antibiotics,

and fluid channels that provide nutrients and oxygen to the colonies and help facilitate movement

of enzymes, metabolites, and waste products found within the biofilm. Once attached to the

surface of our teeth these bacteria will adapt and grow, via cell division, given the parameters of

their environment, such as differences in pH level, temperature, specific nutritional availability

or requirements or other factors (Gehrig, 2003). As long as the proper conditions are met,

bacteria found in dental plaque can grow on other surfaces besides human teeth, such as ceramic,

glass surfaces, or microtiter dish wells. All of these surfaces have been used as models

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when performing experiments on or observing the aspects of dental plaque biofilms. These

models are often used in conjunction with two specific laboratory systems. One is a Continuous

Flow System, which is a complex in vitro system mimicking the conditions in the human mouth.

The other is a Stationary System, which is a simple in vitro test where bacterial growth and acid

production are observed using a medium that contains the anti-cariogenic substance or another

substance and a bacteria culture (Forssten, Bjorklund, & Ouwehand, 2010).

The oral cavity is equipped with specific features to fight off harmful microbes within the

dental plaque, such as the antimicrobial defenses of saliva, which include proline-rich salivary

proteins amylase, statherin, lysozyme, and histatins, as well as the mucosal immune system

properties such as Secretory Immunoglobin A, which neutralizes viruses and binds bacterial

antigens, such as adhesins, toxins and enzymes (Hauser-Gerspach, 2013). Even though these

defense mechanisms are present within the oral cavity there are still some pathogenic organisms

that can overcome them. An example of a microbe that is one of the most common causes of

tooth decay in humans, but is minimally affected by these defense mechanisms is Streptococcus

mutans.

While there many species of bacteria growing within the human mouth, S.mutans is

abundant, covering between thirty to sixty percent of the oral cavity, and is the most damaging

(Allman, 2007). This bacterial species can adapt to its environment, easily resisting changes in

temperature, moisture and pH. It is a Gram positive, nonmotile and facultatively anaerobic

bacteria typically found within the crevices and in-between adjacent teeth. Attachment of S.

mutans to the tooth surface can happen through two methods (J.Loesche, 1986). One method

occurs when S.mutans metabolizes sucrose, obtained from the food we consume, creating a

sticky polysaccharide that aids the bacteria in attachment (Allman, 2007). The other method is by

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cell-cell interaction between the S. mutans and the primary colonizers of dental plaque, during

stage three of the formation of the biofilm (Forssten, Bjorklund, & Ouwehand, 2010). During its

metabolism this bacteria also releases lactic acid. This combination of plaque and acid buildup is

what causes the hard tooth enamel to break down, causing dental decay (Allman, 2007).

Since dental plaque contains harmful, infectious and decay-causing bacteria, it is

important to understand and investigate different methods of both chemical and mechanical

removal of dental plaque. Flossing, teeth brushing, fluoride rinses and regular dentist visits are

basic methods taken to remove dental plaque, reduce the concentration of bacteria within the oral

cavity and help prevent dental decay; however chewing sugar-free gum containing sugar

alcohols may also contribute to these methods by chemically reducing decay causing bacterial

concentrations within dental plaque. The article, Chewing Gum: the Good, the Bad and the Ugly,

suggest that chewing gum stimulates the rate of salivary flow, increasing it, and raises pH levels,

mechanically removes dental plaque from teeth and reduces gingivitis, all of which can

contribute in reducing bacterial concentrations in dental plaque, thereby reducing the amount of

dental decay (Sharman-, Grover & Blaggana, 2008).

Just as fluoride rinses and toothpaste work as chemical methods to reduce bacteria

concentrations within the human mouth, sugar alcohols, found in sugar-free chewing gum, may

be an effective means of chemically reducing the amount of S.mutans within dental plaque

(Allman, 2007). Sugars alcohols are a type of carbohydrate called polyols. They are part sugar

and part alcohol molecules typically added to various foods as a sugar replacement due to their

low calorie quality (Loveren, 2004). Some examples of sugar alcohols include xylitol, mannitol,

erythritol and sorbitol. Given that alcohol is a known antiseptic used to kill microbes, research

on the effectiveness of sugar alcohols to reduce concentrations of a specific species of bacteria

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within dental plaque could show the alcohol portion of the sugar alcohols to be a contributing

factor (Cultivating Organisms, 2013). Multiple studies already suggest that xylitol is an effective

means of reducing the amounts of S. mutans within the dental plaque; however the methods by

which they tested this idea differed with each study and some studies even suggest bias within

their experiment or the results were unclear. However, most experiments described used in vivo

testing or experimental testing that takes place using a living organism.

