Caries activity tests

DR. PRIYA
PG STUDENT
 Getting acquainted with the
terms

 Dental caries-It is defined as a progressive, irreversible microbial

disease of multifactorial nature affecting the calcified tissues of the
teeth, characterized by demineralization of the inorganic portion and
destruction of the organic portion of the tooth.
 Caries activity- refers to increment of active lesions over a stated
period of time.
 Caries susceptibility- inherent tendency of the host and the target
tissue, the tooth, to be afflicted by the caries process.
 Caries activity tests- measures the degree to which the local
environmental challenge favors the probability of occurrence of
carious lesions.
 The impact of caries activity
tests in clinical setting

 To determine the need and extent of the personalized

preventive measures.

 To serve as an index of the success of therapeutic

measures.

 To motivate and monitor the effectiveness of education

progress relating to dietary and oral hygiene procedures.

 To manage the progress of restorative procedures.

 To identify high risk groups and individuals.

 IDEAL REQUISITES (Snyder)

 Correlation between predicted and actual caries

development at maximum.
 Ability to measure the mechanism involved in the caries
process
 Reliability and validity
 Inexpensive
 Easy to evaluate
 Simplicity
 Result obtained within hours or few days
 Non invasive
 Applicability in any clinical setting
The determinants of caries
 CARIES ACTIVITY TESTS-Microbial tests
Test to identify Streptococcus mutans • S.mutans dip-slide method
• Plaque/toothpick method
• Saliva/tongue blade method
• S mutans replicate technique
• S mutans adherens method

Tests for Lactobacilli  Lactobacilli count test(dentocult LB)

 Dip slide method
Tests to check acidogenicity o Snyder test
o Albans test
o The swab test
CARIES SUSCEPTIBILITY TESTS(Tests for evaluating salivary
defense)
Reductase test
Buffer capacity test (Dentobuff test)
Fosdick calcium dissolution test
Dewar test
NEW CARIES ACTIVITY TESTS
Cariostat
Caries risk test
Oratest
 1. LACTOBACILLUS COLONY COUNT TEST

 Hadley in 1933
 Principle- 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 inoculating with a sample of saliva.
 The saliva that
accumulates in Mix well, and dilute to 1:10 dilution
Chew a small the following by pipetting 1 ml of saliva sample
piece of three minute into a 9 ml tube of sterile saline
paraffin period is solution
collected in a
sterile container

The number of Shake well and a

lactobacilli per 0.4ml of each solution of 1:100
mm of saliva is dilution is spread dilution is made
calculated by on the surface of by pipetting 1 ml
Incubated for 3-4
multiplying the an agar plate of the 1:10
days at 37 degree
number of containing 20ml dilution into
Celsius.
colonies on the of cooled another 9ml tube
plate by the of sterile saline
dilution factor of liquefied agar.
its inoculums solution
 Interpretation of the results

No of LB Caries
per ml activity

• 0 -1000 • Little or no
• 1000-5000 • Slight
• 5000 -10,000 • Moderate
• >10,000 • Marked
 DISADVANTAGES
ADVANTAGES
• Inaccurate for predicting the onset
of caries.
• Counting is a tedious procedure.
• Requires relatively complex • Useful for monitoring the
equipment. effectiveness of restorative

• Does not completely exclude the dentistry and care completion

growth of other aciduric • Simple to carry out.

organisms. • Useful as a screening test for

• Few mins for tests, but results are caries activity in large groups.

not available for several days.

TEST TO CHECK ACIDOGENICITY
o Snyder test

o Albans test

o The swab test

 2. COLORIMETRIC SNYDER TEST

• Snyder, 1951
•Measures the ability of salivary
microorganisms to form organic acids from
a carbohydrate medium.
•Medium – indicator dye – Bromocresol
green
PRINCIPLE
•Dye changes colour from green to yellow
in range of pH 5.4 to 3.8.

