CARIES ACTIVITY TEST

INTRODUCTION
Caries activity refers to the increment of active lesion(new and recurrent lesions) over a stated period of time.
Caries activity measures the speed of progression of a carious lesion.

CARIES SUSCEPTIBILITY
It refers to the inherent tendency of the host and target tissue, the tooth to be afflicted by the carious process.
Caries activity measures the degree to which the local environmental challenge favors the

probability of occurrence of carious lesions.

Uses of caries activity test

Identify the high-risks groups and individuals. Determine the need for personalized preventive

measures and motivate the individual. Monitor the effectiveness of the oral health education programs. Ensure a low level of caries activity before any restoration. Aid in selection of patient for caries activity.

IDEAL REQUISITES
Should have sound theoretical basis.
Should have maximum correlation with clinical

status.
Easy to perform. Should be inexpensive and simple.

 Should be adaptable for chair side.

Results should be accurate and reproducible. Results should have good validity, reliability and feasibility.

TYPES OF CARIES ACTIVITY TEST

MICROBIAL TEST FOR MUTANS STREPTOCCOCI

DETECTION.
MICROBIAL TEST FOR LACTOBACILLI DETECTION. SNYDER TEST.

ALBAN TEST.
SALIVARY REDUCTASE TEST.

 SWAB TEST
FOSDICK CALCIUM DISSOLUTION TEST. SALIVARY BUFFER CAPACITY TEST. DIP SLIDE METHOD. ORA TEST.

MICROBIAL TEST FOR MUTANS STREPTOCCOCI DETECTION

Several methods are available to measure the level of Mutans Streptococci in saliva, plaque etc. Types of methods are as follows: 1. Laboratory method. 2. Chair-side method.

3. Survey method. 4. Selective method. 5.Adherence method.

LABORATORY METHOD
Principle: Measures the number of forming unit (CFU)/unit volume of saliva by culturing the plaque samples from discrete sites(occlusal fissures/proximal areas)for detecting and quantitating S.Mutans colonies.
Incubation is done on Mitis Salivarius Agar(MSA) selective streptococcal medium with addition of high concentration of sucrose (20%) and 0.2 U bacitracin/ml (MSB) which supresses the growth of non S.Mutans colonies.

LABORATORY METHOD
Saliva or plaque sample is

collected.
It is then mixed with proper transport medium and

the sample is sent to a microbiological laboratory. i.e(special petri dishes).

Samples are directly placed the selective agar plates The agar plates are incubated at 37C for 48hrs in

95% at 5% CO2 gas mixture.

 Mutans colonies on the plates are counted.

The results are expressed as the number of

colony forming units per ml of saliva.

Most common type of selective agar plates

used is mitis-salivarius-bacitracin agar.

RESULTS FOR LABORATORY METHOD

Caries Risk Or Activity 100,000 (105) S. mutans cells/ml of saliva LOW. 100,000 to 500,000 (105 to 5X105) cells/ml MEDIUM. 500,000 (5X105) or above cells/ml HIGH

CHAIR-SIDE METHOD
Also known as Strip mutans test is based on the ability

of streptococci to grow on hard surfaces.

use of the selective broth (High sucrose concentration in

combination with bacitracin).

The Dentocult SM-strip mutans kit is used for

estimation of mutans streptoccoci in saliva containing test strips, bacitracin discs, test tubes with broth, paraffin for chewing and a standard chart to evaluate the level of mutans after incubation.

 The level of mutans streptococci is given as Class after comparision with chart. Indicating low to high , equivalent to 106 mutans CFU per ml in saliva. The mutans sreptococci colonies as small blue dots but the color can vary from dark blue to pale blue.

DENTOCULT STRIP MUTANS KIT

PROCEDURES
In case of dentocult SM Strip mutans test,bacitracin discs are added to the broth at least for 15 minutes before use.
Then, patient is asked to chew a piece of paraffin for 1 minute. Plastic strip(test strip) is then taken in the mouth to become contaminated.

 The strip is then withdrawn through closed lips, leaving a thin layer of saliva on the strip. The strip then incubated in the selective broth.

After incubation for 48 hour at 35-37 degree centigrade, the strip with attached colonies is compared with the chart.