For example, in the article, Effect of three-year consumption of erythritol , xylitol and

sorbitol candies on various plaque and salivary caries-related variables, the researchers used a

large sample size of students, from seven to eight years old, from one school to test if consuming

sugar alcohol containing candies on school days for three years, would show a significant

reduction of tooth decay and plaque growth (Runnel, et al., 2013). The physical changes of the

children’s teeth throughout the years, such as loss of baby teeth or changes in tooth size, or

dental work done on different individual’s teeth could have impacted their results, so while

sample size was large enough in this experiment to collect a significant amount of data, there

were major areas of variability. Another article entitled, Xylitol-containing products for

preventing dental caries in children and adults, aimed to observe and describe the effects of

xylitol-containing products xylitol toothpaste, lozenges or pacifiers, for prevention of dental

caries in children and adults. This study involved 5903 participants divided into ten groups and

the use of standard deviation techniques to calculate the results of this experiment. The results of

the study revealed that xylitol has a low probability of reducing the amount of dental decay, but

unfortuantly these results may have an element of bias due to missing data that was not

recovered (Riley et al., 2015).

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These articles also demonstrate the issues with using in vivo experiments. There are so

many variables between test subjects such as age, compliance, diets or oral care that can play a

significant role in the effectiveness of sugar alcohols in reducing the amount of dental plaque and

decay .Therefore, isolating the sugar alcohols and testing them in an in vitro experiment will

allow the sugar alcohols to be the only independent variable within the experiment. This will

eliminate the variability seen with in vivo testing and the probability that chewing gum, through

the mechanics of chewing the gum, is a factor in removing the bacteria from the mouth,

(Sharman, Grover, & Blaggana, 2008).

Even though many methods of teeth cleaning have been established, there is a constant

struggle to keep pathogenic bacteria from overriding the natural protective properties seen within

the oral cavity such as saliva proteins and the mucosal immune system and multiplying out of

control, forming a dental plaque biofilm and causing irritating, and painful tooth decay (Hauser-

Gerspach, 2013). S.mutans can be linked to the majority of cases of tooth decay as it is the most

common pathogenic bacteria living in the oral cavity of humans that produces lactic acid during

metabolism of sucrose. While the decay caused by this bacterial species is generally not life

threatening and is treatable, it is a very widespread disease (Allman, 2007). And so it is

important to understand and develop new methods to remove and reduce the concentrations of

this decay causing bacterium growing within dental plaque (Forssten, Bjorklund, & Ouwehand,

2010). This research investigated if Selected Sugar Alcohols, in Sugar-free Chewing gum, are

an effective method of reducing Streptococcus mutans within Dental Plaque, using an in vitro

Closed Chamber System as a Model.

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Materials and Methods

To set-up this investigation, the

environment of the oral cavity was re-

created using a closed flow chamber

system, set-up within the biology lab at

Rogers State University (Williams &

Bloebaum, 2009). The components of the

chamber system were disinfected before

starting each experimental trial, to ensure

there was zero contamination within the

system. The system included three

polypropylene containers, peristaltic tubing,

a peristaltic pump, two magnetic stir plates,

a magnetic stir bar, dental floss and

multiple ceramic discs (see fig 1). The

chamber acting as the mouth had a built-in

platform (see fig 2.) to tie sterile biofilm grown ceramic discs onto with sterilized dental floss

and a magnetic stir bar and it sat on a magnetic stir

plate set between 60 and 200 rpm during each trial.

This chamber was connected to two other chambers

with peristaltic tubing sterilized with 70% isopropyl

alcohol and rinsed with sterile water. To be at the

same height as the mouth chamber the Solution

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Figure 1 Chamber System

Figure 2: Built in platform


chamber was also placed on top of a magnetic stir plate. The 10 rpm peristaltic pump was used to

pump solution from solution chamber to mouth chamber and was set at its maximum speed, so

that 160mL of solution passed through the entire system within each twenty-minute trial.