Indirectly this test is also a measure of

acidogenic and aciduric bacteria.
 Procedure

Saliva is collected
The rate
It is shaken, This is pH indicator and of color
by chewing
then 0.2 ml of allowed to compare to an change
paraffin. A tube of
saliva is solidify and from
Snyder glucose uninoculated
green to
agar is melted and pipetted into then control tube after yellow is
then cooled to 50 tube and mix. incubated at 24, 48 and 72 indicativ
degree Celsius. 37⁰ C. hours of e of the
incubation. degree
of caries
24 hrs 48hrs 72 hrs activity
If yellow If yellow If yellow
Marked caries Definite caries Limited caries
susceptibility susceptibility susceptibility

If green If green If green

Continue to incubate& Continue to incubate & Caries inactive
observe at 48 hrs observe at 72 hrs
 • Relatively simple to carry out.
• Cost is moderate.
Advantages • Only one tube of medium
and no serial dilutions are
required.

• Time consumed is more. Tube 1: Uninoculated Synder

tube
Disadvantages Tube 2: no susceptibility to
• Sometimes the color changes forming dental caries
Tube 3: mild susceptibility to
are not so clear. forming dental caries
Tube 4: moderate susceptibility
to forming dental caries
Tube 5: high susceptibility to
forming dental caries
 3. Alban test - A simplified
substitute for the Snyder test

MAIN FEATURES
I. Use of somewhat softer medium that permits
the diffusion of saliva and acids without the
necessity of melting the medium.
II. Use of a sampling procedure in which the
patient expectorates directly into tubes that
contains the medium.
Snyder test agar

A small scale to measure

60grams

A two liter Pyrex glass to melt ALBAN

the medium. TEST
MEDIUM
A funnel to dispense the
medium into test tubes.

100, 16mm test tubes with

screw caps.
 Procedure
60 grams of Snyder test agar is placed in 1 litre of
water and the suspension is brought to a boil over
a low flame.

When thoroughly melted, the agar is distributed

using about 5ml per tube. These tubes should be
autoclaved for 15 minutes; allowed to cool and
store in refrigerator

2 tubes of Alban medium are taken from the refrigerator

and the patient is asked to expectorate a small amount of
saliva directly into the tubes. The tubes are labelled and
incubated at 98.6 °F(37⁰c) for up to 4 days.

Tubes are observed daily for;

 Change of color from bluish green (pH 5) to definite yellow (pH 4 or below).
 The depth to which the change has occurred.
 The daily results collected for 4 days period should be recorded on the patients
chart.
 Following method is used for final
recording, after 72 or 96 hours of
incubation

1) Reading negative for the entire incubation period are labeled

“negative”.
2) All other readings are labeled positive whether +,++,+++ or++++.
3) Slower change or less color change (compared to previous test) is
labeled “improved”.
4) Faster changes or more pronounced color change (compared to previous
test) is labeled “worse”.
5) When consecutive readings are nearly identical, they are labeled “no
change”.
SCORING

 Scale for Scoring:

o No colour change Negative
o Beginning colour change
(from top of medium down) ‘+’
o One half colour change ‘++’
( from top down)
o Three forth colour change ‘+++’
(from top down)
o Total colour change to yellow ‘++++’
Advantages
Simple
Low cost
Good Diagnostic value
Motivational value (ideal for education)
Good for indicating caries inactivity
Disadvantages
More armamentaria required
Based on subjective evaluation of a colour change
that is often not clear
4. SWAB TEST- GRAINGER (1965)

Interpretation
Swab the buccal The changes in the pH < 4.1 = Marked
surface of the teeth pH following a 48 caries activity
with a cotton hours incubation is
applicator, which is pH 4.2 to 4.4 = Active
read on a pH meter
subsequently or the color change pH 4.5 to 4.6 =
incubated in the is read by the use of slightly active
medium. a color comparator. pH 4.6 and over =
Caries inactive
 Advantages
1) In predicting caries increments,
particularly in children with low
or no previous caries
experience.
2) No collection of saliva is
required.
Test to identify Streptococcus mutans
• MS level in saliva

• S.mutans dip-slide method

• Plaque/toothpick method
MS SCREENING TESTS
• Saliva/tongue blade method
5. STREPTOCOCCUS
MUTANS LEVEL IN SALIVA

Principle:

Measures the number of S. mutans colony forming unit per unit volume of
saliva and culturing of the plaque samples from discrete sites (occlusal
fissure/ proximal area) for detecting and quantitating S. mutans colonized on
teeth.

