Etiology of dental caries/ dental implant courses by Indian dental academy

Etiology of Dental Caries Introduction Definition : Dental caries is a microbial disease of the calcified tissues of the teeth, characterized by demineralization of the inorganic portion and destruction of the organic substance of the tooth. It is the most prevalent chronic disease affecting the human race. Once it occurs, its manifestations persist throughout life even though the lesion is treated.

There are practically no geographic areas in the world whose inhabitants do not exhibit some evidence of dental caries. It affects persons of both sexes in all races, all socio-economic strata and every age group.

In ancient humans caries was usually located at the cemento-enamel junction or in the cementum, whereas in modern man grooves and fissures are the most common sites of decay.

The etiology of dental caries is generally agreed to be a complex problem complicated by many indirect factors, which obscure the direct cause or causes. There is no universally accepted opinion of the etiology of dental caries.

To better understand current concepts of the etiology of caries, earlier theories will be discussed briefly.

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Early theories of caries etiology 1. Worms According to an ancient Sumerian text, tooth ache was caused by a worm that drank the blood of the teeth and fed on the roots of the jaws.

Guy de Cahuliac (1300-1368) the greatest surgeon of the middle ages believed that worms caused dental decay. As a cure he advocated fumigation with seeds of leek, onion and hyoscyamus.

Antony Van Leeuwenhock (1700) the father of modern microscopy wrote a letter to the Royal Society of London describing little worms taken out of a corrupt tooth and said that they caused the pain in tooth ache.

2. Humors The ancient Greeks considered that a persons physical and mental constitution was determined by the relative proportions of the four elemental fluids of the body-blood, phlegm, black bile and yellow bile. All diseases, including caries, could be explained by an imbalance of these humors.

3. Vital theory The vital theory regarded dental caries as originating within the tooth itself, similar to bone gangrene. This theory proposed at the end of the eighteenth century remained dominant until the middle of the nineteenth century.

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4. Chemical theory Parenly (1819) rebelled against the vital theory and proposed that an unidentified “Chemical agent” was responsible for caries. He stated that caries began on the enamel surface in locations where food putrefied and acquired sufficient dissolving power to produce the disease chemically.

5. Parasitic or Septic theory In 1843 Erdl described filamentous parasites in the surface membrane of teeth. Shortly thereafter, Ficinus a Dresden physician observed filamentous microorganisms which he called denticolae, in material taken from carious cavities. He implied that these bacteria caused decomposition of the enamel and then the dentin. Neither Erdl nor Ficinus explained how these organisms destroyed tooth structure.

6. Acidogenic theory Also known as chemo-parasitic theory was proposed by W.D. Miller (1890).

W.D. Miller, probably the best known of the early investigators of dental caries, published extensively on the results of studies, beginning in 1882.

These culminated in the following hypothesis in which he stated : “Dental decay is a chemo-parasitic process consisting of two stages, the decalcification of enamel, which results in its total destruction and the decalcification of dentin, as a preliminary stage, followed by dissolution of the softened residue.

The acid which affects this decalcification is derived from the fermentation of starches and sugar lodged in the retaining centres of the teeth”.

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Miller found that bread, meat and sugar incubated in vitro with saliva at body temperature produced enough acid within 48 hours to decalcify sound dentin. The acid formation could be prevented by boiling, confirming the probable role of bacteria in its production. Subsequently he isolated numerous microorganisms from the oral cavity, many of which were acidogenic and some of which were proteolytic. Since a number of these bacterial forms were capable of forming lactic acid, Miller believed that caries was not caused by any single organisms but rather by a variety of microorganisms.

This theory has been accepted by the majority of investigators in a form essentially unchanged since its inception. The bulk of scientific evidence does implicate carbohydrates, oral microorganisms and acids, and for this reason these deserve further consideration.

7. The proteolytic theory The classical chemo-parasitic theory has not been universally accepted.

Instead, it has been proposed that the organic or protein elements are the initial pathway of invasion by microorganisms. Mature enamel is highly mineralized.

The human tooth contains only about 1.5% to 2% organic material, of which 0.3% to 0.4% is protein. According to the proteolytic theory, the organic component is most vulnerable and is attacked by hydrolytic enzymes of microorganisms. This precedes the loss of the inorganic phase.

