In Vitro Biofilm Formation Of Enterococcus Faecalis In Starving Phase And Antibacterial Effect Of Sodium Hypochlorite

Objective:To create different phases biofilm model of Enterococcus faecalis in vitro, and the focus of acomparative study of antimicrobial about different concentrations of sodium hypochlorite to the Enterococcus Faecalis biofilms sensitivity.Method:Experiments Section One: The Enterococcus faecalis (ATCC29212) was prepared in the form of suspensions, and suspensions were induced in freshly made TSB solutions for different incubating time. The MTT method was used to determine the bioactivity of Enterococcus Faecalis of various samples. Experiments Section Two: Enterococcus Faecalis in different growth phases were incubated on slides. Laser scanning confocal microscope combined with fluorescent Staining technology was introduced while keeping the sample wet was assured. Conformations of Enterococcus Faecalis at different phases were carefully observed with CLSM.Experiments'Section Three: Enterococcus Faecalis with different biofilm set-up models of various growth phases were incubated on culture plate. Samples were exposed to various concentrations of NaClO solutions (m/v 1.0%, 2.5%, 5.25%).The contact time was 30s, 5min and 10min. The reduction of viable cells after treatment was estimated with statistical analysis, following serially dilution by 10-times volume. And direct optical observation of Enterococcus Faecalis was also conducted with CLSM.Result:Section one: During the first 8 hours of incubation, the cells were in exponential phase; Between 6 hours and 24 hours, the cells were in the stationary phase; after 48 hours, the cells were in the starvation phase. Enterococcus faecalis can survival in the starvation phase, even though not as active as in the other two phases.Section two: Biofilm models for Enterococcus faecalis of different growth phases have various spatial conformations, which was confirmated by CLSM. And the density of biofilm formed in starving phases was lowest.Section three: Biofilms of starved cells were more resistant to NaClO than the ones formed in exponential phase and stationary phase (p<0.05). With all the sample of different contact time, 5.25% sodium hypochlorite solutions were proved to be the most effective antibacterial reagents. However, complete elimination of living Enterococcus Faecalis could not be accomplished in 10mins.Conclusion:1.Enterococcus Faecalis can survive in the starving environment.2.Biofilm can also be formed in Enterococcus Faecalis cells at starving phases, and the biofilms tend to pose an irregular conformation with typical spatial properties. There are fewer live bacteria on the surface of biofilms.3.Compared with the cells in the exponential phase and stationary phase, the starving ones could form less but more stable biofilms. And it means that starving ones will have stronger susceptibility against antibacterial reagents under uniform situations. Indeed, 5.25% sodium hypochlorite solutions were proved to be the most effective antibacterial reagents in our experiments.