In this post we shall discuss various Caries Activity and Caries Susceptibility Tests
LACTOBACILLUS COLONY COUNT TEST
Introduced by HODLEY IN 1933 PRINCIPLE: This caries activity & caries susceptibility 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 after inoculation with a sample of saliva. Selective media favoring the growth of aciduric lactobacilli is the basis for the test.
EQUIPMENTS: saliva collecting bottles, paraffin, 29ml tubes of saline, 2 agar plates, 2 bent rods, incubator, Quebec counter and pipettes
PROCEDURE: patients chews a piece of paraffin before breakfast. Then saliva get accumulated and collected and then mix it. Saliva sample is diluted to 1:10 by pipetting 1 ml of the saliva.
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. Its only takes few minutes to do the test result are not several days.
COLORIMETRIC SYNDER TEST
Principle: This caries activity & caries susceptibility measures the ability of salivary microorganism to form organic acids from a carbohydrate medium. The medium contains an indicator dye, bromocresol green this dye changes of ph 5.4 to 3.8.
PROCEDURE • Saliva is collected by chewing paraffin. • A tube of synder glucose agar is melted n then cooled to 50’C. • 0.2 ml of saliva is pitted into the tube n mix it. • The agar is solidified & incubated. • Amount of acid produced by acidogenic organism is detected by changes in the ph indicator n then compared to the uninoculated control tube against a white background after 24,48 and 72 hours. • The rate of color change from green to yellow is indicative of degree of caries activity
ADVANTAGES: 1. Relative simple to carry out 2. Cost is moderate.
DISADVANTAGES: 1. Time consuming 2. Color changes are not very clear
Several modifications of Snyder test have been proposed to further simplify the method for use in dental office— A smaller volume(0.2ml) of culture media is inoculated with saliva using calibrated wire loop. This avoids use of pipettes and saves medium and space. Alban’s method uses less agar in the medium so that tubes do not require melting. The buccal surfaces are swabbed and the cotton applicator incubated in semi-fluid Snyder medium. This has advantage of culturing directly from plaque.
THE SWAB TEST Grainger et al in 1965
PRINCIPLE: This caries activity & caries susceptibility measures the ability of salivary microorganism to form organic acids from a carbohydrate medium. The medium contains an indicator dye,bromocresol green this dye changes of ph 5.4 to 3.8.
PROCEDURE: Swab the buccal surface of the teeth with cotton applicator. And it is subsequently incubated in the medium
ADVANTAGES: 1. No collection of saliva is required 2. Predicts caries increment
S.MUTANS LEVEL IN SALIVA:–
PRINCIPLE: This caries activity & caries susceptibility measures no. of S.mutans colony forming units for detecting and quantitating S.mutans colonized on teeth .
PROCEDURE: Sample is obtained by the use of tongue blades(wooden spatulas). They are pressed against S.mutans selective Mitus Salivarius Bacitracin(MSB) The agar plates are incubated at 37degree C for 48 hours in 95%at 5% CO2 gas mixture.
ADVANTAGES: Frequency of isolation of S.mutans is high so this test is utilized as an adjunct in caries management.
DISADVANTAGES: • S.mutans are located at specific site • Difficulty to differentiate between carrier state and cariogenic bacteria • S.Mutans may constitute less than 1%of total flora of plaque.
Dip slide method for S.Mutans count:– Estimates S.mutans levels in saliva
PROCEDURE:- Undiluted paraffin stimulated saliva is poured on special plastic slide that is coated with MSA(mitis salivarius agar) containg 20%sucrose. The agar surface is thoroughly moistened and excess saliva is allowed to drain off. Two discs containing 5ug of bacitracin are placed on the agar 20mm apart. The slide is tightly screwed into cover tube after inserting a CO2 tablet Incubated at 37degree C for 48 hours .
SALIVARY BUFFER CAPACITY
PRINCIPLE: Buffer quantitated using either a Ph meter or color indicatocapacity can be r. This test measures the Number of milliliters of acid requires 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 color indicators to their end point.
EQUIPMENTS: The needed equipments include a pH meter, titration equipment ,0.05 N lactic acid,0.05N base, paraffin, and sterile glass jars containing a small amount of oil.
PROCEDURE: • Ten ml of stimulated saliva is collected under oil at least 1 hour after eating. • 5ml of this is measured into a beaker. After correcting 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 litre.
EVALUATION: There 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.
ADVANTAGES: Simple to carry out
DISADVANTAGES: Does not correlate adequately with caries activity.
SALIVARY REDUCTASE TEST
PRINCIPLE:: This caries activity & caries susceptibility measures the activity of the reductase enzyme present in salivary bacteria.