 Incubation is done on Mitis Salivarius Agar (MSA), selective streptococcal

medium with addition of high concentrated sucrose (20%) and 0.2 U
bacitracin per ml (MSB) suppress the growth of most non- S.mutans colonies.
 Procedure:

 Sample of organism is obtained by the use of tongue blades (wooden spatulas)

which are then pressed against Streptococcus Mutans selective MSB ( Mitis
Salivarius Bacitracin) Agar in special petri dishes.

 The agar plates are incubated at 37⁰Celsius for 48 hours in 95% carbon
dioxide gas mixture.
Interpretation
 Level of S. mutans > 105 / ml of saliva = unacceptable.
 Colonization of a new surface does not occur readily unless the level of
S. Mutans reaches :
4.5 × 104 / ml for smooth surface and 103 /ml for occlusal fissures.
 • Since the frequency of isolation of S.mutans
is high prior to initiation of lesions as
Advantages contrasted to lactobacilli, so the clinician
utilizes this count as an adjunct in caries
management.

• Difficulty of distinguishing between a

carrier state and cariogenic infection.
• S. mutans may constitute less than 1% of
Disadvantages total flora of plaque.
• S. mutans tend to be located at specific sites
only.
• Plates have short shelf-life of 1week.
6. Dip- Slide Method for S.
mutans count- the estimation of
Streptococcus Mutans levels in saliva

• Undiluted paraffin – stimulated saliva is poured on a special plastic

slide that is coated with MSA (Mitis Salivaris Agar ) containing 20%
sucrose

• Agar surface is thoroughly moistened and excess saliva is allowed to

drain off.

• Two discs containing 5mg of Bacitracin are placed on the agar 20mm
apart.
• Slide is tightly screwed into a cover tube and incubated at 37°C for
48hrs in a sealed candle jar.
The colonies are discrete The colonies are
and could be readily discrete and the
counted at 15X number in the zone of
magnification with the inhibition is more than
total count of CFU inside 200 at 32X
the inhibition zone less magnification.
than 200.

The colonies are tiny

and almost completely
or totally cover the
inhibition zone with
the number of colonies
uncountable even with
32X magnification.
 7. STREPTOCOCCUS MUTANS
SCREENING TEST

1. Plaque/ tooth pick method:

a)Action:
Simple screening of dilute plaque sample streaked on a selective culture
media.
b)Equipment:

Sterile tooth Platinum Incubator

picks loop Mitis Salivarius
Agar plates
Sterile (MSA)
Ringer’s containing
sulphadimetine
solution(5ml)
Procedure:

Plaque samples are collected from the gingival 3rds of

buccal tooth surfaces one from each quadrant and placed in
Ringer’s solution.

The sample is shaken until homogenized.

The plaque suspension is stretched across

MSA plates.

After aerobic incubation at 37°C for 72 hours,

cultures are examined and total colonies in 10
fields are recorded.
 2. Saliva/ Tongue blade
method

Action:
The test estimates the number of S. mutans in mixed paraffin- stimulated saliva
when cultured in Mutans Salivarius Bacitracin(MSB) agar.

Equipment:
Paraffin wax, Sterile tongue Blades, Disposable contact petri dish containing MSB
agar.
 PROCEDURE

•The subjects chew a piece of paraffin wax for one minute to displace
plaque micro-organisms, thereby increasing the proportions of plaque
micro-organisms in saliva.

•Using sterile tongue blade, which they rotate in their mouth 10 times
so that both sides are thoroughly inoculated by subjects flora, which are
then pressed into a MSB agar in a disposable petri dish.

•Incubation is done at 37°C .