PROCEDURE

RESULTS FOR CHAIR SIDE METHOD

Strips for plaque (Squaretipped)

Test strips for stimulated saliva (round-tipped)

SELECTIVE METHOD
Described by Kristoffersson and Bratthall.
This method is used for demonstration of S.mutans at

the specific sites of oral cavity.

This test involves simple screening of diluted plaques

sample streaked on the selective culture media.

Equipments required are sterile toothpicks, sterile ringer

solution, platinum loops, mitis-salivarius agar plates and incubator.

PROCEDURES
Plaques sample are collected from gingival 3rd of the

tooth surface. Toothpicks are inserted into the approximal spaces. Then it is placed in Ringers solution. It is then shaken homogeneously. Contaminated slides are then pressed directly against selective agar plates. Incubation is done at 37 degree centigrade for 72 hours. Then sites with or without mutans sreptococci can be identified.

ADHERENCE METHOD
It categorizes salivary samples based on ability of S.

mutans to adhere to glass surfaces when grown in sucrose containing broth.

Equipment required are tube to collect saliva, rack to

hold the culture tubes, disposable pipettes, incubator and MSB(mitis- salivarius bacitracin ) broth.

PROCEDURES
Unstimulated saliva(0.1 ml) is inoculated in MSB broth.
Inoculated tubes are set at 60 degree angle and

incubated aerobically at 37 degree centigrade for 24 hours.

After the growth has been observed, the supernatent

medium is removed and the cells adhering to the glass surface are examined microscopically and scored.

RESULT OF ADHERENCE METHOD

VALUE (-) (+) (++) (+++) INFERENCE No growth expressed A few deposits ranging from 1-10. Scattered deposits of smaller size. Numerous minute deposits.

MICROBIAL TEST FOR LACTOBACILLI DETECTION

Introduced by Hadley in 1933.
-Introduced by Hadley in 1933.

Principle:
Estimates the number of acidogenic and aciduric bacteria in patients saliva by counting the number of colonies appearing in tomato peptone agar plates(pH 5.0) after inoculation with sample of saliva.

PROCEDURES
Before breakfast the patient is asked to chew a paraffin and the saliva accumulated is collected in a sterile container and shaken for 2 mins to mix it.
saliva sample is diluted to 1:10 dilution by pipetting 1 ml of saliva into a 9 ml of sterile saline solution. It is again diluted to 1:100.

 0.4 ml of the diluted solution is spread on the agar plate and incubated at 37C for 3-4days. Lactobacillus colonies are then counted using a colony counter or Quebec counter. The number of lactobacilli/ml saliva is calculated by multiplying the number of colonies by the dilution factor.

The colony count as related to caries susceptibility is shown n the table:

Number lactobacilli per ml saliva

0-1000 1000-5000 5000-10000 >10000

Caries activity
Little or none Slight Moderate Marked

COLORIMETRIC SNYDER TEST

It measures the ability of salivary micro-organisms to

form organic acid from carbohydrate medium.

Principle:

The Snyder test measures the rapidity of acid formation when a sample of stimulated saliva is inoculated into glucose agar adjusted to pH 4.7 to 5 with the help color indicator named bromocresol green.

EQUIPMENTS
Saliva collecting bottles
Paraffin A tube of snyder glucose agar containing

bromocresol green Pipettes Incubator

PROCEDURE:
Paraffin stimulated saliva is collected before breakfast.
A tube of Snyder glucose agar is melted, cooled at 50C,

shaken and 0.2 ml of saliva is pipetted in the tube.

The agar is solidified and incubated at 37C. Amount of acid produced is detected by pH indicator and

compared to an uninoculated control tube after 24, 48 & 72 hrs of incubation.

RESULT OF SNYDER TEST

Time Time Time

24 hours
Color Caries activity Color Caries activity Yellow Marked activity Green Continue test

48 hours
Yellow Definite activity Green Continue test

72 hours
Yellow Limited activity Green inactive

ALBAN TEST
Is a simplified version of the Snyder test. Main features: 1. Softer medium are used that diffuse saliva and acids without the necessity of melting the medium.
2. The patient simply has to spit directly into the tubes that contain the medium.

Materials required to prepare the Alban test medium: 1. Snyder agar 2. A small scale to measure 60 grams 3. A 2 litre Pyrex glass to melt the medium 4. A funnel to dispense the medium into test tubes 5. Hundred 16mm test tubes with screw caps.

