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1. 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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2. 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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3. 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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4. 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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5. 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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6. 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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7. 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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8. 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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9. 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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10. 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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11. 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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12. 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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13. 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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14. - 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 α-hemolytic streptococci. IgA isolated from human parotid secretions
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15. 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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16. 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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17. 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

18. 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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19. 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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20. 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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21. 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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22. 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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23. 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.
23

24. 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.
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 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 incubated at 37°C. The color change of the indicator is observed after 24, 48 and 72 hours of
incubation.
24

25. 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
Blue 15 min 1 Non conducive
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26. 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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27. 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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28. 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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