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OBJECTIVES The objective of this study was to evaluate the efficiency of SB-20 M culture medium to perform differential morphological identification of S. mutans and S. sobrinus compared to biochemical identification and to proteomic identification by the MALDI-TOF mass spectrometry technique. MATERIAL AND METHODS Unstimulated saliva samples from 266 dental students were seeded on SB-20 M culture medium by the wooden spatula technique. After incubation, S. mutans and S. sobrinus colonies were identified by stereomicroscopy based on their differential morphological characteristics. Following these procedures, 135 colonies with characteristic morphology of S. mutans (89 colonies) and S. sobrinus (46 colonies) were randomly selected, submitted to biochemical identification (biotyping) and proteomic identification by the MALDI-TOF mass spectrometry technique. The results were compared using the Kappa test, with a 5% significance level. RESULTS All (100%) S. mutans colonies were correctly identified after culture in SB-20 M medium compared to biotyping and proteomic identification. For S. sobrinus, morphological identification in SB-20 M medium was correct for 43 colonies (93.5%) compared to biotyping and proteomic identification. However, there was no statistically significant difference when comparing the capacity to identify S. mutans and S. sobrinus of the three techniques (p < 0.001; K = 0.951). CONCLUSIONS It was concluded that the SB-20 M culture medium for morphological identification of S. mutans and S. sobrinus was highly reliable, being comparable to the MALDI-TOF mass spectrometry technique. CLINICAL RELEVANCE The efficiency evaluation of identification methods of S. mutans and S. sobrinus is clinically relevant in order to determine caries risk and activity of patients.