PROCEDURE
60 gms of Snyder test agar is placed in 1 litre

water and the suspension is boiled over a low flame.

The melted agar is then distributed among the

tubes. About 5ml/tube.

These tubes are autoclave for 15 mins, cooled and

stored in a refrigerator.

 2 tubes of Alban medium are taken out and the

patient is asked to expectorate saliva directly into the tubes.

The tubes are labelled and incubated at 98.6F up

to 4 days.
The tubes are observed daily for the colour

change from blue to green to definite yellow with decrease in pH.

RESULT OF ALBAN TEST

Color change Score

No color change
Beginning of color change One half color change Three fourths color change Total color change to yellow

+ ++ +++

++++

SALIVARY REDUCTASE TEST

This test measures the activity of the reductase enzyme present in the salivary bacteria. Action: This test measures the rate at which the indicator molecule Diazoresorcinol changes from blue to red to colourless on reduction by the mixed salivary flora.

PROCEDURE
The patient is asked to chew a paraffin and the saliva accumulated is collected directly into the collection tube(5ml).
Then sample collected is mixed with a fixed amount of diazoresorcinol. Change in colour after 30 seconds and after 15 mins is taken as a measure of caries activity.

RESULT OF SALIVARY REDUCTASE TEST

COLOR BLUE ORCHID RED RED PINK OR WHITE TIME 15 minute 15 minute 15 Immediately Immediately SCORE 1 2 3 4 5 CARIES ACTIVITY Non conductive Slightly conductive Moderately conductive Highly conductive Extremely conductive

SWAB TEST
Grainger et al developed this test in 1965 Principle is similar to that of Snyder test.

Procedure: Buccal surfaces of the teeth are swabbed with a cotton applicator.
The sample collected is incubated in the medium.
The change in the pH after 48 hrs incubation period is

read on a pH meter or using a color comparator.

RESULT OF SWAB TEST

pH 4.1 4.2-4.4 4.5-4.6 Over 4.6 Caries activity Marked caries activity Active Slightly active Caries active

FOSDICK CALCIUM DISSOLUTION TEST

Principle: 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 host tubes, test tube agitation equipment, and equipment for determining the calcium content of the saliva.

PROCEDURE
The patient is asked to chew paraffin wax and 25 ml of the

saliva is collected and part of it is analysed 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 tube is sealed and shaken for 4 hours at body

temperature, after which it is again analysed for calcium content.

The use of paraffin to stimulate saliva increases the concentration of the glucose by 5%. The amount of enamel dissolution increases as the caries activity increases.

SALIVARY BUFFER CAPACITY TEST

Principle: It measures the number of millimetres of acid required to lower the pH of saliva through an arbitrary pH interval, such as from pH 7.0 to 6.0 or the amount of acid or base necessary to bring colour indicators to their end point.

EQUIPMENT
pH meter Titration equipment 0.05 N lactic acid and 0.05 base Paraffin Sterile glass jars containing oil.

1. 2. 3. 4. 5.

LABORATORY METHOD
10 ml of stimulated saliva is collected under oil after 1 hour of eating. 5 ml is then measured in the beaker.
pH of the saliva is adjusted to 7.0 by addition of

lactic acid or base using a pH meter in room temperature.

 The level of lactic acid in graduated cylinder is rerecorded. pH of the sample is brought down to 6.0 by adding lactic acid. The amount (no of millimetres) of lactic acid needed to reduce the pH from 7 to 6 is the measure of buffer capacity.

CHAIRSIDE METHOD
This system differentiates between buffer capacities. This was developed by Ericson and Bratthall.

SALIVARY BUFFER KIT

PROCEDURES
A new and simplified method to estimate the salivary buffer capacity .
Dentobuff Strip, consists of a pH indicator paper

that has been impregnated with acid. A small volume of saliva is added to the strip and after 5 min the color of the strip is compared with a chart. The colors have been chosen to indicate low, medium, or good buffer capacity.

RESULT OF BUFFER CAPACITY TEST

BUFFER CAPACITY
Low buffer capacity Intermediate buffer capacity Normal buffer capacity

FINAL pH VALUE
4.0 or less 4.5 to 5.5 6.0 or more

COLOR CHANGE
Yellow color Green color Blue color

ORA TEST
Developed by Rosenberg et al in 1989 for estimating oral microbial level. Principle:
Based on the 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.