At the start of each trial, dental plaque was grown on ceramic disc using a saliva solution

prepared by obtaining five mL of saliva bacterial culture and mixing it with 60mL of sucrose

broth diluted 1:10 using 534 mL of sterile water (M.Altlas & Parks, 1993). To make the sucrose

broth recipe was halved in volume and divided into two solutions. Solution A contained 7.5

grams(g) of pancreatic digest of casein, 6.0 g of sodium acetate, 5 g of sodium diphosphate, 2.75

g of glucose, 2.5g of yeast extract,1.25 g of sodium chloride, 0.25 g of L-cystine, and was

brought up to 250mL using deionized water(DI water). Solution B of the sucrose broth contained

25 grams of ultrapure sucrose and was brought to 250mL using DI water. Both solutions were

autoclaved prior to making the saliva solution. The 60mL of sucrose broth was made by mixing

30mL or Solution A with 30mL of solution B. Sodium thioglycate was omitted from the recipe,

since the bacteria tested can grow with or without oxygen and the function of this chemical is to

remove oxygen, therefore the S.mutans was grown aerobically (M.Altlas & Parks, 1993). To

grow dental plaque, two dental floss stringed ceramic discs were placed in a sterile glass beaker,

covered in saliva solution and incubated at 37 degrees Celsius for four days. The remaining 1:10

diluted saliva solution was divided into two sterile 500mL media bottles and incubated at the

same temperature and during the same 4 day time period as the ceramic discs to promote growth

of S.mutans. Saliva solution provides a pedicle for bacteria to attach to the ceramic discs and an

incubation temperature of 37 degrees Celsius is to mimic the temperature of the human mouth.

New dental plaque and diluted saliva solutions were grown and prepared at the start of each trial.

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After the four day incubation period two biofilm grown dental floss stringed discs were

tied to the platform of the mouth chamber, using forceps sterilized with 70% alcohol, the

sterilized peristaltic tubing was attached to the containers and the pump. About half of the saliva

solution was poured into the mouth chamber, so that the ceramic disc would be submerged in

some of the solution, while the remaining saliva solution was poured into the solution chamber.

The purpose of having a solution chamber is to stimulate salivary flow, once the sugar enters

human mouth. After the saliva solution was poured into the chamber system a member of a

selected sugar was tested by placing a solution of 5% concentration of sterilized xylitol,

mannitol, erythritol, sorbitol or control (sucrose) into solution chamber along with the remaining

saliva solution. The polyols were sterilized using bottle top sterilization process, since it is

unknown whether or not they would survive autoclaving and the sucrose solution was sterilized

using the autoclave. Each sugar alcohol solution was made into ten percent solutions by adding

ten grams of the sugar and dissolving it into 100mL of DI water before sterilizing. For each trial

five percent of sugar solution was measured from these ten percent sugar solutions. After the

solution and sugar had been poured into the chamber system the magnetic stir plate and

peristaltic pump were immediately turned on. The incoming solution of sugar mixed with saliva

solution was pumped at a rate of 8mL per minute, into the mouth chamber, for a time period of

20 minutes during each trial. After the trial, ceramic discs were taken out of the mouth chamber,

placed separately into two sterile containers, one tube labeled sample one and the other labeled

sample two. Each disc was rinsed with sterile water to remove what had not adhered to the discs

and 25mL of normal saline was added to each container containing a ceramic disc and put into a

sonciator for two minutes to shake off the biofilm.

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After the biofilm was removed, two 15ml plastic sterile tubes were labeled sample one

and two, and weighed. The weight of each tube was recorded and 10mL of the biofilm from

sample one was added to one of the pre-weighed 15mL tubes and 10mL of the biofilm solution

from sample two was added to the other 15mL tube. Both 15mL tubes containing 10mL of

biofilm were then centrifuged for 15 minutes at 3800rpm until a small white pellet was formed.

The supernant was then poured off and the tubes were weighed again. The original weight of the

15ml tube was subtracted from the weight of the tube containing the pellet to obtain the weight

of each pellet. Next each pellet underwent a 1:10 serial dilution using normal saline, sterile tips

and sterile microcentrifuge tubes and diluted out to 10-9. Each dilution was plated onto sucrose

agar using the spotting technique. Sucrose agar was used as a differential media to allowed living

S.mutans to grow. The 1 L recipe was accommodated to make 200mL of sucrose agar and

contains three grams of agar powder, 10g of ultrapure sucrose, two grams of tryptose, one gram

of sodium chloride, and 187mL of beef heart infusion these components were mixed together,

autoclaved and poured into sterile dishes (M.Altlas & Parks, 1993). The beef heart infusion was

made in the lab, and was filtered using both a cheesecloth and vacuum filter system to obtain a

clearer infusion (Krause, 1957). Sucrose agar was used because it allows only organisms that can

metabolize it to grow; other forms of bacteria present in a sample will not be able to grow since

sucrose is the only available carbohydrate in the plate. This is important since S. mutans

metabolizes sucrose for energy and therefore the sucrose agar allows this bacterium to grow

given the sucrose-rich environment of the plates.

