Biofilms were collected from the maxillary dentures of each participant at five time points, i.e., before using the solutions (baseline) and after using them. Before using each solution, the internal surfaces were stained by a disclosing solution (1% neutral red), cleaned by researchers with a specific brush (Bitufo®, Itupeva, SP, Brazil) and neutral liquid soap (Pleasant, Perol Commercial and Industrial Ltda., Ribeirão Preto, SP, Brazil), and then returned to the patients in the same clinical initial condition. Biofilms were collected in an aseptic zone by placing each complete maxillary denture in a sterile Petri dish. Dentures were rinsed with 10 mL saline solution, and their internal surfaces were brushed (Tek, Johnson & Johnson Brazil’s Industry and Commerce Healthcare Products Ltda., São José dos Campos, SP, Brazil) for 2 min. The biofilm suspension obtained was vortexed for 2 min and diluted in decimal series (100 - 10-3). Then, 50 µL aliquots from the decimal dilutions were cultured in Petri dishes containing Mitis salivarius agar base (more bacitracin solution and 20% sucrose), MacConkey agar, and CHROMagar® Candida for detecting S. mutans, gram-negative microorganisms, and Candida spp., respectively. Culture media were incubated at 37°C according to each respective condition: candle jar for 48 - 72 h (mutans group), or aerobiosis for 48 h (gram-negative and Candida spp).
After incubation, the number of colonies for each dilution was counted. Based on the dilution that provided 1 - 300 colonies, colony forming units (CFUs) were determined using the formula CFU/mL=number of colonies x 10n/q, where “n” is the absolute value of the dilution (0, 1, 2, or 3) and “q” is the quantity of plated suspension (0.05 mL).
For Candida spp., the number of colonies was counted and the species were identified based on the chromogenic properties of the medium. Each identified species was confirmed using yeasts kit (Candifast®, ELITech Microbio, Signes, France); in addition, the resistance to antifungal agents, such as amphotericin B, nystatin, flucytosine, econazole, ketoconazole, miconazole, and fluconazole, was evaluated.
In order to conceal the ones involved, the solutions were dispensed in identical dark flasks and delivered without specific identification, and in the quantity to be used for a period of seven days. Each cleanser solution was used by the participants in a random sequence. Researcher P1, who was not involved in the other operational phases of the study, used a computer program to obtain a list of random numbers corresponding to the possible sequences of the treatment. Researcher P2 received these numbers and distributed the solutions to the participants. Researcher P3 implemented hygiene instructions and collected the prostheses. Researcher P4 collected the biofilms while researcher P5 washed the prostheses to ensure the complete elimination of the biofilms. Researcher P2 obtained variable information and provided it to researcher P1, who performed statistical analyses. Thus, during the study, all researchers, as well as the participants, were blinded to the applied solutions.
After incubation, the number of colonies for each dilution was counted. Based on the dilution that provided 1 - 300 colonies, colony forming units (CFUs) were determined using the formula CFU/mL=number of colonies x 10n/q, where “n” is the absolute value of the dilution (0, 1, 2, or 3) and “q” is the quantity of plated suspension (0.05 mL).
For Candida spp., the number of colonies was counted and the species were identified based on the chromogenic properties of the medium. Each identified species was confirmed using yeasts kit (Candifast®, ELITech Microbio, Signes, France); in addition, the resistance to antifungal agents, such as amphotericin B, nystatin, flucytosine, econazole, ketoconazole, miconazole, and fluconazole, was evaluated.
In order to conceal the ones involved, the solutions were dispensed in identical dark flasks and delivered without specific identification, and in the quantity to be used for a period of seven days. Each cleanser solution was used by the participants in a random sequence. Researcher P1, who was not involved in the other operational phases of the study, used a computer program to obtain a list of random numbers corresponding to the possible sequences of the treatment. Researcher P2 received these numbers and distributed the solutions to the participants. Researcher P3 implemented hygiene instructions and collected the prostheses. Researcher P4 collected the biofilms while researcher P5 washed the prostheses to ensure the complete elimination of the biofilms. Researcher P2 obtained variable information and provided it to researcher P1, who performed statistical analyses. Thus, during the study, all researchers, as well as the participants, were blinded to the applied solutions.
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