Optimization of an automatic counting system for the quantification of Staphylococcus epidermidis cells in biofilms

Biofilm formation is recognized as the main virulence factor in a variety of chronic infections. In vitro evaluation of biofilm formation is often achieved by quantification of viable or total cells. However, these methods depend on biofilm disruption, which is often achieved by vortexing or sonication. In this study, we investigated the effects of sonication on the elimination of Staphylococcus epidermidis cell clusters from biofilms grown over time, and quantification was performed by three distinct analytical techniques. Even when a higher number of sonication cycles was used, some stable cell clusters remained in the samples obtained from 48- and 72-h-old biofilms, interfering with the quantification of sessile bacteria by plate counting. On the other hand, the fluorescence microscopy automatic counting system allowed proper quantification of biofilm samples that had undergone any of the described sonication cycles, suggesting that this is a more accurate method for assessing the cell concentration in S. epidermidis biofilms, especially in mature biofilms.This work was funded by the Fundacao para a Ciencia e a Tecnologia (FCT) and the COMPETE grants PTDC/BIA-MIC/113450/2009 and FCOMP-01-0124-FEDER-014309