The plating process required sucrose agar plates, the dilution set for each sample, sterile

tips, and micropipetors. In this process, each plate was divided into nine sections and had five,

five microliter spots spotted onto each section, a total volume of 25 microliters plated per

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section. These plates were incubated at 37 degrees Celsius overnight before they were taken out

and examined for S. mutans. Clear bacterial colonies on the countable plates were counted on

each section of the plate and were charted into a data chart for each dilution set (See fig.3)

After the data was collected it was quantitatively analyzed by calculating a dilution factor

of each sample dilution and then calculating the concentration factor in CFU/mL or colony

forming units per milliliter per each sample, an estimate of how many viable bacterial cells, are

in each original sample. The dilution factor is a ratio of final volume/aliquot volume and

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Figure 3 Control sample


concentration factor is a ratio that is the inverse of the dilution factor (Cappuccino & Sherman,

2014). A comparison graph was made comparing concentration factor in CFU/mL and sugar

type to determine how effective each sugar alcohol was in reducing the amount of Streptococcus

mutans compared to the control. After this experiment was completed, 13 trials had been

performed, three xylitol, three sorbitol, two erythritol, two mannitol and two sucrose trials.

However, due to human error only 8 of the 13 trials contained usable data. Giving a total of 16

samples collected during the entire process( 2 samples per each trial).Therefore, only 2

samples( one trial each) were collected for both sorbitol and xylitol and 4 samples( two trials

each) were collected for each erythritol, mannitol, and sucrose experimental groups.

ResultsPellet weight recorded was different for every sample collected from this experiment, so

standard deviation was performed on each sample set for each sugar to statistically show how

wide the variability is for each sugar group (See Table 1).

Table 1: Pellet Weight Standard DeviationSugar Sample size Standard Deviation for each pellet

weight(g)Average

Pellet Weight for each Sugar

Sucrose 4 0.020615528 0.10Erythrito

l4 0.023804761 0.11

Mannitol 4 0.020615528 0.11Sorbitol 2 0.007071068 0.11Xylitol 2 0.014142136 0.12

When comparing those sugars with the same number of samples, standard deviation of

pellet weight is very small, meaning there is little variation between the differences in pellet

weight for each sugar. And so, pellet weight does not play a significant factor in relation to the

amount of bacterial growth on each plate. Collectively, the colony forming units per milliliter

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maximum value was held by mannitol at 9.36 X 1012 CFU/mL and the minimum value was held

by erythritol at zero CFU/mL. When viewing the plates and counting colonies from each sample

the sugar that had samples with the least amount of S.mutans was erythritol, however this was

only true for the first two samples (See figure 4) the other erythritol trials produced samples with

high amounts of S.mutans.

Comparing the control to the sugar alcohols, sucrose exhibited the least amount of

colonies on each 10^- 9 section of each sample, with the exception of two erythritol samples.

Comparing the samples of all five sugars, all four sucrose samples had the lowest amount of

colony forming units per milliliter with the exception of two erythritol samples (See fig 5).

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Figure 4: Erythritol samples showing least amount of Growth


Xylitol

Sorbitol

Mannitol

Erythritol

Sucrose

0.00E+00 1.00E+12 2.00E+12 3.00E+12 4.00E+12 5.00E+12 6.00E+12 7.00E+12 8.00E+12 9.00E+12 1.00E+13

4.88E+12

5.36E+12

4.20E+12

4.80E+11

2.64E+12

7.08E+12

8.92E+12

4.20E+12

0.00E+00

2.64E+12

9360000000000

6640000000000

3160000000000

8720000000000

9160000000000

2200000000000

Sugar vs. Concentration Factor(CFU/mL)

Sample 1 Sample 3 Sample 2 Sample 4

Concentration Factor(CFU/mL)

Suga

r

And so, based on the data only 16% of sugar alcohol samples showed the possibility of being

effective at reducing the concentration of S.mutans within dental plaque.

Conclusion

Since sugar-free gum can mechanically remove plaque and food debris from teeth it

already has the potential to be a tool of bacterial removal or reduction. Alcohol is an antiseptic

which kills infectious microbes and can be used on living tissue, but not within a living organism

because of its toxicity. Given that sugar alcohols have alcohol as a part of their structure and are

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Figure 5. Comparison graph


present in safe amounts in sugar-free gum, it is thought to be a potential tool in the reduction of

S.mutans in dental plaque (Loveren, 2004, Cultivating Organisms 2013).

