CARIES ACTIVITY/ SUSCEPTIBILITY TESTS

CARIES ACTIVITY/ SUSCEPTIBILITY TESTS



INTRODUCTION:-
Caries activity refers t the increment of active lesions (new and recurrent lesions) over a stated period of time. Caries activity is a measure of the speed of progression of a carious lesion.
Caries susceptibility refers to the inherent tendency of the host and target tissue, the tooth, to be afflicted by the caries process.
Caries activity tests measure the degree to which the local environmental challenge (e.g. dietary effect on microbial growth and metabolism) favors the probability of occurrence of carious lesions.

A Caries activity test helps to:
• Identify high-risk groups and individuals.
• Determine the need for personalized preventive measures and motivate the individual.
• Monitor the effectiveness f oral health education programs by establishing an initial baseline level of cariogenic pathogens as a basis for future evaluation
• Ensure a low level of caries activity before starting any extensive restorative procedure.
• Serve as an index of the success of therapeutic measures by monitoring patient behavior towards reducing the number of S. Mutans and lactobacilli as part of counseling to curtail sucrose intake.

IDEAL REQUISITES OF A CARIES ACITIVTY TEST: given by Snyder
• Should have a sound theoretical basis.
• Should show maximum correlation with clinical status.
• Should be accurate with respect to duplication of results
• Should be simple
• Should be inexpensive
• Should take little time.

SOME CARIES ACTIVITY AND SUSCEPTIBILITY TESTS:
1. Lactobacillus colony count test
This caries activity test was introduced by Hadley in 1933.

Principle involved:
This test estimates the number of acidogenic and aciduric bacteria in the patient’s saliva by counting the number of colonies. Appearing on tomato peptone agar plates (pH 5.0) after inoculation with a sample of saliva. A selective media favoring the growth of aciduric lactobacilli is the basis for the test.

Procedure:-
• The patient chews a small piece of paraffin, before breakfast in the morning saliva accumulated in the following 3-min period (5-10 ml) is collected in a sterile container and shaken for 2 minutes to mix it.
• Saliva sample is diluted to 1:10 dilution by pipetting 1 ml of the saliva sample in to 9 ml of tube sterile saline solution and shaken.
• It is again diluted (1:100) by pipeting 1 ml of the 1:10 dilution into another 9 ml tube of sterile sample solution and mixed thoroughly.
• 0.4 ml of each dilution is spread on the surface of agar plate and incubated for 3-4 days at 37 0c.
• The numbers of lactobacillus colonies that develop are counted using a colony counter with bright light and a large magnifying glass or the quebec counter.

The lactobacilli colony counts in saliva as related to caries susceptibility is shown in the table.

Number of lactobacilli/ml Carries Activity
0-1000
1000-5000
5000-10000
>10000 Little or no activity
Slight Activity
Moderate Activity
Marked Activity

Disadvantages
1) Inaccurate for predicting the onset of caries.
2) It does not completely exclude the growth of other relatively aciduric organisms.
3) Requires relatively complex equipment,
4) It only takes few minutes to do test, but the result are not available for several days.
5) Counting is a tedious procedure.


2. COLORIMETRIC SNYDER TEST

Snyder devised this colorimetric caries activity test in 1951.

Principle:
It measures the ability of salivary microorganism to form organic acid from a carbohydrate medium. The medium contains an indicator dye, Bromocresol green.
Procedure:
Saliva is collected by chewing paraffin before breakfast in the morning. A tube of snyder glucose agar is melted and then cooled to 50 0c. it is then shaken and 0.2 ml of saliva is pipetted into the tube and thoroughly mixed. The agar is allowed to solidify and then incubated at 37 0c.
Amount of acid produced by acidogenic organisms is detected by change in the PH indicator and compared to an uninoculated control tubes against a white background after 24,48 and 72 hours of incubation. The rate for color change from green to yellow is indicative of degree of carries cavity.

Time
24 hours 48 hours 72 hours
Color Yellow Yellow Yellow
Carries Activity Marked Activity Definite Activity Limited Activity
Color Green Green Green
Carries Activity Continue Test Continue Test Inactive

Advantages:
• Relative simple to carry out.
• Cost is moderate.
Disadvantages:
1) Time Consumed is more.
2) Sometime the color changes are not very clear.
3) Measures acidogenic potential but is limited in predictive value because these salivary organisms may not be representative of those in plaque.

