In situ quantification of biocide efficacy using GFP transformed Aureobasidium pullulans.

To develop a real-time in situ method to quantify loss of viability of Aureobasidium pullulans PRAFS8 cells attached to plasticized polyvinyl chloride (pPVC) with incorporated biocides, and to use the method to compare biocide efficacy in situ. A. pullulans PRAFS8, transformed with green fluorescent protein (GFP), was used to quantify the efficacy of a range of biocides incorporated into pPVC. Experimentally, it was found that a density of 1.53 x 10(6) yeast cells per cm(2) of pPVC was optimal as increasing the density of the yeast cells to 6.12 x 10(6) cm(-2) attached to pPVC containing the biocide 2-n-octyl-4-isothiazolin-3-one (OIT) decreased the rate of fluorescence loss. A strong positive correlation between fluorescence and viable yeast cell number was observed and fluorescence was used as a direct indicator of cell viability. The effectiveness of five commercial biocides, commonly incorporated into pPVC at their in-use concentrations, was tested against yeast cells attached to the pPVC surface. The loss of fluorescence and hence viability in situ was quantified using image analysis. The biocides N-(trichloromethylthio) phthalimide (NCMP), 10,10'-oxybisphenoxarsine (OBPA), OIT and 2,3,5,6-tetrachloro-4-(methylsulphonyl) pyridine (TCMP) caused complete loss of fluorescence within 30-50 h. In contrast the biocide dichloro-octyl-isothiazoline caused only 55 +/- 15% fluorescence loss after 50 h. Starvation of the yeast cells in suspension for 24 h prior to attachment reduced their initial sensitivity to OBPA, NCMP, OIT and TCMP by 15-20%, but eventually the fluorescence was also completely lost. The use of A. pullulans expressing cytosolic GFP enables the in situ quantification of loss of viability when cells are attached to pPVC with incorporated biocides. GFP fluorescence was used as a real-time indicator of cell viability and thus can be applied for direct quantification of the effectiveness of a broad range of biocides, incorporated into the polymer mass and used to protect a variety of plastics or other materials from microbial growth.