Certain of the enamel structures are made up of organic material such as enamel lamella, enamel rod sheaths. Bodeker suggested that these lamella may be important in the progress of dental caries since they could serve as a pathway for microorganisms through the enamel.

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According to Gottlieb (1944) caries is essentially a proteolytic process.

The microorganisms invade the organic pathways and destroy them. Acid formation accomplished the proteolysis. Gottlieb said that yellow pigmentation was characteristic of caries and this was due to pigment production of proteolytic organisms.

Frisbie (1944) also described caries as a proteolytic process involving depolymerization and liquifaction of the organic matrix of enamel. The less soluble inorganic salts could then be freed from their “organic bond� favoring their dissolution by acidogenic bacteria that secondarily penetrate along widening paths of ingress.

Pincus (1949) proposed that enamel proteins are mucoproteins yielding sulfuric acid upon hydrolysis. Certain gram negative bacilli capable of producing the enzyme sulfatase has been isolated from the oral cavity. This enzyme releases sulfuric acid from the mucoprotein. The liberated acid then dissolves the enamel combining with the calcium to form calcium sulfate. This compound has been found in carious enamel.

Manley and Hardwick (1951) attempted to reconcile the two chief theories of etiology of dental caries. Many bacteria produce acid from an appropriate carbohydrate substrate. Some bacteria may even degrade protein in the absence of carbohydrate. On this basis it has been proposed that there may be two types of carious lesions. In one type, microorganisms invade enamel lamellae, attack enamel and involve the dentin before there is clinical evidence of caries. In the other, no enamel lamellae are present, and there is alteration of the enamel prior to invasion by microorganisms. This alteration is produced through decalcification of the enamel by acids found by bacteria in a dental

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plaque overlying the enamel. The early lesions produced are those typically described as “Chalky” enamel.

8. The proteolysis chelation theory This theory was proposed by Schatz and his co-workers to explain the cause of dental caries. Most of their publications have dealt with theoretic discussions of the dental disease and the chemical aspects of chelation with little direct evidence given for proteolysis – chelation as a mechanism in the caries process.

Chelation is a process involving the complexing of a metallic ion to a complex substance through a covalent bond, which results in a highly stable compound. In other words a chelating agent is a molecule capable of seizing and holding a metal ion in a claw-like grip and forming a heterocyclic ring.

In biology there are well known chelates, including hemoglobin (containing

iron),

chlorphyll

(containing

magnesium),

vitamin

B-12

(containing cobalt) etc.

Chelation is independent of pH. Numerous naturally occurring biologic chelating agents exist example citrates, amino acids, lactate.

The proteolysis – chelation theory states that the bacterial attack on enamel results in a break down of the protein and other organic components of enamel, chiefly keratin. This results in the formation of substances which may form soluble chelates with the mineralized component of the tooth and thereby decalcify the enamel at a natural or even alkaline pH. Enamel also contains other organic components besides keratin, such as mucopolysaccharide lipids and citrate which may be susceptible to bacterial attack and act as chelators.

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The proteolysis chelation theory resolves the arguments as to whether the initial attack of dental caries is on the organic or inorganic portion of enamel by stating that both may be attacked simultaneously.

But several questions still remain in unanswered. These include : 1. The observation of increased caries incidence with increased sugar consumption.

2. The observation of increased lactobacillus counts with high caries activity.

3. The observation of decreased caries incidence following topical or systemic administration of fluoride.

Increased caries incidence due to increased carbohydrate consumption can be explained through the action of carbohydrate in : i.

Increasing proteolysis.

ii.

Producing conditions under which keratinous proteins are less stable.

iii.

Complexing calcium.

Increased caries incidence accompanying increased lactobacillus counts

might be explained by the microorganisms being the result of the caries process rather than its cause. Thus Schaltz has suggested that : i.

Proteolysis may provide ammonia which prevents a pH drop that would tend to inhibit growth of the lactobacilli.

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ii.

The release of calcium from hydroxyapatite by chelation might encourage the growth of lactobacilli. Since calcium has been reported to produce this effect.

iii.

Calcium exerts a vitamin sparing action on some lactobacilli.

Reduced caries incidence due to administration of fluoride might occur

through formation of fluorapatite, which strengthens the linkages between the organic and inorganic phases of the enamel, thereby preventing or reducing their complexing.

