The detailed description of the method development, particular application conditions and its use were published by Linhova et al., (2010a). The main idea of the staining is based on fact that clostridia are usually stained according to Gram as G+ after germination from spores (motile, juvenile cells) and as G — when the cells started to sporulate. The change in Gram staining response corresponds to metabolic switch from acids to solvents formation and also with an alteration in a cell membrane composition i. e. thinning of peptidoglycan layer (Beveridge, 1990). Therefore the cells of C. pasteurianum were labelled with a combination of fluorescent probes, hexidium iodide (HI) and SYTO 13 that can be considered a fluorescent alternative of Gram staining. Cells of C. pasteurianum forming mainly acids fluoresced bright orange-red as G+ bacteria and the solvent producing, sporulating cells exhibited green-yellow fluorescence as G- bacteria (see Fig. 2). The red colour of labelled young cells was a result of a fact that green fluorescence of SYTO13 was quenched by that of HI while bright green-yellow colour of sporulating and/or old cells was caused by staining only by SYTO13 when HI did not permeate across the cell wall. Jones et al., (2008) used different combination of dyes (propidium iodide and SYTO 9) for labelling C. acetobutylicum ATCC 824 during time course of batch cultivation but attained the same conclusion.

Then, flow cytometry enabling quantification of fluorescent intensities of labelled clostridial populations was used for monitoring of physiological changes during fed-batch cultivation (Linhova et al., 2010a). For flow cytometry measurement, the cells were stained only by HI and the signal of fluorescent intensity acquired in a channel FL3 (red colour) was related to forward scatter signal (FSC) which corresponded to cell size in order to gain data independent on cell size. The data measured for C. pasteurianum were compared with those for typical G+ and G — bacteria i. e. for Bacillus megatherium and Escherichia coli and there was a striking difference between the values of FL3/FSC for C. pasteurianum on one hand and those for B. megatherium and E. coli on the other hand. While the values for B. megatherium (G+) and

E. coli (G-) oscillated ±0.1 and ±0.2, respectively, in time course of 32 h in which they were sampled, the values for C. pasteurianum dropped from 3.1 to 0.8 during the cultivation. It was

also evident that acidogenic phase had a very short duration and both metabolic phases overlapped. Further experiments are necessary to assess unambiguously the acquired data, however it is tempting to hypothesize that C. pasteurianum NRRL B-598 has a different pattern of acids and solvents formation when solvents production is connected rather with exponential growth phase than the well-known solventogenic strain C. acetobutylicum ATCC 824 in which solvents production is generally assembled with stationary growth phase.