5 h after eating, drinking, or tooth cleaning. Saliva samples were collected in sterile 50 mL polypropylene tubes, chilled in an ice bath or frozen at −20 °C. After 500 mL saliva had been collected, it was pooled and centrifuged (30 min, 4 °C, 27,000 × g); the supernatant was pasteurized (60 °C, 30 min) and re-centrifuged in sterile tubes. The resulting supernatant was stored into sterile 50 mL polypropylene tubes at −80 °C. The efficiency of the process was assessed Selleck NLG919 by plating processed saliva samples onto BHI agar; after 72 h at 37 °C no CFUs were observed on incubated

plates. Streptococcus mutans biofilms were grown on 96-wells microtiter plates through a methodology developed by Stepanovicet al. 33 and Islam et al. 34 with some modifications. In a first moment, 100 μL of processed saliva plus 100 μL of carbonate buffer pH 9.3 were added to each well and incubated at 4 °C for 2 h. After this period the wells were washed tree times with saline phosphate buffer pH 7.6. In sequence,

100 μL of sterile BHI were distributed in a 96-wells polypropylene tissue culture plates HA-1077 in vitro (Orange Scientific®, Braine-l’Alleud, Belgium) (with flat-bottom) followed by placement of 100 μL of DC in concentrations that were prepared using a procedure similar to the one used in the antimicrobial activity tests (MIC) with same initial bacterial cells concentration. All the plates were incubated at 37 °C, CO2 10%, during 24 h for biofilm development. After biofilm growth in the presence or absence of CD concentrations, the content of each well was removed and the biofilms were washed twice RG7420 in vitro with 200 μL of sterilized water, to remove cells weakly adhered. The attached biofilm mass was quantified using crystal violet staining.35 Briefly, the plates containing

the biofilms were left to air dry for 30 min, and 200 μL of a solution sodium acetate/formalin 2% were distributed in each well, in order to fix the adhered cells, and left for 15 min. After this time, the solution sodium acetate/formalin 2% was removed and 200 μL of crystal violet 1% (Gram colour-staining set for microscopy – Merck©) were added to each well for 5 min. Following the staining step, the washing procedure, with sterile water, was repeated and the plates were left at room temperature for 1 h. To re-solubilize the dye bounded to biofilms, 200 μL of 95% ethylic alcohol (Merck©) were added to each well and submitted to agitation for 15 min. The crystal violet (CV) solutions obtained were transferred to a new sterile flat bottom 96-wells plate and the optical density of the content was measured using a microtiter plate spectrophotometer (Biotrak II Plate Reader – Amersham Biosciences©) at 570 nm. The biofilms were generated as described above and after 24 h of incubation at 37 °C, CO2 10%, the plates were washed twice using sterile distilled water to remove cells weakly adhered.