Live / Dead discrimination of biofilm bacteria from a drinking water pilot dsitribution system
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chair:
Varela Villarreal, J. / Jungfer, C. / Obst, U. / Schwartz, T. (2011)
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place:
VAAM 2011, Karlsruhe, Deutschland, 3.-6. April, 2011
- Date: 2011
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Varela Villarreal, J. / Jungfer, C. / Obst, U. / Schwartz, T. (2011): „Live / Dead discrimination of biofilm bacteria from a drinking water pilot dsitribution system “. In: VAAM 2011, Karlsruhe, Deutschland, 3.-6. April, 2011
Abstract
Formation of biofilms in drinking water distribution networks, including pipelines of households and food industries, are of great concern. Biofilms are potential habitats for all kinds of bacteria, including pathogens, and may be responsible for contaminations of bulk water systems.
Nowadays, DNA-based methods are used for the detection and characterization of bacteria. One of the major disadvantages of these techniques is that they can not distinguish between DNA from live and dead cells. A battery of methods to face this problematic is presented in this work.
Conditioned surface water disinfected with ozone/ClO2 flowed through a pilot scale built up with different pipe materials for biofilm formation. Bacterial population analysis was done by PCR-DGGE, comparing direct samples (total DNA) and samples pre-treated with Propidium monoazide or DNase I (DNA from live cells). Shifts in the DNA patterns observed after DGGE analysis, demonstrated: (i) the applicability of PMA and DNase I treatment in natural biofilm investigation; (ii) detection of DNA from dead bacteria and eDNA was blocked by pretreatment with PMA or DNase I; and (iii) DNase I treatment demonstrated a clearer effect on live/dead differentiation.
Traditional cultivation methods and qPCR completed the biofilm analysis. The results of the bacterial population analysis and the results of the quantification methods that provide an overview of the different physiological states of bacteria: live cells, total amount of cells, and cultivable cells, are presented here.
Nowadays, DNA-based methods are used for the detection and characterization of bacteria. One of the major disadvantages of these techniques is that they can not distinguish between DNA from live and dead cells. A battery of methods to face this problematic is presented in this work.
Conditioned surface water disinfected with ozone/ClO2 flowed through a pilot scale built up with different pipe materials for biofilm formation. Bacterial population analysis was done by PCR-DGGE, comparing direct samples (total DNA) and samples pre-treated with Propidium monoazide or DNase I (DNA from live cells). Shifts in the DNA patterns observed after DGGE analysis, demonstrated: (i) the applicability of PMA and DNase I treatment in natural biofilm investigation; (ii) detection of DNA from dead bacteria and eDNA was blocked by pretreatment with PMA or DNase I; and (iii) DNase I treatment demonstrated a clearer effect on live/dead differentiation.
Traditional cultivation methods and qPCR completed the biofilm analysis. The results of the bacterial population analysis and the results of the quantification methods that provide an overview of the different physiological states of bacteria: live cells, total amount of cells, and cultivable cells, are presented here.