Animal studies by Zipkin and Larson have shown that the incorporation of a chelating agent such as EDTA into the cariogenic diet resulted in an increase in severity of dental caries as well as a difference in the distribution pattern of the lesions.

9. Current concepts of Caries Etiology According to the current concepts dental caries is a multifactorial disease in which there is an interplay of three principal factors; the host factor (saliva and teeth) the microflora, the substrate or diet. In addition a fourth factor time is to be included. Diagramatically these factors can be portrayed as four overlapping circles.

For caries to occur, conditions within each of these factors must be favorable. In other words caries requires a susceptible host, a cariogenic oral flora, a suitable substrate that must be present for a sufficient length of time.

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Contributing factors in Dental Caries The mere presence of microorganisms and a suitable substrate at a given point on a tooth surface is insufficient to establish a carious lesion in all cases.

It is reasonable to assume that variations in caries incidence exist because of a number of possible indirect or contributing factors.

Indirect factors that might influence the etiology of caries are : A. Tooth 1. Composition.

2. Morphologic characteristics.

3. Position.

B. Saliva 1. Composition

Inorganic Organic

2. pH 3. Quantity 4. Viscosity 5. Antibacterial factors C. Diet 1. Physical factors : quantity of diet 2. Local factors Carbohydrate content Vitamin content Fluorine content

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Tooth Factor Morphology and position A susceptible host is one of the factors required for caries to occur.

Tooth morphology is one of the determinant. For example attempts to induce caries in dogs have been unsuccessful mainly because of the wide spacing and the conical shape of their teeth.

Pit and fissure areas of the posterior teeth are susceptible to caries. Food debris and microorganisms readily impact in the fissures. Investigations have shown a correlation between caries susceptibility and the depth of the fissure.

Certain surfaces of a tooth are more prone to decay, whereas other surfaces rarely show decay. Eg : in mandibular I molar the most prone areas in order are occlusal, buccal, mesial, distal and lingual, whereas in maxillary first molars the order is occlusal, mesial, lingual, buccal and distal. On maxillary lateral incisors the lingual surface is more susceptible to caries than the buccal surface. These differences in decay rates of various surfaces on the same tooth are in part due to morphology, for example the buccal pit on mandibular molars, lingual groove of maxillary incisors. The distal surface of Ist permanent molars is freely accessible to saliva for about 4 to 5 yrs until second molars erupt at age of 10 or 11, proximal plaque may form on the mesial surface shortly after euption at age 6 or 7.

An intraoral variation exists in susceptibility to caries between different tooth types. The most susceptible permanent teeth are the mandibular first molars, closely followed by the maxillary first molars and the mandibular and maxillary second molars. The second premolars, maxillary incisors and first

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premolars are the next in sequence. Mandibular incisors and canines are the least likely to develop caries.

Irregularities in arch form, crowding and overlapping of the teeth also favor the development of carious lesions. Experimentally this has been verified by the gold plate technique in which stagnation areas are deliberately created on selected tooth surfaces. These surfaces develop “white spot� lesions within a few weeks. The white appearance is due to optical phenomenon associated with increased enamel porosity.

Composition of the tooth In a number of studies on the relation of caries to the chemical composition of teeth, such as those by Armstrong no differences were found in the calcium, phosphorous, magnesium and carbonate contents of enamel from sound and carious teeth. Significant differences in fluoride content of sound and carious teeth however have been reported by these same workers. They have found the fluoride content of enamel and dentin from sound teeth to be 410ppm and 873ppm respectively but only 139ppm and 223ppm respectively in carious teeth.

Studies of the chemical composition of enamel by Brudevold and his associates indicate that surface enamel is more resistant to caries than subsurface enamel. Surface enamel is more highly mineralized and tends to accumulate greater quantities of fluoride, zinc, lead and iron than the underlying enamel. The surface enamel is lower in carbon dioxide, dissolves at a slower rate in acids, contains less water and has more organic material than subsurface enamel. These factors contribute to caries resistance and are partly

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responsible for the slower disintigration

of surface enamel than of the

underlying enamel in initial carious lesions.

The Saliva factor The fact that the teeth are in constant contact with and bathed by the saliva suggests that the environmental agent influences the state of oral health of a person including the dental caries process.

The complex nature of saliva and the great variation in its composition pose difficulties in establishing which factors may directly influence dental health.

The composition of saliva varies between persons. Mixed saliva (Resting + stimulated) is of no fixed composition, varies according to the circumstances under which the saliva is collected.