For field studies, the plates can be kept in plastic bags containing
expired air, which are then sealed and incubated at 37°C.
 CARIES SUSCEPTIBILITY TESTS(Tests for
evaluating salivary defense)

• Reductase test

• Buffer capacity test (Dentobuff test)

• Fosdick calcium dissolution test

8. SALIVARY BUFFER CAPACITY

Measures the no: of mm of acid

required to lower the pH of saliva
through an arbitrary pH interval

pH meter or
colour Amount of acid
indicator or base
necessary to
PRINCIPLE bring colour
indicators
 PROCEDURE

10ml of
After The number of
stimulated
correcting the The level of millimeter of
saliva is
pH meter to lactic acid is lactic acid
collected at
room then added to needed to
least one hour
temperature, the sample reduce pH
after eating ;
the pH of until a pH of 6 from 7.0 to 6.0
5ml of this is
saliva is is reached. is a measure of
measured into
adjusted to 7.0 buffer capacity.
a beaker.
Buffering
Capacity

EVALUATION
Caries
Activity

Advantages
• Simple to carry out
Disadvantages
• Does not correlate adequately with caries activity
9. Salivary Reductase Test
(Susceptibility Test)- Rapp 1962

Saliva PROCEDURE
collected by
chewing
paraffin and
expectorated Mix with Caries
directly into the dye conduciven
collection Diazo- ess reading
tube. resorcinol or colour
change is
done after
15 minutes.
 Evaluation

Color Time score Caries activity

Blue 15min 1 Non Conducive

Orchid 15min 2 Slightly

Conducive

Red 15min 3 Moderately

Conducive
Red Immediate 4 Highly Conducive

Pink or White Immediately 5 Extremely

Conducive
Evaluation Advantages Disadvantages
• The evaluation is • No incubation • Test results vary
based on the required. with time after
colour changes food intake and
• Quick results
and the caries after brushing.
conduciveness.
10. Fosdick Calcium Dissolution
Test

25 milliliters of this

Saliva is stimulated by
having the patient chew PRINCIPLE
saliva is collected and part
of it is analyzed for
gum or paraffin.
Measures the mg of powdered
calcium enamel
content.
The remaining dissolved
saliva isin 4 hours by acid formed
placed in an 8 when
inch sterile The tube is sealed and
the patient’s saliva shaken
is mixedfor with
4 hours at body
test tube with about 0.1
gram of powdered human
glucose temperature
and powdered enamel. - calcium
enamel. content analyzed again

The chewing gum The amount of dissolution

stimulate the saliva to increases as the caries
activity increases.
produce sugar.
In limited study the
correlation reported
is good. Test is not simple and
requires complex equipment.
Test is expensive and
requires trained personnel.
 11. Oratest- Rosenberg et al in 1989 for
estimating oral microbial levels

 Aerobic
Principle: is baseddehydrogenase
on the rate of oxygen depletion by micro organisms in
transfersmilk
expectorated electron
samples. or protons to
oxygen.

Once O2 get utilized – methylene

blue acts as an electron acceptor
– Leucomethylene blue

Reflects the metabolic activity

of aerobic organisms.
 PROCEDURE

Mouth is rinsed vigorously with 10

ml of sterile milk for 30s and the
expectorate is collected

3ml transferred to screw cap tube

0.12 ml of 0.1% methylene blue-

mixed-placed on a stand

Tubes observed every 10 mins at

the bottom-colour change

Time taken-initiation of colour

change-within 6mm ring
Less time consuming
Lack of
Specificity
Economic

Non-toxic vehicle
Cannot accurately
Can be easily learnt differentiate btwn
by auxiliaries. between initial and
progressive carious
lesion
 LIMITATIONS OF CARIES
ACTIVITY TESTS

 None of the tests are highly reliable as indicators of expected caries

increments.
 Measures a single parameter such as acid produced or colony counts of
bacterial species.
 Tests do not encompass those factors involved in determining caries
resistance such as fluoride exposure, maturation of enamel or immune
protection.
 CONCLUSION

 Caries activity test are a valuable adjunct for patient motivation and
prognosis of the carious process.
 To run an effective caries prevention program it is of utmost
importance.
 There still has to be a lot research to happen in this area to come up
with an ideal caries activity test.
 Presently, a combined use of several selected tests will give a picture of
the caries activity of an individual.