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.

1

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.

2

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”.

3

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.

4

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

5

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.

6

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.

7

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.

8

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

9

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

10

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

11

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.

12

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.

13

- 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 -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.

15

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.

16

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.

18

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 (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 (green) or (nochange) and occasionally .

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.

19

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.

20

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.

21

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

22

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.

I. 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.

23

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 37°C for 3 to 4 days. A count of

the number of colonies is then made by using the quebec counter.

II. 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 50°C. 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

24

incubated at 37°C. The color change of the indicator is observed after 24, 48

and 72 hours of incubation.

Time in hours

24 48 72

Color

Caries activity

Yellow

Marked

Yellow

Definite

Yellow

Limited

Color

Caries activity

Green

Continue test

Green

Continue test

Green

Inactive

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

25

Blue 15 min 1 Non conducive

Orchid 15 min 2 Slightly conducive

Red 15 min 3 Moderately conducive

Red Immediately 4 Highly conducive

Pink or white Immediately 5 Extremely conducive

IV. 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

26

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.

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

27

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.

Conclusion

Numerous dental research investigators for more than a century have

studied various aspects of the dental caries problems. Despite this extensive

28

investigation, many aspects of etiology are still obscure, and efforts at

prevention have been only partially successful.

29