EQUIPMENT
1. Sterile beakers
2. Sterilized milk 3. Screw cap test tubes

4. 0.1% aqueous solution of methylene blue

5. 10 ml disposable syringes 6. Pipette 7. Mirror 8. Stop watch and test tube

PROCEDURES
Mouth is rinsed vigorously with 10 ml of sterile milk for 30 secs and the expectorate is collected. 3 ml of this is transfered to the screw cap tube with the help of a disposable syringe. To this, 0.12 ml of 0.1% methylene blue is added, mixed and placed on a stand.

 The tubes are observed every 10 mins for any color change at the bottom using a mirror. The time taken for the initiation of color change within 6 mm ring is recorded.

Higher the infection, lesser time taken for the color change reflecting higher oral microbial levels.

DIP SLIDE METHOD FOR S.MUTANS COUNT

Devised for the estimation of S. mutans levels in saliva. Procedure:
Saliva stimulated with paraffin is poured on a special plastic slide that is coated with MSA (Mitis salivarious agar) containing 20% sucrose.

 The agar surface is moistened and excess saliva is allowed to drain. Two discs containing 5 g of Bacitracin are placed on the agar plate 20 mm apart.

The slide is tightly screwed into a cover tube after inserting CO2 tablet and incubated at 37C for 48 hours in a sealed candle jar.

EVALUATION FOR DIP SLIDE METHOD

Score Caries Activity
1

Description
The colonies are discrete and could be readily counted at 15X magnification with the total count of CFU inside the inhibition zone less than 200
The colonies are discrete and the number in the zone of inhibition is more than 200 at 32X magnification

Low

Medium

Score
3

Caries Activity

Description

High

The colonies are tiny and almost completely or totally cover the inhibition zone with the number of colonies uncountable even with 32X magnification.

CARIOGRAM
It is a computer program showing a graphical picture that illustrates a possible overall caries risk scenario. It expresses to what extent different etiological factors of caries affect caries risk. The cariogram identifies the caries risk factors for the individual and provides examples of preventive

and treatment strategies to the clinician.

HOW IS A CARIOGRAM CREATED?

The patient is examined and the data is collected for some factors of direct relevance for caries including bacteria,diet and susceptibilty related factors.
The factors/variables are given score according to a predetermined scale and entered in a computer program. The program presents a pie diagram which represents a following sectors.

1. The dark blue sector (DIET) is based on a combination of diet contents and diet frequency.
2. The red sector (BACTERIA) is based on the amount of plaque and mutans streptoccoci.

3. The light blue sector (SUSCEPTIBILITY) is based on a combination of fluoride program, saliva secretion and salivary buffer capacity.
4. The yellow sector (CIRCUMSTANCES) is based on a combination of caries experience and related diseases.

5. The green sector shows an estimation of (CHANCE OF AVOIDING CARIES). When the chance of avoiding caries is high, the caries risk is small and vice-versa.

CARIOGRAM

CONCLUSION
Till date no single caries activity test is sufficiently accurate or reliable to serve as a predictor of potential caries activity for the

individual patient. Since, caries activity tests measure a single parameter such as acid production, colony counts etc, the best predictors of caries activity would therefore result from the combined use of several selected tests.

REFERENCE
Nikhil Marwah 2nd edition. Textbook Of Pediatric Dentistry
Shova Tandon 2nd edition.Textbook Of

Pedodontics Soben Peter 4th edition. Essentials Of Preventive And Community Dentistry Internet sources

 WHAT ARE THE USES OF CARIES ACTIVITY TEST?

 Identify the high-risks groups and individuals.

Determine the need for personalized preventive measures and motivate the individual.

Monitor the effectiveness of the oral health education programs.

Ensure a low level of caries activity before any

restoration. Aid in selection of patient for caries activity.

 WHAT IS A CARIOGRAM?

 It is a computer program showing a

graphical picture that illustrates a possible overall caries risk scenario.

IDENTIFY THE PICTURE.

 WHAT IS THE MOST COMMAN TYPE OF AGAR USED FOR DETECTION OF MUTANS STREPTOCOCCI?

 mitis-salivarius-bacitracin agar(MSB)