Novel yeast cell dehydrogenase activity assay in situ.
The aim of this research was to develop a suitable method of succinate dehydrogenase activity assay in situ for different industrial yeast strains. For this purpose different compounds: EDTA, Triton X-100, sodium deoxycholate, digitonin, nystatin and beta-mercaptoethanol were used. The permeabilization process was controlled microscopically by primuline staining. Enzyme assay was conducted in whole yeast cells with Na-succinate as substrate, phenazine methosulfate (PMS) as electron carrier and in the presence one of two different tetrazolium salts: tetrazolium blue chloride (BT) or cyanoditolyl tetrazolium chloride (CTC) reduced during the assay. In comparabile studies of yeast vitality the amount of intracellular ATP was determined according to luciferin/luciferase method. During the succinate dehydrogenase assay in intact yeast cells without permeabilization, BT formazans were partially visualized in the cells, but CTC formazans appeared to be totally extracellular or associated with the plasma membrane. Under these conditions there was no linear relationship between formazan color intensity signal and yeast cell density. From all chemical compounds tested, only digitonin was effective in membrane permeabilization without negative influence on cell morphology. Furthermore, with digitonin-treated cells a linear relationship between formazan color intensity signal and yeast cell number was noticed. Significant decreasing of succinate dehydrogenase activity and ATP content were observed during aging of the tested yeast strains.
Berłowska
Joanna
J
Institute of Fermentation Technology and Microbiology, Technical University of Lódź.
Kregiel
Dorota
D
Klimek
Leszek
L
Orzeszyna
Bartosz
B
Ambroziak
Wojciech
W
D002463:Cell Membrane Permeability; D004072:Digitonin / Q000737:chemistry*; D005562:Formazans / Q000737:chemistry / Q000493:pharmacokinetics*; D007218:Industrial Microbiology / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013385:Succinate Dehydrogenase / Q000032:analysis / Q000378:metabolism*
D005562:Formazans; D013385:Succinate Dehydrogenase; D004072:Digitonin