PROCEDURE: • Saliva is collected by chewing paraffin and expectorated directly into the collection tube. • The sample is then mixed with the dye diazo-resorcinol. • The “Caries Conduciveness” reading or color change is done after 15 minutes. No incubation procedures are required.
ADVANTAGES: • No incubation required. • Quick results.
DISADVANTAGES: • Test results vary with time after food intake and after brushing.
ALBAN TEST It is a simplified substitute for the snyder test. Main features: Use of a somewhat softer medium that permits the diffusion of saliva and acids without the necessity of melting the medium. Use of a simpler sampling procedure in which the patient expectorates directly into tubes that contain the medium. To prepare the Alban test medium ,the following materials are required: Snyder test agar. A small scale to measure 60 grams. A 2litre Pyrex glass to melt the medium. A funnel to dispense the medium into test tubes. Hundred 16mm test tubes with screw caps.
Procedure: 60grams of Snyder test agar is placed in 1liter 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 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 at 98.6 degree Fahrenheit for up to 4 days. The tubes are daily observed for: Change of color from bluish green (pH 5) to definite yellow(pH 4 or below). The depth in 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 readings are labeled “positive” o 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 readings 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 color change that may not be clear cut.
STREPTOCOCCUS SCREENING TEST:
A. Plaque/Toothpick method
ACTION: The test involves a simple screening of diluted plaque sample streaked on a selective culture media.
EQUIPMENTS: • Plague samples are collected from the gingival thirds 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 degree Celsius for 72 hours ,cultures are examined and total colonies in 10 fields are recorded. • This test is an attempt to semi quantitatively screen the dental plaque for a specific group of caries inducing streptococci Mutans.
B. SALIVA /TONGUE BLADE METHOD ACTION: This test estimates the number of s mutans in mixed paraffin –stimulated saliva when cultured in Mutans salivarius bacitracin (MSB) agar. This was developed for use in large number of school children. EQUIPMENTS: paraffin wax ,sterile tongue blades ,disposable contact Petri-dish containing MSB agar incubator.
Procedure: The subjects chew a piece of paraffin wax for one minute to displace plaque microorganisms , thereby increasing the proportions of plaque microorganisms in saliva. Sterile tongue blades are then rotated in the patients mouth 10 minutes so that both the sides are thoroughly inculcated by the oral flora. It is then pressed onto MSB agar in a disposable contact petri-dish . Incubation is done at 37degree Celsius . For field studies ,the plates can be plastic bags containing expired air, which are then sealed and incubated at 37 degree Celsius. Counts of more than 100 colony forming units by this method are proportional to greater than 10 colony forming units of S. Mutans per ml of saliva by conventional methods.
Advantages: This is a simplified and practical method for field studies. Avoids the necessity of collecting saliva. It requires no transport media /dilution steps.
FOSDISK CALCIUM DISSOLUTION TEST
PRINCIPLE: This test measures the milligrams of powdered enamel dissolved in 4 hours by acid formed when the patient’s saliva is mixed with glucose and powdered enamel.
PROCEDURE: Saliva is stimulated by having the patient chew gum paraffin . 25 ml of this saliva is collected and part of it is analyzed for calcium content. oThe remaining saliva is placed in an 8-inch sterile test tube with about 0.1gm of powdered human enamel .
The test 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 equipments. The test is expensive and requires trained personnel.
This test was developed by Rosenberg et al 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, the aerobic dehydrogenase transfers electrons or proton to oxygen . • Once oxygen gets utilized by the aerobic organisms, ethylene blue acts as an electron acceptor and gets reduced to leucomeyhylene blue. This reflects the metabolic activity of the aerobic organisms.
Equipments: Sterile beakers, sterilized milk, screw cap test tubes ,0.1% aqueous solution of methylene blue ,10ml disposable syringes ,pipette, mirror, stopwatch and test tube stand.
PROCEDURE: • Mouth is rinsed vigorously with 10ml of sterile milk for 30 seconds and the expectorate is collected. • 3ml of this is transferred to the screw cap tube with the help of a disposable syringe. • To this ,0.12ml of 0.1# methylene blue is added, thoroughly mixed and placed on a stand in a well illuminated area. 36. o The tubes are observed every 10 minutes for any color change at the bottom using a mirror. o The time taken for the initiation of color change within 6mm ring is recorded. o The higher the infection ,lesser was the time taken for the change in color of the expectorate reflecting higher oral microbial levels.
Advantage: Less time consuming. Economic. Non-toxic vehicle. Can be easily learnt by auxiliary personnel.
DISADVANTAGES: Lack of specificity.