This research investigated if Selected Sugar Alcohols, in Sugar-free Chewing gum, are an

effective method of reducing S. mutans within Dental Plaque, using an in vitro Closed Chamber

System as a Model. The importance of testing this research problem is because dental caries is

one of the most widespread diseases amongst humans that are expensive to fix, painful and can

lead to cardiovascular disease if the decay causing bacteria ,S. mutans, travels through the

bloodstream to the heart (Allman, 2007; Burks, 2014).Given that the structure of these sugar

alcohols is part alcohol and that alcohol is often used as an antiseptic to kill microbes, it was

hypothesized that the sugar alcohols would be effective in reducing the concentration of

S.mutans within dental plaque. This hypothesis was tested through a series of experimental trials

that involved preparing diluted saliva solution, growing biofilm on ceramic disc, placing both

components into an in vitro closed chamber system along with a 5% concentration of a selected

sugar group, running this system for twenty minutes each trial, taking samples of the biofilm,

diluting and plating those samples, counting colonies as data and quantitatively analyzing this

data. For this hypothesis to be true it would be expected to see CFU/mL for sugar alcohols to be

lower than the control group samples. However this was true for 16 percent of the sugar alcohol

samples taken. The other 84% of sugar alcohols samples did not give any indication of being

effective in the reduction of S.mutans, since each of their concentration factor values exceeds the

controls. Differences in pellet weight was ruled out as variable in this experiment, since the

standard deviation of each sugar group compared to another experimental groups with the same

amount of samples was very small. Given that there were only two samples taken for xylitol and

sorbitol and half of erythritol of samples showed effectiveness in lowering the concentration of

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S.mutans the results for xylitol, sorbitol and erythritol are inconclusive. However these results

show that mannitol is not effective at reducing the S.mutans concentration within dental plaque.

Although these results do not show that sugar alcohols are an effective method to reduce

the concentration of S.mutans within dental plaque it is important to continue research on this

subject given the abundance and detriment S.mutans can have on worldwide dental and possibly

cardiovascular health (Allman, 2007, Burks, 2014).

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Works CitedAtlas, R. M., & Parks, L. C. (1993). Handbook of microbiological media. Boca Raton: CRC

Press.

Allman, A. (2007). Streptococcus mutans: Tooth decaying Bacteria. Retrieved from Streptococcus mutans: http://bioweb.uwlax.edu/bio203/s2007/allmann_ambe/interactions_with_other_species.htm

Burks, B. (2014). Does cariogenic Streptococcus mutans play a role in Cardiovascular Disease? Undergraduate Reveiw: A journal of undergraduate Research, 1-3.

Cappuccino, J. G., & Sherman, N. (2014). Microbiology: A Labratory Manual tenth edition. Glenview,IL: Pearson Education Inc.

(2013). Cultivating Organisms. In D. R. Wessner, C. Dupont, & T. C. Charles, Microbiology (pp. 1-867). Hoboken,NJ: Wiley.

Forssten, S. D., Bjorklund, M., & Ouwehand, A. C. (2010). Streptococcus mutans, caries and Simulation Models. Nutrients, 290-298.

Gehrig, J. S. (2003). Dental Plaque Bioflilms. 1-6. Asheville, North Carolina, United States.

Hauser-Gerspach, I. (2007, June 26). Adhesion of Oral Streptococci to enamel and dental materials- Studies using a flow chamber and microcalorimetry. University of Basal, pp. 1-89.

J.Loesche, W. (1986). Role of Streptococcus mutans in Human Dental Decay. Mircobiology Reviews, 353-380.

Krause, R. M. (1957). Studies on bacteriophages of Hemolytic streptococci : I. Factors influencing the interaction of phage and susceptible host cells. The Journal of Experimental Medicine, 106(3), 365–384.

Loveren, C. (2004). Sugar Alcohols: What is the Evidence for Caries-Therapeutic Effects? Caries Research, 286-293.

Riley, P., Moore, D., Ahmed, F., Sharif, O. M., & Worthington, H. V. (2015, March 26). Xylitol-containing products for preventing dental caries in children and adults. Cochrane Library.

Runnel, R., Makinen, K. K., Honkala, S., Olak, J., Makinen, P.-L., Nommela, R, Saag, M. (2013). Effect of Three Year Consumption of Erythritol, xylitol and Sorbitol Candies on various plaque and Salviary caries-related variables. Journal of Dentistry, 1236-1244.

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Sharman, Grover, & Blaggana. (2008, September). Chewing Gum: the Good the Bad and the Ugly. Journal of Pierre Fauchard Academy (India Section), 105-114.

Williams, D. L., & Bloebaum, R. D. (2009). Observing the Biofilm Matrix of Staphylococcus epidermidis ATCC 35984 Grown Using the CDC Biofilm Reactor. Cambridge Journals, 143-152.

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