3. THE SWAB TEST

Grainger et al developed this test in 1965. it has an advantage over test in that no collection of saliva is necessary. So it is valuable in evaluating carries activityin very young children.
Principle:
It is based on the same principle as the snyders test.
Procedure:
• The oral flora is sampled by swabbing the buccal surface of the teeth with a cotton applicator,
• It is subsequently incubated in the medium.
The change in the pH following a 48 hours incubation period is read on pH meter.
Advantages:
• The test is of voice in predicting caries increment, particularly in children with low or no previous caries experience
• No collection of saliva is required.

4. S MUTANT LEVEL IN SALIVA

Principle:
The test measure the number of S. Mutants colony forming units (CFU)/unit volume of saliva by culturing the plaque samples from discrete sites (occlusal fissure / proximal area) for detecting and quantifying S. Mutans colonized on teeth.

Procedure:-
• The sample of organisms is obtained by the use of tongue blades (wooden spatulas)
• They are then pressed organist streptococcus mutans selective MSB (Mitus Salivarius Bacitracin) Agar in special perti dishes.
• The agar plates are incubated at 370C for 48 hrs in 95% at 5% CO2 gas mixture.

Interpretation:
• Levels of streptococcus mutans> 100 / ml of saliva = unacceptable.
• Colonization of a new surface does not occur readily unless the level of S. Mutans reaches 4.5X104 / ml for smooth surface and 103/ml for occlusal fissures.

Advantages:-
Since the frequency of isolation of S. Mutans is high prior to initiation of lesions as contrasted to Lactobacilli, this test can be utilized as an adjunct in caries management.

Disadvantages:-
• Difficulty of distinguishing between a carrier state and cariogenic infection.
• S. Mutans may constitute less than 1% of total flora of plaque.
• S. Mutans tends to be located of specific sites only.
• Plates have a self life of only about a week, therefore not convenient for chirside tests.

5. DIP SLIDE METHOD FOR S. MUTANS COUNT

This method was developed for the estimation of streptococcus Mutans levels in saliva.

Procedure:-
• Undiluted paraffin stimulated saliva is poured on a special plastic slide that is coated with MSA (Mitis Salivarius Agar) containing 20% sucrose.
• The agar surface is thoroughly moistened and excess saliva is allowed to drain off.
• Two discs containing 5 µg of Bacitrac in are placed on the agar 20 mm apart.
• The slide is tightly screwed into a cover tube after inserting a CO2 tablet and incubated at 370C for 48 hours in a sealed cadle jar.

6. SALIVARY BUFFER CAPACITY TEST :-

Principle
Buffer capacity can be quantitated using either a pH meter or color indicator. This test measures the number of milliliters of acid required to lower the pH of saliva through an orbitary 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 point.

Procedure:-
• Ten ml of stimulated saliva is collected under oil at least 1 hour after eating.
• 5 ml of this is measured into a beaker. After carrecting the pH meter to room temperature, the pH of saliva is adjusted to 7.0 by addition of lactic acid or base.
• Lactic acid is then added to the sample until a pH of 6.0 is reached.
• The amount of lactic acid needed to reduce the pH from 7.0 to 6.0 is a measure of buffer capacity. This number can be converted to milli equivalents per liter.

Evaluation:-
Thee is an inverse relationship between buffering capacity of saliva and caries activity the saliva of individuals whose mouths contain a considerable number of carious lesions frequently have a lower acid-buffering capacity than the saliva of those who are relatively caries free.

Advantage:-
Simple to carry out

Disadvantages:
Does not correlate adequately with caries activity.

7. SALIVARY REDUCTASE TEST:-
Principle:-
This test measures the activity of the reeducates enzyme present in salivary bacteria.

Procedure:-
• Saliva is collected by chewing paraffin and expectorates directly into the collection tube.
• The sample is then mixed with the dye Diaxo resorcinol
• The “Caries Conduciveness” reading or color change is done after 15 minute. No incubation procedures are required.

Advantages:-
No incubation required
Quick results

Disadvantages:-
Test results vary with time after food intake and after brushing.

8. ALBAN TEST

Procedure:-
• 60 grams of Snyder test agar is placed in 1 liter of water and the suspension is brought to a boil over a low flame.
• When thoroughly melted, the agar is distributed using about 5 ml per tube.
• These tubes should be autoclaved for 1.5 minutes, allowed to cool and stored in a 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 labeled and incubated a 98.60F (370C) for up to 4 days.

