Application of glutaraldehyde for the staining of esterase-active cells with carboxyfluorescein diacetate

• Published in: Biotechnology letters Vol. 26; no. 5; pp. 379 - 383
• Main Authors: MORONO, Yuki, TAKANO, Suguru, MIYANAGA, Kazuhiko, TANJI, Yasunori, UNNO, Hajime, HORI, Katsutoshi
• Format: Journal Article
• Language: English
• Published: Dordrecht Springer 01.03.2004; Springer Nature B.V
• Subjects: Bacteria; Biological and medical sciences; Biotechnology; Cell Membrane Permeability - drug effects; Cell Survival; Colony Count, Microbial - methods; Esterases - metabolism; Fluoresceins - pharmacokinetics; Fundamental and applied biological sciences. Psychology; Glutaral - pharmacology; Gram-negative bacteria; Gram-Negative Bacteria - cytology; Gram-Negative Bacteria - drug effects; Gram-Negative Bacteria - metabolism; Image Enhancement - methods; Permeability; Staining and Labeling - methods; Staining; Hydrolases; Esterases; Enzyme; Application
• ISSN: 0141-5492; 1573-6776
• DOI: 10.1023/B:BILE.0000018255.89810.1a

Staining of esterase-active bacteria with carboxyfluorescein diacetate (CFDA) has been used to evaluate the viability of various types of cell. However, the outer membrane of Gram-negative bacteria prevents CFDA from permeating into the cell. Although EDTA can increase the permeability of the outer membrane allowing CFDA to enter the cells, it was experimentally confirmed that there is still considerable difficulty in visualizing viable cells due to passive diffusion of carboxyfluorescein (CF), a hydrolyzed product of CFDA, out of the cells. We found that glutaraldehyde enhances the discriminative recognition of esterase-active Gram-negative bacteria under microscopic observation by improving the efficacy of staining. We believe the successful staining in the presence of glutaraldehyde is due to two separate effects: an increase in the permeability of CFDA into the cell and prevention of leakage of CF out of the cell.