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.
Linhová
M
M
Department of Fermentation Chemistry and Bioengineering, Institute of Chemical Technology Prague, 166 28, Prague, Czechia. Michaela.Linhova@vscht.cz
Patáková
P
P
Lipovský
J
J
Fribert
P
P
Paulová
L
L
Rychtera
M
M
Melzoch
K
K
D020001:1-Butanol / Q000378:metabolism*; D000096:Acetone / Q000378:metabolism*; D003013:Clostridium / Q000378:metabolism*; D000431:Ethanol / Q000378:metabolism*; D005434:Flow Cytometry / Q000379:methods*; D005840:Gentian Violet; D010457:Peptidoglycan / Q000032:analysis*; D010619:Phenazines; D013194:Staining and Labeling / Q000379:methods
C035863:Gram's stain; D010457:Peptidoglycan; D010619:Phenazines; D000096:Acetone; D000431:Ethanol; D020001:1-Butanol; D005840:Gentian Violet