Because the negative control hybridizations with probe NonEUB388 and the subsequent measurements in flow cytometer did not
show any fluorescent cells, the absence of cross hybridization effects for UASS samples find more is indicated (Figure 5C). The low hybridization rates observed for bacteria in UASS samples and C. thermocellum could be caused by a lower metabolic activity of parts of these cells. Microorganisms in the environment often do not grow at their optimal rate and could show different metabolically stages: active, inactive, starved, and dormant. Generally, microbial cells with metabolic activity have a sufficient number of 16S rRNA molecules which were usually used as targets for fluorescently labeled FISH probes. In consequence, a sufficient number of 16S rRNA molecules is required for strong fluorescence signals in flow cytometry or fluorescence BIBW2992 datasheet microscopy, respectively [7, 8, 37]. Determination of the microbial metabolic state Because of the low hybridization rate partially observed for some samples (Figure 5), the metabolic cell activity was determined by examination of dehydrogenase activity visualized by 5-Cyano-2,3-ditolyl tetrazolium chloride (CTC) reduction in microbial
cells. CTC is reduced to CTC formazan by electron transfer through respiratory activity and accumulates Thymidine kinase as red fluorescent crystals inside the cell [38–40]. This enables the detection of active cells by flow cytometry as well as by fluorescence microscopy. Therefore, a regular sampling within 24 h from the UASS biogas reactor as well
as growth series of E. coli and C. thermocellum were performed. At anaerobic conditions an abiotical reduction of CTC is possible . Hence, inactivated samples from the UASS reactor as well as E. coli and C. thermocellum cultures were used as negative controls to exclude possible false positive fluorescence signals. No fluorescence signals could be detected from any inactivated samples after CTC incubation indicating that no abiotical reduction of CTC occurred at the apparent experimental conditions (data not shown). The evaluation of UASS samples after CTC incubation was difficult. Because it could not be ruled out that the CTC formazan crystals will be washed out of the cells during purification procedure as described above, we decided to pass on the sample pretreatment. Hence, measurement by flow cytometry could not be conducted and cell counts in UASS samples were estimated by microscopic field analysis. Because of background fluorescence of unpurified UASS samples a reliable quantification of total cell count as well as of CTC-formazan positive cells was not possible. In general, the activity of cells in UASS reactor samples was low according to CTC-formazan selleck screening library staining.