**3.3.2 Lactobacillus**

Lactobacillus is considered secondary invaders, can contribute to tooth demineralization once they are established carious lesions. Lactobacilli can be found in the mouth before the teeth erupt, even when the diet is rich in fermentable carbohydrates and no active carious lesions.

They are present in small numbers on the plaque and tend to be found in saliva and in deep carious lesions. Weakly bind to the enamel surfaces, are lazy in their nutritional requirements, are acidogenic and aciduric. They are located in the undercut areas of the tooth, such as defects or margins of fillings or orthodontic bands (Ansai et al., 1994; Heintze et al., 1999; Menaker et al., 1986; Tanzer et al., 2001).

The development of caries should be considered in two-stage process: in Streptococcus mutans involved in lesion initiation and lactobacilli in progression it.

The standard method for determining the presence of lactobacilli is through the selective medium Rogosa SL agar. An alternative was established by Larmas in 1975 with the introduction of the test Dentocult LB®. The advantage is that it can be used in the dental office and the results can be shown to make the patient aware of the presence of lactobacilli in the mouth (Fig. 9).

Fig. 9. Dentocult LB® test.

Values greater than 100,000 CFU / mL indicate a high risk of caries (Heintze et al., 1999; Larmas, 1992).

Dentocult® LB3 procedure (Fig. 10) consists in pour the collected saliva over both agar surfaces, ensuring that they are well moistened. If the saliva is very viscous, the sample can also be applied using a sterile swab, then screw the slide tightly back into the tube and place

<sup>3</sup> Dentocult® LB (Orion Diagnostica, Helsinki, Finland)

the tube in an incubator for 72 hours at 37° C. To obtain a colony count remove the slide from the tube and compare the colony density with the model chart provided in the kit:


166 Contemporary Approach to Dental Caries

Incubate the vial at 37° C for 48 hours with the cap one quarter of a turn open. Interpretation

Lactobacillus is considered secondary invaders, can contribute to tooth demineralization once they are established carious lesions. Lactobacilli can be found in the mouth before the teeth erupt, even when the diet is rich in fermentable carbohydrates and no active carious lesions. They are present in small numbers on the plaque and tend to be found in saliva and in deep carious lesions. Weakly bind to the enamel surfaces, are lazy in their nutritional requirements, are acidogenic and aciduric. They are located in the undercut areas of the tooth, such as defects or margins of fillings or orthodontic bands (Ansai et al., 1994; Heintze

The development of caries should be considered in two-stage process: in Streptococcus

The standard method for determining the presence of lactobacilli is through the selective medium Rogosa SL agar. An alternative was established by Larmas in 1975 with the introduction of the test Dentocult LB®. The advantage is that it can be used in the dental office and the results can be shown to make the patient aware of the presence of lactobacilli

Values greater than 100,000 CFU / mL indicate a high risk of caries (Heintze et al., 1999;

Dentocult® LB3 procedure (Fig. 10) consists in pour the collected saliva over both agar surfaces, ensuring that they are well moistened. If the saliva is very viscous, the sample can also be applied using a sterile swab, then screw the slide tightly back into the tube and place

of results according following score:

2 105 to 106 CFU/mL, 3 >1 000 000 CFU/mL

**3.3.2 Lactobacillus** 

in the mouth (Fig. 9).

Fig. 9. Dentocult LB® test.

3 Dentocult® LB (Orion Diagnostica, Helsinki, Finland)

Larmas, 1992).

0 negative or < 100 000 CFU/mL, 1 100 000 to 1 000 000 CFU/mL,

et al., 1999; Menaker et al., 1986; Tanzer et al., 2001).

mutans involved in lesion initiation and lactobacilli in progression it.


Actually exists the CRT bacteria test (Ivoclar Vivadent AG, Schaan, Liechtenstein) which allows clearly identify and semi-quantitatively determine both cariogenic bacterias.

Prognosis of caries becomes more effective when the lactobacilli and streptococci tests are combined.

Fig. 10. Collect the lactobacillus sample, manufactured chart.

### **3.4 Plaque pH**

Acidogenic bacteria in dental plaque metabolize carbohydrates rapidly getting acid as final product. The result is a change in pH of the plaque, as it relates to the time called the Stephan curve as a scheme to bring it takes a curve. The pH decreases rapidly in the first few minutes to gradually increase; it suggests that in 30 minutes should return to normal levels.