It has been reported that the calcium and phosphorus content of saliva is low in caries active persons. There are numerous other inorganic components of saliva such as sodium, magnesium, potassium, carbonate, chloride and fluoride. With the exception of fluoride, these substances have not been thoroughly investigated. Thiocyanate has also been isolated from saliva and at one time was thought to inhibit the microorganisms associated with dental caries. It is now found that thiocyanate has no effect either on the bacterial flora or on dental caries.

The organic constituent of saliva contains salivary cholesterol, mucin etc. its significance is not clear.

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The ammonia and urea content of saliva has been studied by many workers. Several investigators have noted that saliva of caries immune persons exhibited a greater ammonia content than saliva from persons with caries. High ammonia concentration retared plaque formation and neutralized acid to some extent.

A number of different enzymes have been isolated from saliva. The most prominent and important oral enzyme is amylase or ptyalin a substance responsible for the degradation of starches.

The relation between amylase activity and dental

caries has been

studied by numerous investigators with conflicting results. Some studies say that high amylolytic activity is associated with low caries some other studies have found low amylolytic activity with low caries and some studies have found no correlation.

Studies dealing with the pH of saliva and its relation to dental caries have shown no positive correlation.

The quantity of saliva secreted influences caries incidence. Humans suffering from decreased or lack of salivary secretion called xerostomia often experience an increased rate of dental caries and rapid tooth destruction.

Xerostomia may be seen in the following conditions.

-

Sarcoidosis which causes reduced salivary gland functions.

-

Sjogrens syndrome

-

Radiation to the head and neck can cause progressive atrophy and fibrosis of salivary glands.

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-

Surgical removal of salivary glands for neoplasms

-

Chronic administration of anticholinergic drugs.

-

In diabetes mellitus

-

Patients with Parkinson’s disease

-

Acute viral infection involving salivary glands results in temporary xerostomia.

-

Anxiety, mental stress and depression may temporarily decrease salivary flow.

The viscosity of saliva has been suggested to be of some significance in accounting for differences in caries activity between different persons.

The antibacterial properties of saliva have been investigated by numerous workers in an attempt to explain the wide variation in caries incidence among different persons. Using lactobacillus acidophilus as test organism numerous studies have shown that the saliva of caries free persons have a greater inhibiting effect on these microorganisms than from caries active persons.

Immunoglobulins: The major immunoglobins in saliva is secretory IgA. Antibodies against specific bacteria have been reported in human saliva. Purified salivary IgA and IgG fractions have been found with agglutinating activity against oral isolates of Îą-hemolytic streptococci. IgA isolated from human parotid secretions 14

specifically inhibits the adherence of certain strains of streptococci to human buccal epithelial cells, facilitating their elimination from the oral cavity by swallowing.

Role of Microorganisms Although there are differences of opinion as to how and which microorganisms produce carious lesions, it is agreed that caries cannot occur without microorganisms. The evidence implicating microorganisms in the etiology of caries can be summarized as 1. Germ – free animals do not develop caries 2. Antibiotics fed to animals are effective in reducing the incidence and severity of caries 3. Totally unerupted and unexposed teeth do not develop caries, yet when exposed to the oral environment and microflora can become carious.

4. Oral bacteria can demineralize enamel and dentin in vitro and produce caries like lesions.

5. Microorganisms have been histologically demonstrated invading caries enamel and dentin. They can be isolated and cultivated from caries lesions.

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Localization of the oral flora related to caries Reports on caries indicates that different organisms display some selectivity as to which tooth surface they attack • Pit and fissure caries  This is the most common carious lesions found in modern humans.

Many organisms can colonize in fissures, which provide mechanical retention for the bacteria. S mutans, S salivarius, S sanguis, L acidophilus, L casei, A viscous, A nalsundii, Actinomyces israelii develop fissure lesions. A wide variety of microbes may be able to initiate pit and fissure caries.

• Smooth surface caries  A limited number of organisms have proved able to colonize smooth surfaces in large enough numbers to cause decay in test animals. Streptococus mutans is very significant in this respect.

• Root caries  In rodents, gram – positive filamentous rods, including actinomyces species have been associated with this type of lesion. Strains of Nocardia and S. sanguis may also cause root caries.

