Development of flow cytometry technique for detection of thinning of peptidoglycan layer as a result of solvent production by Clostridium pasteurianum

Clostridium pasteurianum forms acetic and butyric acids in an initial growth phase, which is a typical feature of clostridial acetone-butanol fermentation where an initial accumulation of acids is followed by production of solvents 1-butanol, acetone and ethanol. The initiation of the solvent production coupled with endospore formation leads to decrease of cell-wall thickness; thinner cell wall is more resistant against solvents and dyes. These changes can be observed by the method based on adaptation of Gram staining. The cell wall of G + bacteria allows the entry of hexidium iodide and rhodamine 123, whereas the outer membrane of G − bacteria does not allow the uptake and therefore G + bacteria are stained with higher fluorescence intensity than G − bacteria. The ratio of fluorescence intensity (FI) to forward scatter (FSC) was determined to correspond to G + bacteria when clostridia were producing less solvents. The significant drop of the ratio FI to FSC to the level corresponding to G − bacteria is detected after initiation of solvent production.