Back ground: Prevention of dental caries requires new researches utilizing experimental set up due to strict ethical issue. Aim: In vitro creation of monospecies (Streptococcus mutans) biofilm induced microscopic human enamel demineralization using a batch culture technique and evaluating it using scanning electron micrograph and Energy Dispersive X-ray analysis. Study design: Human tooth enamel sections of Group 1 (control group n=nine), Group 2 (Study group n= Nine) were incubated in fresh BHI broth with 5 % sucrose for fifteen days. Test tube with Group 2 is only inoculated with streptococcus mutans every day for fifteen days. SEM-EDAX aided structural analysis for surface morphology and elemental analysis for calcium, phosphorus, oxygen and fluorine content of both control and study groups performed before and at the end of fifteen days. Result: A thick, whitish coating of biofilm was visible only on group 2 (Study group), at the end of fifteen days. SEM - EDAX of enamel surface of group 2(Study group) showed loss of superficial structure and decrease in the concentration of calcium, phosphorus ions indicating demineralization unlike group 1 which shows no change in structural and elemental concentration after the incubation period. ANOVA test showed that difference of mean in two groups (1&2) were statistically significant (p<0.0001) in calcium, phosphorus, content.
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