• Deep dentinal caries  Because the environment in deep dentinal lesions is different from that at other locations the flora here is also different. The predominant organism is lactobacillus.

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Transmissibility of the cariogenic flora The specificity of the cariogenic flora has been demonstrated by animal experiments, fulfilling one of Koch’s postulates

that the infections agent

should be able to produce the disease when transmitted to another animal.

In 1960 Keyes demonstrated that caries inactive hamsters failed to develop extensive lesions unless they were caged with caries rampant animals.

The role of the Dental Plaque The dental plaque is a structure of vital significance as a contributing factor to at least the initiation of the carious lesion.

There is general agreement that enamel caries begins beneath the dental plaque. The presence of a plaque, however, does not necessarily mean that a caries lesion will develop at that point. Extensive study of the bacterial flora of the dental plaque has indicated a heterogeneous nature of the structure.

Filamentous microorganisms which grow in long interlacing threads and have the property of adhering to smooth enamel surfaces predominate. Smaller bacilli and Cocci then become entrapped in this reticular meshwork. Aciduric and acidogenic streptococci and lactobacilli are also found in large numbers.

Stephan (1940) using an antimony microelectrode measured the pH in a dental plaque insite. The pH of plaques in different persons varied but averaged about 7.1 in caries free persons to 5.5 in persons with extreme caries activity.

Stephan also studied the pH in dental plaque after rinsing of the mouth with 10% glucose or sucrose solution. Within two to five minute after the rinse, the pH in the plaque had fallen to between 4.5 and 5.0 and gradually returned to the initial pH level within one to two hours. Studies indicated differences in 17

reduction in pH in caries free and caries active subjects. The plaque pH in the caries free group did not fall below 5.0 units after the glucose rinse, while the pH in the caries active group fell below 5 units, after the glucose rinse. The maxillary anterior teeth exhibited a greater pH drop in the plaque than the mandibular anterior teeth indicating that the saliva influences plaque acid production.

An important discovery was the recognition that certain cariogenic and highly acidogenic strains of streptococci, espicially S. mutans have the ability to metabolize dietary sucrose and synthesize glucan. This glucan is an insoluble, sticky or slimy gel, which is resistant to bacterial hydrolytic enzyme.

It causes plaque to adhere to tooth surfaces.

Studies by Gibbons has demonstrated that certain cariogenic bacteria are capable of storing intracellular polysaccharides which may act as a reserve source of carbohydrate for fermentation and maintenance of acid production in the plaque during periods when the diet of the individual is sugar free.

Oral streptococci Irrespective of the age of plaque and the diet, the predominant organisms are gram - positive cocci of the genus streptococcus which form about 50% of the total colony forming units recovered from young plaque.

These streptococci have been divided into various groups based on their colonial morphology and physiological characteristics. Oral streptococci have been isolated on mitis-salivaries agar a selective medium that permits isolation from mixed flora.

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Streptococcus sanguis.

This is one of the predominant groups of streptococci colonizing on the teeth. Formerly it was called streptococcus s. b. e because of its involvement in sub acute bacterial endocarditis. Caries from this strain occurs primarily in sulci and is significantly less extensive than S. mutans. On blood agar S. sanguis causes α (green) hemolysis.

Streptococcus mutans.

In 1924 Clarke isolated a streptococcus that predominated in many human carious lesions and he named them streptococcus mutans because of its varying morphology.

Characteristics of S. mutans include the following – They are nonmotile, Catalase negative, gram positive cocci in short or medium chains. On mitis – salivarius agar they grow as a cushion shaped colony. These colonies are opaque; the surface resembles frosted glass. Hemolysis of blood agar is variable and may be α (green) or γ (nochange) and occasionally β.

Streptococcus mutans exhibit several important properties i.

It colonizes on tooth surfaces.

ii.

It is more aciduric than other streptococci.

iii.

It is a homofermentative lactic acid former.

iv.

It synthesizes insoluble polysaccharides from sucrose.

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Evidence of the ecology of S. mutans indicates that this organism can survive in the mouth only when solid surfaces such as teeth or dentures are present.

Streptococci Salivarius They have been found in the plaque, throat, nasopharynx and oral mucosa, but their natural habitat in the dorsum of the tongue. In humans they have only a minor degree of cariogenic significance.

Oral Lactobacilli Lactobacilli are gram positive, non-spore forming rods that grow best under microaerophilic conditions. Lactobacilli represents about 1% of the oral flora. A favorite habitat of lactobacilli is in the dentin of deep carious lesions.

