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9% NaCl and individually transferred to microcentrifuge tubes containing see more 1?mL of 0.9% NaCl. The tubes were sonicated at 7?W for 30?s (Branson, Sonifier 50, Danbury, CT, USA) to detach the biofilms formed on the microscope glass, according to Aires et al. [24]. The glass slides were carefully removed from the suspension and discarded. Aliquots of the suspension were used to determine biofilm bacterial viability and biomass (dry weight). 2.6. Biomass Determination Biofilm dry weight was determined according to Koo et al. [22] from 200?��L of the suspension. For the dry weight determination, three volumes of cold ethanol (?20��C) were added to cell suspension, and the resulting precipitate was collected (10?000?g for 10?min, 4��C). The supernatant was discarded, and the cell pellet was washed twice with cold ethanol, dried, and had the weight checked. 2.7. Bacterial Viability An aliquot of 100?��L of the suspension formed after sonication was diluted in 0.9% NaCl in series up to 10?7 and 2 drops of 20?��L of each dilution were inoculated on BHI agar (BD, Sparks, USA) SERCA to determine the number of viable microorganisms and assess the successfulness of the sonication procedure [25]. The plates were incubated for 24?h at 37��C and 10% CO2 (IG 150, Jouan incubator). CFU were counted and the results were expressed as CFU/mg of biofilm dry weight [24]. 2.8. Statistical Analysis The descriptive analysis, in statistical analysis, was carried out presenting measures of position and dispersion for continuous variable. The level of significance adopted for the statistical tests was 5%. ANOVA's test was used to compare the parameters, considering the factors used. The variables were transformed into ranks due to the sample size. Multiple comparisons had been carried through by the test of Tukey and the test of profile for contrasts. The Jonckheere-Terpstra http://www.selleckchem.com/products/NVP-AUY922.html test was used for comparing numerical measures among groups with ordinance and magnitude (tendency). Capital letters indicate statistical differences between culture media (UTYEB, MH, and MHS), and lowercase letters indicate statistical differences according to the treatments using each culture medium (UTYEB, MH, or MHS), by ANOVA and Tukey tests (p