The caries activity test Cariostat®**4**, developed by Shimono, is used to measure the decrease of pH caused by bacterial action in the plaque. It has been reported positive correlations between caries activity test score and the counts of *SM* and *LB*.

Not only can determine whether establishing new carious lesions, but also diagnose active or chronic lesions present (Nishimura et al., 1988; Lara-Carrillo et al., 2010b).

<sup>4</sup> Cariostat® (Dentsply-Sankin KK, Tokyo, Japan)

Clinical, Salivary and Bacterial Markers on the Orthodontic Treatment 169

It has become the leukocytes counts, in these salivary markers as an indicator of periodontal disease, since most of the salivary leukocytes entering to the oral cavity through the crevicular fluid when exist gingival inflammation, but these cells vary from person to person and even in one person can change during the day (Kaufman & Lamster, 2000).

Actually exists another colorimetric salivary test used as indicator at inflammation which involves determining occult blood derived from the gingival tissue for evaluates periodontal disease in initial stages, called Salivaster®5 (Hashimoto et al., 2006; Niwa, & Fukuda, 1989). The Salivaster® is a colorimetric test based on a catalytic reaction of hemoglobin in saliva inducing the formation of different colors ranging from yellow to dark green. The principle of the color reaction is similar to the test for blood in urine, but was developed for the

Fig. 12. Initial indicator of periodontal disease through occult blood in saliva (Salivaster®)

detection of gingival inflammation (Kaufman & Lamster, 2000).

Llight blue 1.0 mg/dL (incipient periodontal disease) Dark blue 2.5 mg/dL (periodontal disease present).

5 Salivaster ® (Showa Yakuhin Kako Co. LTD, Tokyo, Japan)

It is reported that this method has a sensitivity of 75.9% and a specificity of 90.5% for the

The procedure involves dipping the test paper in stimulated saliva for 2-3 seconds and then

judging by comparing to the standard color change chart, divided into 3 levels:

Yellow 0.0 mg of blood per dL of saliva (no periodontal disease)

particular viscosity of saliva (Fig. 12).

Munshi et al. (1999) reported a Cariostat® sensitivity of 96.7% and a specificity of 93.3%.

The procedure of Cariostat® is by changing color as a result of increased production of acids produced by fermentation of bacteria, is very sensitive and its relevance lies in its ability to predict the presence of caries in the future (Munshi et al., 1999).

It contains a high concentration of sucrose tryptose growth inhibitor of Gram-negative bacteria, with two types of indicators (green bromocresol and purple) to reveal visually the pH decrease in dental plaque (Ansai et al., 1994; Kornman, 2005; Nishimura et al., 1988a, 2008b).

A pH range of 4.0 ± 3 is considered high risk or marked caries activity.

Plaque is collected from buccal surfaces of first upper molars, using a sterilized cotton swab supplied in the kit, which was put into a test medium and incubated 48 hours at 37° C. The test color change is compared with the pattern provided by the manufacturer as follows:


#### **3.5 Occult blood in saliva**

The use of saliva for periodontal diagnosis has been subject to several investigations, which have been proposed for disease markers including proteins, cells, hormones, volatile components, ions, bacteria and bacterial products, among others.

The procedure of Cariostat® is by changing color as a result of increased production of acids produced by fermentation of bacteria, is very sensitive and its relevance lies in its ability to

It contains a high concentration of sucrose tryptose growth inhibitor of Gram-negative bacteria, with two types of indicators (green bromocresol and purple) to reveal visually the pH decrease in dental plaque (Ansai et al., 1994; Kornman, 2005; Nishimura et al., 1988a,

Plaque is collected from buccal surfaces of first upper molars, using a sterilized cotton swab supplied in the kit, which was put into a test medium and incubated 48 hours at 37° C. The test color change is compared with the pattern provided by the manufacturer as follows:

components, ions, bacteria and bacterial products, among others.

The use of saliva for periodontal diagnosis has been subject to several investigations, which have been proposed for disease markers including proteins, cells, hormones, volatile

Munshi et al. (1999) reported a Cariostat® sensitivity of 96.7% and a specificity of 93.3%.

predict the presence of caries in the future (Munshi et al., 1999).