Oral Actinomyces It is a gram positive, non motile, non spore forming organism occurring as rods and filaments. It is a good plaque former. It is the most common group of organisms isolated from the subgingival microflora and from plaque of human root surface caries.

The diet factor The physical nature of the diet has been suggested as one factor responsible for the difference in caries experience between primitive and modern man.

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The diet of the primitive man consisted generally of raw unrefined foods containing a lot of roughage which cleanses the teeth of adherent debris during mastication. In the modern diet, soft refined foods tend to cling to the tooth and are not removed because of the general lack of roughage.

The carbohydrate content of the diet has been universally accepted as one of the most important factors in the dental caries process.

The prevalence of caries among native populations such as Australian Aborigines, New Zealand Maoris, Eskimos, ghanaians etc was very low to their exposure European type diets. Native diets did not contain any sucrose other than the relatively small amounts found in fruits and vegetables. As their diets changed to include products containing sugar caries prevalence increased.

Surveys in Europe and Japan have demonstrated that caries was dramatically reduced during and after World war II due to rationing of sucrose and the decreased between meal eating. In the post war years, as rationing eased and sugar became more readily available the caries rate rose again. These findings on the effects of wartime dietary restrictions indicate that the caries process can be influenced by diet.

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Interventional human studies Vipeholm Vipeholm is a mental institution in Southern Sweden. Adult patients followed for several years on a nutritionally adequate diet, were found to develop caries at a slow rate. The patients were divided into nine groups to compare the effects of various modifications of carbohydrate intake. Sucrose was included in the diet as toffee, chocolate, caramel, in bread or in liquid form. Caries increased when sucrose containing foods were ingested between meals. In addition, not only the frequency but also the form in which sucrose was ingested was important. Sticky or adhesive forms of sucrose containing foods which can maintain high sugar levels in the mouth were more cariogenic than those forms that were rapidly cleared. The Vipeholm study demonstrated that it was possible to increase the average sugar consumption with very little increase in caries provided the additional sugar was consumed at meals in solution form.

Hope wood house Interesting findings on the effect of diet on dental caries have emerged from the study of institutionalized children at the Hope wood House in Australia. Babies were either born at the home or taken into it in the first few weeks of life. From the beginning sugar and other refined carbohydrates were excluded from the childrens diet.

Dental surveys of these children during the ages of 5 and 13 revealed an average def and DMFT score of 1% or about 10% of the caries prevalence in the general population of that age group. It should be noted that the water

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supply contained insufficient fluoride (0.1ppm) and the childrens oral hygiene was poor, about 15% of them suffered from gingivitis. It follows that caries can be reduced to a minimal level by dietary means alone in spite of unfavorable hygiene and fluoride levels. As the children grew older they were relocated and no longer adhered to the original diet that had limited caries completely. A steep increase of DMFT experience occurred in the children above 13 years of age. This indicates that these teeth had not acquired any permanent resistance to caries. The reason that the teeth had not decayed between 5 to 13 yrs of age was that the local oral environment was favorable and little cariogenic foods were ingested.

Industrial risk : Workers in sugar factories experienced more caries than among textile industry.

Children taking syrup medicines for long term have more caries experience than children who either did not take any medicine or took tablets.

Caries Activity tests Caries activity tests have been used in dental research for many years.

There is no ideal test in existence at the present time. Some of the commonly done tests are.

1. Lactobacillus colony count Action : This test estimates the number of acidogenic and aciduric bacteria in the patients saliva by counting the number of colonies appearing on tomato peptone agar plates (pH 5.0) after inoculation with a sample of saliva.

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Equipment : The necessary equipment includes saliva collecting bottles, paraffin, two 9 ml tubes of saline, two agar plates, two bent glass rods, facilities for incubating, a quebec counter and pipettes.

Procedure : Saliva is collected by having the subject chew paraffin before breakfast and then collecting the saliva in a bottle. A 1:10 dilution is prepared by pipetting 1 ml of the saliva sample into a 9 ml tube of sterile saline solution.

This is shaken and a 1:100 dilution is made by pipetting 1 ml of the 1:10 dilution into another 9 ml tube of sterile salt solution. The 1:100 dilution is mixed thoroughly and 0.4ml of each dilution is spread on the surface of an agar plate with a bent glass rod.

