Evaluation in vitro the influence of hygiene solutions on cell viability of microorganisms composing the biofilm overdentures

• Grant number: 16/16934-0
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: September 01, 2016
• End date: August 31, 2017
• Field of knowledge: Health Sciences - Dentistry
• Principal Investigator: Cláudia Helena Lovato da Silva
• Grantee: Camila Borba de Araujo
• Host Institution: Faculdade de Odontologia de Ribeirão Preto (FORP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil

Abstract

The aim of the study is to evaluate (in vitro) the effect of sanitizer solutions (Triclosan, Chloramine T, R. communis 10% and 2% and sodium hypochlorite at 0.25%) on the cell viability using epifluorescence microscopy> Denture biofilme will be evaluated. Initially, the minimum inhibitory concentration (MIC) of Triclosan and Chloramine-T solutions will be determine by the method of micro dilutions in cell culture plates with 96 wells, in duplicate. The variable will be tested for eight microorganisms (C. albicans, C. glabrata, C. tropicalis, S. mutans, S. aureus, E. coli, P. aeruginosa and E. faecalis). The immersion medium (solution) will be considered in 5 levels: GHS = sodium hypochlorite solution 0.25%; GT = Triclosan solution; GCT = Chloramine T solution; GRc10: R. communis 10% solution; GRc2: R. communis 2% solution. Control groups (n = 6) will be formed to verify the formation of biofilms and to prove sterilization specimen (n = 6). Biofilms will be formed on circular specimens (13mm diameter x 4mm thick) of thermally activated acrylic resin and they will be immersed in the solutions for 20 minutes, washed with PBS and immersed in liquid culture medium (Letheen) for processing. The cell viability of biofilms after exposure to solutions will be evaluated by the epifluorescence microscope. The experimental procedure will be summarized on the dilution of solutions of the Live/Dead® BacLight " it to 0.3%, according to the manufacturer's instructions. Then the specimen (n = 3) exposed to the hygiene protocols will be transferred to a new 24 well plate containing 4ml of PBS, pH 7, in each well. 300 uL of the solution of A component of the kit (0.3%) + 300 uL of solution of component B (0.3%) will be applied to the surface of the specimen and the plates will be incubated, protected from light and kept at room temperature for 15 minutes. The dye will be drained and the specimen will be observed in epifluorescence microscope with appropriate filters. The reading is based on determining the viability of the microorganisms from the difference in membrane integrity of the incorporated cells, where living microorganisms with intact membranes and dead organisms with damaged membranes will have a difference in fluorescence. The analysis will be performed in triplicate. The data will be submitted to the normal analysis (Shapiro-Wilks test) and homoscedasticity (Levene test) for the definition of the relevant statistical test. The analyzes will be conducted at a 5% significance level. (AU)