Blue negative value = pH 5.8-7.2 Green one positive value = pH 5.4 + 0.3 Yellow greenish two positives value = pH 4.8 + 0.3 Yellow three positives value = pH < 4.4 (Fig. 11).

Fig. 11. Caries activity test Cariostat®.

**3.5 Occult blood in saliva** 

A pH range of 4.0 ± 3 is considered high risk or marked caries activity.

2008b).

It has become the leukocytes counts, in these salivary markers as an indicator of periodontal disease, since most of the salivary leukocytes entering to the oral cavity through the crevicular fluid when exist gingival inflammation, but these cells vary from person to person and even in one person can change during the day (Kaufman & Lamster, 2000).

Actually exists another colorimetric salivary test used as indicator at inflammation which involves determining occult blood derived from the gingival tissue for evaluates periodontal disease in initial stages, called Salivaster®5 (Hashimoto et al., 2006; Niwa, & Fukuda, 1989).

The Salivaster® is a colorimetric test based on a catalytic reaction of hemoglobin in saliva inducing the formation of different colors ranging from yellow to dark green. The principle of the color reaction is similar to the test for blood in urine, but was developed for the particular viscosity of saliva (Fig. 12).

Fig. 12. Initial indicator of periodontal disease through occult blood in saliva (Salivaster®)

It is reported that this method has a sensitivity of 75.9% and a specificity of 90.5% for the detection of gingival inflammation (Kaufman & Lamster, 2000).

The procedure involves dipping the test paper in stimulated saliva for 2-3 seconds and then judging by comparing to the standard color change chart, divided into 3 levels:


<sup>5</sup> Salivaster ® (Showa Yakuhin Kako Co. LTD, Tokyo, Japan)

Clinical, Salivary and Bacterial Markers on the Orthodontic Treatment 171

They have developed several methods of toothbrushing and most are identified by a single name like Bass, Stillman, Charters, or by a term indicating the main action to be taken: as

Although the brush is the main mechanical means of plaque removal, often required of dental auxiliaries to remove residual plaque present in the proximal surfaces, among which include: dental floss, interdental cleaners, oral irrigators, mouth rinses buccal interdental

However, the majority of the population is disabled, unmotivated or are unaware of the need to devote time to remove the plaque from all tooth surfaces, or using products not

To know the habits of the patient is recommended to apply a questionnaire which included a series of questions regarding daily brushing and eating habits, focusing on behavioral risk

We recommended that patients brush their teeth three times a day using the Bass modified

technique with toothpaste containing fluoride after placement of the appliances.

spinning or massage.

The objectives of brushing are:

Stimulate the gingival tissues.

sticks, and others (Klaus et al., 1991).

factors for dental disease. (Fig. 13).

 Remove the plaque and stop the growth of it. Clean the teeth of food, debris and stains.

suitable for removing plaque on the site crucial, or both.

Fig. 13. Filling the questionnaire dietary and hygiene habits

**4. Collection of samples** 

### **3.6 Hygienic-dietary habits**

This section covers those data on the frequency and quality of oral hygiene and consistency of diet, time and frequency of food intake.

There is little doubt that the change in lifestyle of civilization was resulting in an increase in the prevalence of dental caries, referring mainly to the increase of the diet of soft foods that contain carbohydrates.

Certain features of sugary foods and the conditions under which they are ingested, are more important in determining the cariogenic potential than amount of sugar (Moynihan, 2005).

The factors that establish the potential cariogenicity of sugary foods are:


The severity of the above is that the sugars are rapidly degraded by bacteria in acidic metabolic end products, which will result in a greater demineralization process than remineralization with subsequent carious lesions.

But also, there are certain foods that can protect against the formation of dental caries by the substances that they contain in their structure, either because they are fibrous, fatty or protein, etc. which reduces their cariogenic potential, and when mixed with sugary foods, reduce the potential of the latter, these are called protective foods, among which we mention the cheese. It has been shown to finish a meal with cheese for dessert, reduces the acidity of the plaque and therefore tooth decay (cariostatic).

The cheese prevents enamel demineralization by two different mechanisms: by stimulating the flow of saliva, which buffers the plaque and by increasing concentrations of calcium and phosphorus in dental plaque, which promotes remineralization (Saroglu, 2007).

