Methods of sampling
As mentioned earlier, the correlation between salivary MS counts and the number of MS-colonized tooth surfaces is relatively good (Lindquist et al, 1989), and simple salivary sampling methods are a more convenient and realistic means of assessing the severity of MS infection than sampling from individual tooth surfaces.
Laboratory methods. Saliva is collected, mixed with a proper transport medium, and forwarded to a microbiologic laboratory. After incubation using a selective medium, mutans colonies are counted and the results are expressed as the number of colonyforming units per milliliter of saliva.
Several selective media are available, and their properties are not identical: This fact must be taken into consideration in assessing the results. A common type of selective medium for plating mutans streptococci is the mitis-salivarius-bacitracin agar (Gold et al, 1973). With the exception of the rare serotype a, all types of mutans streptococci grow on this medium. Bacitracin is the main selective ingredient. Because the plates have a shelf life of only about 1 week, they are not convenient for chairside tests.
For screening surveys using agar plates, the following simplified method has been described, eliminating the need for transportation, dilution, and plating of saliva (Kohler and Bratthall, 1979; Newbrun et al, 1984): Wooden spatulas are contaminated with saliva and immediately pressed against selective agar plates. After incubation, the number of colonies on a predetermined area of the plate is calculated.
Chairside method. The so-called Strip Mutans test, described by Jensen and Bratthall (1989), is based on the ability of mutans streptococci to grow on hard surfaces and the use of a selective broth (high sucrose concentration in combination with bacitracin). Because the bacitracin can be added to the broth just before use, the shelf life of the test can be prolonged considerably compared to that of agar plates.
The test set is used as follows: A bacitracin disc is taken from the vial with forceps or a needle. The cap is reclosed tightly. The bacitracin disc is put in the culture broth vial and allowed to stand for at least 15 minutes. The vial is shaken gently after 15 minutes. When more than one Strip Mutans test is to be run, the bacitracin discs can be added to the vials beforehand. However, only one Strip Mutans test can be performed in each vial, and vials to which bacitracin has been added must be used on the same day.
The patient is given a Dentocult paraffin pellet and instructed to chew it for 1 minute.
The stimulated saliva should be swallowed or spat out.
A test strip is removed from the Strip Mutans container, so that only the square end is touched. About two-thirds of the strip is placed in the patient’s mouth and rotated on the surface of the tongue about 10 times. The strip is removed from the mouth, pulled between the patient’s closed lips to remove any excess saliva. The strip is placed in the culture vial containing the well-mixed bacitracin-broth solution. The cap is reclosed tightly (Fig 35). The patient data is written on the label, and the label is attached to the vial. The strip is incubated for 2 days at 35°C to 37°C (95°F to 99°F). The strip is removed from the culture vial and allowed to air dry. The treated side, which is marked with a line, can be examined immediately or later (Fig 36). After it is dried, the test strip can be stored for future reference in a plastic bag, plastic wrap,
autoclave plastic, or other similar material.
The bacitracin and the higher sugar content inhibit the growth of practically all microorganisms, other than S mutans, that grow in mitis-salivarius medium. In proportion to their actual amount in saliva, S mutans in the specimen will adhere to the treated side of the strip, and grow as small, dark or light blue colonies, 1 mm in diameter, or considerably less, when growth is very dense. The amount of S mutans per milliliter of saliva is estimated by comparing the colony density on the strip with the standard charts included in the instructions.
If the number of S mutans is very high, the treated side of the test strip will turn blue, and separate colonies will be indistinguishable. A test strip without S mutans may have a blue shade as a result of precipitation of the color indicator present in the culture medium. A magnifying glass or a microscope should be used to verify questionable cases.
The Strip Mutans method should not be used within 12 hours of an antibacterial mouthwash (eg, chlorhexidine) or 2 weeks of a course of antibiotics to avoid falsenegative results.
