Funding:

Project management / project work:

Prof. Andreas Tiehm (TZW), Dr. Bernd Bendinger / Anne-Madeleine Trimbach, Dorota Bruniecka-Sulewski

Situation:

Flow cytometry (FCM) is a powerful method for the quantification of all bacteria in a water sample (total cell count, TCC). After staining with a DNA-specific fluorescent dye every single cell is excited by a laser beam and its light scattering and fluorescence intensity is detected by optical sensors. The combination of different signals gives a specific fingerprint of a water sample showing e.g. the proportion of small cells with a low DNA content (LNA) to big cells with a high DNA (HNA) content.

The measurement of a TCC takes only a few minutes. In contrast culture methods detect only 0.01 to 0.1 % of the TCC and the determination of colony counts according to the german drinking water ordinance require at least 48 hours. The time saving and the availability of mobile and recently also of online-instruments are important arguments for a promising application of FCM in water analysis.

Additionally, FCM can differentiate between live (membrane intact) and dead (membrane damaged) cells which allows the detailed investigation of the efficacy of disinfection methods. Several publications propose the general application of FCM for the surveillance of disinfection methods. However, this has to be regarded critically due to different mechanisms of action of disinfectants - especially with regard to UV-disinfection.

The objective of the project is to determine the potential and the frontiers of this forward-looking method. The FCM should be further developed and validated for different fields of application:

• Fast measurement of total cell counts (TCC) in different matrices
• Fast characterization of raw water and drinking water
• Specific detection of fecal indicators
• Surveillance of the efficacy of disinfection

The comparison of the results from FCM with classical culture methods should reveal the relevance of FCM data for the assessment of the hygienic quality of the water. For an as far as possible close-to-practice development of flow cytometry real water samples of preferably different compositions from water utilities will be analyzed.

The DVGW-Forschungsstelle TUHH performs the following work program in the overall project:

• Participation in interlaboratory tests for the determination of TCC, LNA/HNA-ratio and proportion of live (membrane intact) cells
• Analysis of the microbial dynamics in treatment processes of different raw waters and during distribution (long-distance water main)   
• Surveillance of disinfection: investigation of the effect of chlorine dioxide on the live/dead differentiation of cells in laboratory experiments with different drinking water qualities
• Measurement of the effect of different water matrices (e.g. particles, dissolved compounds) on bacterial FCM-signals
• Hygienic relevance of the data: interpretation of FCM-data and evaluation of their hygienic relevance by comparison with the results from routine culture techniques
• Adaptation of standard operation procedures (SOPs) for sampling, sample fixation, instrument settings and quality control