In recent years, has also increased the use of sweeteners and sugar substitutes; investigations have focused mainly on sugar alcohols (sorbitol, mannitol, maltitol and xylitol), starch hydrolysates (Lycasin), protein (Monellina) and synthetic chemicals (saccharin, cyclamate and aspartic). Unlike sugar, these are poorly metabolized by oral bacteria or metabolized by pathways that lead to acid formation. Even some of them reduce the bacterial metabolism and consequently the development of plaque on the oral tissues. Nutrition education is very important, also correct oral hygiene with effective brushing after every meal is basically in oral health, while considering preventive periodic revisions to the dentist.

The useful life of a toothbrush is determined more by the brushing method that the length of use. Its half-life is approximately three months; however, this estimate may vary due to differences in the brushing habits.

They have developed several methods of toothbrushing and most are identified by a single name like Bass, Stillman, Charters, or by a term indicating the main action to be taken: as spinning or massage.

The objectives of brushing are:

170 Contemporary Approach to Dental Caries

This section covers those data on the frequency and quality of oral hygiene and consistency

There is little doubt that the change in lifestyle of civilization was resulting in an increase in the prevalence of dental caries, referring mainly to the increase of the diet of soft foods that

Certain features of sugary foods and the conditions under which they are ingested, are more important in determining the cariogenic potential than amount of sugar (Moynihan, 2005).

The physical consistency of the diet: food adhesives are more retentive than non-

 Time of ingestion: sugary foods are more dangerous when consumed between meals, as the natural defense mechanisms operate at maximum during meals. The worst time to cariogenic food is just before bedtime, because the mouth is dry by the circadian

 The frequency: sugar intake reduces the pH of dental plaque that facilitates demineralization and promotes tooth decay, so that the more frequent the intake, more

The severity of the above is that the sugars are rapidly degraded by bacteria in acidic metabolic end products, which will result in a greater demineralization process than

But also, there are certain foods that can protect against the formation of dental caries by the substances that they contain in their structure, either because they are fibrous, fatty or protein, etc. which reduces their cariogenic potential, and when mixed with sugary foods, reduce the potential of the latter, these are called protective foods, among which we mention the cheese. It has been shown to finish a meal with cheese for dessert, reduces the acidity of

The cheese prevents enamel demineralization by two different mechanisms: by stimulating the flow of saliva, which buffers the plaque and by increasing concentrations of calcium and

In recent years, has also increased the use of sweeteners and sugar substitutes; investigations have focused mainly on sugar alcohols (sorbitol, mannitol, maltitol and xylitol), starch hydrolysates (Lycasin), protein (Monellina) and synthetic chemicals (saccharin, cyclamate and aspartic). Unlike sugar, these are poorly metabolized by oral bacteria or metabolized by pathways that lead to acid formation. Even some of them reduce the bacterial metabolism and consequently the development of plaque on the oral tissues. Nutrition education is very important, also correct oral hygiene with effective brushing after every meal is basically in oral

The useful life of a toothbrush is determined more by the brushing method that the length of use. Its half-life is approximately three months; however, this estimate may vary due to

phosphorus in dental plaque, which promotes remineralization (Saroglu, 2007).

health, while considering preventive periodic revisions to the dentist.

The factors that establish the potential cariogenicity of sugary foods are:

**3.6 Hygienic-dietary habits** 

contain carbohydrates.

cariogenic.

of diet, time and frequency of food intake.

rhythm of saliva during sleep.

remineralization with subsequent carious lesions.

the plaque and therefore tooth decay (cariostatic).

cariogenic foods become.

differences in the brushing habits.


Although the brush is the main mechanical means of plaque removal, often required of dental auxiliaries to remove residual plaque present in the proximal surfaces, among which include: dental floss, interdental cleaners, oral irrigators, mouth rinses buccal interdental sticks, and others (Klaus et al., 1991).

However, the majority of the population is disabled, unmotivated or are unaware of the need to devote time to remove the plaque from all tooth surfaces, or using products not suitable for removing plaque on the site crucial, or both.

To know the habits of the patient is recommended to apply a questionnaire which included a series of questions regarding daily brushing and eating habits, focusing on behavioral risk factors for dental disease. (Fig. 13).

Fig. 13. Filling the questionnaire dietary and hygiene habits
