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

Abstract

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.

Biotimer assay: A reliable and rapid method for the evaluation of central venous catheter microbial colonization

“…Previously, BTA was proven reliable to count microbial biofilm adherent to abiotic surfaces (Pantanella et al, 2008;De Giusti et al, 2011;Berlutti et al, 2014). Of note, BTA is performed without sample manipulation thus overcoming the bias related to CVC intra-luminal colonization and to vortex or sonication procedures (Freitas et al, 2014;Pantanella et al, 2013;Srivastava and Bhargava, 2016). In particular, vortex procedures do not guarantee the detachment of all microbes as confirmed by the data reported here (Fig.…”

“…Unfortunately, no one of these methods is fully reliable. Indeed, the rollplate technique does not determine the intra-luminal colonization of CVCs (Dobbins et al, 2003;Mermel et al, 2009;Guembe et al, 2016;Gominet et al, 2017) and the vortex or sonication methods do not ensure the detachment of all microbes (Pantanella et al, 2013;Freitas et al, 2014). It is evident that a rapid and reliable microbiological method for the early determination of CVC colonization is extremely important.…”

Evaluation of Antibacterial Effect of Diode Laser 980 Nm, Endovac and Passive Ultrasonic Irrigation During Final Irrigation

“…The count of bacteria in biofilm in the current study was achieved by CFU protocol after detaching them from the surface on which they adhere by vortexing or sonication. However, these procedures often do not obtain homogeneous cell suspensions, thus introducing a relevant bias in the results (57) . In the present study, microorganisms were collected with sterile paper points from the root canal system.…”

Assessing and Reducing Sources of Gene Expression Variability in Staphylococcus Epidermidis Biofilms

“…In brief, biofilm formation capability was determined by A 640 as described elsewhere (16). The presence of the genes of interest was determined by PCR using DreamTaq PCR Master Mix (Thermo Scientific, Waltham, MA).…”