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

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Abstract

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.

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References

  • Amann RI, Ludwig W, Schleifer KH (1995) Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol. Rev. 59: 143–169.

    Google Scholar 

  • Diaper JP, Edwards C (1994) The use of fluorogenic esters to detect viable bacteria by flow cytometry. J. Appl. Bacteriol. 77: 221–228.

    Google Scholar 

  • Forrest TM, Wilson GE, Pan Y, Schaefer J (1991) Characterization of cross-linking of cell walls of Bacillus subtilis by a combination of magic-angle spinning NMR and gas chromatographymass spectrometry of both intact and hydrolyzed 13C-and 15N-labeled cell-wall peptidoglycan. J. Biol. Chem. 266: 24485–24491.

    Google Scholar 

  • Fraud S, Hann AC, Maillard JY, Russell AD (2003) Effects of orthophthalaldehyde, glutaraldehyde and chlorhexidine diacetate on Mycobacterium chelonae and Mycobacterium abscessus strains with modified permeability. J. Antimicrob. Chemother. 51: 575–584.

    Google Scholar 

  • Hoefel D, Grooby WL, Monis PT, Andrews S, Saint CP (2003) A comparative study of carboxyfluorescein diacetate and carboxy-fluorescein diacetate succinimidyl ester as indicators of bacterial activity. J. Microbiol. Meth. 52: 379–388.

    Google Scholar 

  • Hughes RC, Thuman PF (1970) Cross-linking of bacterial cell walls with glutaraldehyde. Biochem. J. 119: 925–926.

    Google Scholar 

  • Kawai M, Yamaguchi N, Nasu M (1999) Rapid enumeration of physiologically active bacteria in purified water used in the pharmaceutical manufacturing process. J. Appl. Microbiol. 86: 496–504.

    Google Scholar 

  • McDougald D, Rice SA, Weichart D, Kjelleberg S (1998) Nonculturability: adaptation or debilitation? FEMS Microbiol. Ecol. 25: 1–9.

    Google Scholar 

  • Nikaido H (1996) Outer membrane. In: Neidhardt FC, Curtiss R, Ingraham JL, Lin ECC, Low KB, Magasanik B, Reznikoff WS, Riley M, Schaechter M Umbarger HE, eds. Escherichia coli and Salmonella. Washington D.C: American Society for Microbiology, pp. 29–47.

    Google Scholar 

  • Park JT (1996) The murein sacculus. In: Neidhardt FC, Curtiss R, Ingraham JL, Lin ECC, Low KB, Magasanik B, Reznikoff WS, Riley M, Schaechter M, Umbarger HE, eds. Escherichia coli and Salmonella. Washington D.C.: American Society for Microbiology, pp. 48–57.

    Google Scholar 

  • Reasoner DJ, Geldreich EE (1985) A new medium for the enumeration and subculture of bacteria from potable water. Appl. Environ. Microbiol. 49: 1–7.

    Google Scholar 

  • Reithmeier RA, Bragg PD (1977) Cross-linking of the proteins in the outer membrane of Escherichia coli. Biochim. Biophys. Acta 466: 245–256.

    Google Scholar 

  • Tanaka Y, Yamaguchi N, Nasu M (2000) Viability of Escherichia coli O157:H7 in natural river water determined by the use of flow cytometry. J. Appl. Microbiol. 88: 228–236.

    Google Scholar 

  • Vaara M (1992) Agents that increase the permeability of the outer membrane. Microbiol. Rev. 56: 395–411.

    Google Scholar 

Download references

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Authors and Affiliations

  1. Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama, 226-8501, Japan

    Yuki Morono, Suguru Takano, Kazuhiko Miyanaga, Yasunori Tanji, Hajime Unno & Katsutoshi Hori

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  1. Yuki Morono
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  2. Suguru Takano
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  3. Kazuhiko Miyanaga
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  4. Yasunori Tanji
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  5. Hajime Unno
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  6. Katsutoshi Hori
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Morono, Y., Takano, S., Miyanaga, K. et al. Application of glutaraldehyde for the staining of esterase-active cells with carboxyfluorescein diacetate. Biotechnology Letters 26, 379–383 (2004). https://doi.org/10.1023/B:BILE.0000018255.89810.1a

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  • DOI: https://doi.org/10.1023/B:BILE.0000018255.89810.1a

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