Enzymatic breakdown of biofilm matrix to allow flow cytometry viability analysis of Clostridium beijerinckii cells.

Abstract

Flow cytometry (FC) is a good way to enumerate the number of viable cells in suspension but is not adapted to mature biofilm analysis. The aim of this study is to investigate the effect of mechanical treatment coupled with enzymatic hydrolysis of biofilm matrix on FC viability analysis of biofilm cells. Biofilm was grown for 300h of continuous fermentation on polyurethane foams. Fermentation was stopped, and the biofilm was detached by agitating the foams in PBS buffer with vortex agitation for 2 min. The best enzymatic hydrolysis consisted of sequential use of DNase I and proteinase K incubated for 1h at 34°C. Biofilm cells detached from polyurethane foams were stained with both propidium iodide (PI) and carboxyfluoresceine diacetate and analyzed by FC. FC analysis performed after vortex agitation revealed the presence of high non-fluorescent events (78.9%±3.3%). After enzymatic treatment, a cell population was extracted from background noise and could be observed on FSC-SSC profile. The non-fluorescent events of this cell population decreased drastically to 41.9%±6.6%, and the percentage of viable cells was enhanced from 2.6%±0.9% to 38.2%±4.0% compared to analysis performed after mechanical treatment alone. Consequently, protease and nuclease activity are essential to hydrolyze extra polymeric substances prior to FC viability analysis in mature biofilm formed by Clostridium beijerinckii.