The tubes are observed daily for:
• Change of color from bluish green (pH5) t definite yellow (pH 4 or below)
• The depth n the medium to which the change has occurred.

The daily results collected for a 4 day period should be recorded on the patient’s chart. The following method is used for final recordings, after 72 or 96 hours of incubation,
• Readings negative for the entire incubation period are labeled “negative”
• All other reading are labeled “positive” whether +, + +, + + + or + + + +.
• Slower change or less color change (compared to previous test) is labeled “improved”.
• Faster change or more pronounced color change (compared to previous test) is labeled “worse”.
• When consecutive reading are nearly identical, they are labeled “no change”

Advantages:-
Simple
Low cost
Diagnostic value when negative results are obtained.
Its motivational value (ideal for education)

Disadvantages:-
More armamentaria required
Based on subjective evaluation of a color change that may not be clear cut.

9. STREPTOCOCCUS MUTANS SCREENING TEST
A. Plaque / tooth pick method:

Action:-
The test involves a simple screening of diluted plaque sample streaked on a selective culture media.

Procedure:-
• Plaque samples are collected from the gingival thirds of buccal tooth surfaces one from each quadrant and placed in Ringers solution.
• The sample is shaken until homogenized.
• The plaque suspension is stretched across MSA plates.
• After aerobic incubation at 370C for 72 hours, cultures are examined and total hours, cultures are examined and total colonies in 10 fields are recorded.

B. Saliva / Tongue blade method
Action
This test estimates the number of S. mutans in mixed paraffin-stimulated saliva when cultured in Mutans Salivarious Bacitracin (MSB) agar. This was developed for use in large number of school children.

Procedure:-
• The subjects chew a piece of paraffin was for one minute to displace plaque microorganisms, thereby increasing the proportions of plaque microorganisms in saliva.
• Sterile tongue blades are then rotated in the patient’s mouth 10 times so that both the sides are thoroughly inoculated by the oral flora.
• It is then pressed into MSB agar in a disposable contact petri dish.
• Incubation is done at 370C, for field studies, the plates can be plastic bags containing expired air, which are then sealed and incubated at 370c.

Advantages:-
This is a simplified and practical method for field studies.
Avoids the necessity of collecting saliva
It requires no transport media/ dilution steps.

10. FOSDICK CALCIUM DISSOLUTION TEST

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

Procedure:-
• Saliva is stimulated by having the patient chew gum or paraffin. 25 ml of this saliva is collected and part of it is analyzed for calcium content.
• The remaining saliva is placed in an 8 inch sterile test tube with about 0.1 gm of powdered human enamel.
• The remaining a liva is placed in 0.1 gm 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 paraffin is used. A concentration of about 5% glucose is added. The amount of dissolution increases as the caries activity increases.

Advantages:-
In limited studies, the correlation reported is good.

Disadvantages:-
• The test is not simple and requires complex equipment.
• The test is expensive and requires trained personnel.


11. ORA TEST
This test was developed by Rosenberg et at in 1989 for estimating oral microbial levels.

Principle:-
It is based on the rate of oxygen depletion by microorganisms in expectorated milk samples. In normal conditions the bacterial enzyme, aerobic dehydrogenase transfers electrons or protons to oxygen. Once oxygen gets utilized by the aerobic organisms, methylene blue acts as an electron acceptor and gets reduced to leucomethylene blue. This reflects the metabolic activity of the aerobic organisms.

Procedure:-
• Mouth is rinsed vigorously with 10 ml of sterile milk for 30 seconds and the expectorate is collected.
• 3 ml of this is transferred to the screw cap tube with the help of a disposable syringe.
• To this, 0.12 ml of 0.1% methylene blue is added, thoroughly mixed and placed on a stand in a well illuminated area.
• The tubes are observed every 10 minutes for any color change at the bottom using a mirror.
• The time taken for the initiation of color change within 6mm ring is recorded.
The higher the infection, lesser was the time taken for the change in color of the expectorate reflecting higher oral microbial levels.

Advantages:-
• Less consuming
• Economics
• Non-toxic vehicle
• Can be easily learnt by auxiliary personnel

Disadvantages:-
• Lack of specificity





CONCLUSION
None of the tests are highly reliable as indicator of expected caries increments. Dental caries is a multi-factorial disease and caries predictive tests do not encompass all those factors involved in determining caries resistance such as fluoride exposure, maturation of enamel or immune protection.







CARIES ACTIVITY/ SUSCEPTIBILITY TESTS