The plates are labelled and incubated at 37째C for 3 to 4 days. A count of the number of colonies is then made by using the quebec counter.

2. Snyder test Action : Measures the rapidity of acid formation when a sample of stimulated saliva is inoculated into glucose agar adjusted to pH 4.7 to 5 and with bromcresol green as color indicator.

Indirectly the test is also a measure of acidogenic and aciduric bacteria.

Equipment : Saliva collecting bottles, paraffin, a tube of Snyder glucose agar containing bromcresol green and adjusted to pH 4.7 to 5 pipettes and incubator.

Procedure : Saliva is collected before breakfast by having the subject chew paraffin. A tube of Snyder glucose agar is melted and then cooled to 50째C. The saliva specimen is shaken vigorously for 3 minutes. Then 0.2 ml of saliva is pipetted into the tube. The agar is allowed to solidify in the tube and is incubated at 37째C. The color change of the indicator is observed after 24, 48 and 72 hours of incubation.

24

Time in hours 24

48

72

Color

Yellow

Yellow

Yellow

Caries activity

Marked

Definite

Limited

Green

Green

Green

Continue test

Continue test

Inactive

Color Caries activity

III.

Reductase test

Action : The test measures the rate at which an indicator molecule diazoresorcinol, changes from blue to red to colorless or leukoform on reduction by the mixed salivary flora.

Equipment : The reductase test comes in a kit that includes calibrated saliva collection tubes with reagent on the inside of the tubes cap plus flavoured paraffin.

Procedure : Saliva is collected by chewing paraffin and expectorating directly into the collection tube. When the saliva reaches the calibration mark the reagent cap is replaced. The sample is mixed with a fixed amount of diazoresorcinol, the reagent upon which the reductase enzyme is to react. The change in color after 30 seconds and after 15 minutes is taken as a measure of caries activity.

Color

Time

Score

Caries activity

Blue

15 min

1

Non conducive

25

Orchid

15 min

2

Slightly conducive

Red

15 min

3

Moderately conducive

Red

Immediately

4

Highly conducive

Pink or white

Immediately

5

Extremely conducive

4. Buffer capacity test Action : Buffer capacity can be quantitated using either a pH meter or color indicators. The test measures the numbers of millimeters of acid required to lower the pH of saliva through an arbitary pH interval such as from pH 7.0 to 6.0 or the amount of acid or base necessary to bring color indicators to their end point.

Equipment : pH meter, titration equipment, 0.05N lactic acid, 0.05N base, paraffin, sterile glass jars containing a small amount of oil.

Procedure : 10 ml of stimulated saliva are collected under oil at least 1 hour after eating. 5ml of this are measured into a beaker. The pH of the saliva is adjusted to 7.0 by addition of lactic acid or base. The level of lactic acid in the graduated cylinder is re-recorded. Lactic acid is then added to the sample until a pH of 6.0 is reached. The no. of mls of lactic acid needed to reduce pH from 7.0 to 6.0 is a measure of buffer capacity.

There is an inverse relationship between buffering capacity of saliva and caries activity.

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V.

Fosdick calcium dissolution test

Action : The test measures the milligrams of powdered enamel dissolved in 4 hours by acid formed when the patients saliva is mixed with glucose and powdered enamel.

Equipment : Powdered human enamel, saliva collection bottles, sterile test tubes, test tube agitation equipment, equipment for determining the calcium count of the saliva. Saliva is stimulated by having the subject chew gum or paraffin.

Procedure : Twenty five millimeters of gum stimulated saliva are collected.

Part of this analysed for calcium content. The rest is placed in an 8 inch sterile test tube with about 0.1g of powdered human enamel. The tube is sealed and shaken for 4 hours at body temperature after which it is again analyzed for calcium content. The chewing of gum to stimulate the saliva produces sugar; if the paraffin is used a concentration of about 5% glucose is added. The amount of enamel dissolution increases as the caries activity increases.

VI.

Dewar test

Action : This test is similar to the Fosdick calcium dissolution test except that the final pH after 4 hours is measured instead of the amount of calcium dissolved.

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Conclusion

Numerous dental research investigators for more than a century have studied various aspects of the dental caries problems. Despite this extensive investigation, many aspects of etiology are still obscure, and efforts at prevention have been only partially successful.

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