and Methanosarcina spp. [22, 23]. This hampers any cell counting attempt by microscopy as well as flow cytometry. In addition, some of these cell associations can reach a thickness that inhibits the penetration of FISH probes into deeper layers of cell clusters. In consequence, only the surface Selleckchem ON-01910 cells are hybridized with FISH probes and are detectable by Flow-FISH. Hence, samples from this environment have to be pretreated to purify and to isolate all microbial cells of the whole biogas reactor biocenosis. Despite the number of different selleck inhibitor pretreatment approaches developed for a variety of samples of different environmental origins [24–28],

up to now no procedures are published for the purification of samples from biogas reactors leading to preparations suited for the measurement of the microbial community by Flow-FISH. To overcome these technical limitations, the aim of this study was to establish a high-throughput technique for the

detection and the quantification of process relevant, active microorganisms in anaerobic digestion using the process liquor of an upflow anaerobic solid-state (UASS) biogas reactor as test material [29]. Therefore, a purification technique was primarily optimized to fulfill the following requirements: (1) detachment of cells from organic and inorganic particles, (2) disbandment of cell aggregates, (3) no or low cell loss, and (4) a rapid implementation. Furthermore, a modified Flow-FISH

protocol based on different already published see more protocols [12, 20, 30] was developed and tested regarding following influencing parameters: (1) type of fixative used for cell fixation directly after sampling, (2) possible cell losses by centrifugation during FISH procedure, and (3) cell activity. Results and discussion Optimization of the purification technique The application of flow cytometry for the analysis of the microbial community in biogas reactors requires previous sample purification due to its high content of organic and inorganic particles and the presence of huge cell aggregates and biofilms. The capillary within the flow cytometer could clog due to such large particles. Moreover, the microbes bound in aggregates and biofilms are hardly detectable and countable with the Flow-FISH. In this study, six purification procedures with in total 29 modifications (-)-p-Bromotetramisole Oxalate were tested (Table 1). These six purification strategies are based on the use of a detergent to dissolve cell aggregates and to detach cells from different surfaces in soils [24–26, 28] or turbid seawater [27]. A current method to increase the effect of detergent is the ultrasonic treatment [31] and homogenization of the sample with a dispersion unit [26]. The concentration of the used detergent and the settings of ultrasound and homogenization should be adjusted because these treatments can also destroy the cell wall of microbes.