Skip to main content

Comparison Between Fluorescent Probe and Ion-Selective Electrode Methods for Intracellular pH Determination in Leuconostoc mesenteroides

Abstract

The intracellular pH (pHin) of Leuconostoc mesenteroides subsp. mesenteroides 19D was evaluated by two different methods, fluorescent probe and ion-selective electrode. Two fluorescent probes 5 (and-6)-carboxyfluorescein diacetate succinimidyl ester (cFDASE) and 5 (and-6)-carboxy-2′,7′-dichlorofluorescein diacetate succinimidyl ester (cDCFDASE) were tested to evaluate the intracellular pH (pHin) of living cells at a medium pH (pHex) ranged from 5.0 to 6.5. Salicylic acid was used as a probe for the ion-selective electrode method. Cells kept 60–80% of cFDASE probe at all pHex values against 5–10% of cDCFDASE probe at pHex ≤ 6.0. The pHin values measured by the ion-selective electrode were higher by 0.1–0.6 pH units at pHex ranged from 5.0 to 6.5 than those determinated by fluorescent probe method. The possibility to study the intracellular pH at a wide external pH range using a single probe, and the simplicity of the material and experimental protocol may make the ion-selective electrode method most useful and easy to measure the intracellular pH of lactic acid bacteria compared with the other techniques like fluorescent probes.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2

References

  1. Carlin F, Nguyen-The C, Cudennec P, Reich M (1989) Microbiological spoilage of fresh, ready-to-use grated carrots. Sci Aliments 9:371–386

    Google Scholar 

  2. Hatcher WS, Weihe JL, Splittstoesser DF et al (1992) Fruit beverages. In: Speck ML (ed) Compendium of methods for the microbiological examination of foods. American Public Health Association, Washington, pp 953–960

    Google Scholar 

  3. Korkeala H, Suortti T, Makela P (1988) Ropy slime formation in vacuum-packed cooked meat products caused by homofermentative Lactobacilli and Leuconostoc species. Int J Food Microbiol 7:339–347

    CAS  Article  PubMed  Google Scholar 

  4. Murdock DI (1977) Microbiology of citrus products. In: Nagy S, Shaw PE, Veldhuis MK (eds) Citrus science and technology. Avi Publishing Company, Westport, pp 445–527

    Google Scholar 

  5. Daeschel MA, McFeeters RF, Fleming HP (2018) Bacterial starter cultures for food. In: Gilliland SE (ed) Bacterial starter cultures for food. CRC Press, Boca Raton

    Google Scholar 

  6. Belguendouz T, Cachon R, Diviès C (1997) pH homeostasis and citric acid utilization: differences between Leuconostoc mesenteroides and Lactococcus lactis. Curr Microbiol 35:233–236

    CAS  Article  Google Scholar 

  7. Padan E, Schuldiner S (1986) Intracellular pH regulation in bacterial cells. Methods Enzym 125:337–352

    CAS  Article  Google Scholar 

  8. Booth IR (1985) Regulation of cytoplasmic pH in bacteria. Microbiol Rev 49:359–378

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Kashket ER (1985) The proton motive force in bacteria: a critical assessment of methods. Annu Rev Microbiol 39:219–242

    CAS  Article  PubMed  Google Scholar 

  10. Rottenberg H (1979) The measurement of membrane potential and deltapH in cells, organelles, and vesicles. Methods Enzym 55:547–569

    CAS  Article  Google Scholar 

  11. Siegumfeldt H, Rechinger KB, Jakobsen M (1999) Use of fluorescence ratio imaging for intracellular pH determination of individual bacterial cells in mixed cultures. Microbiology 145(7):1703–1709

    CAS  Article  PubMed  Google Scholar 

  12. Chitarra LG, Breeuwer P, Van Den Bulk RW, Abee T (2000) Rapid fluorescence assessment of intracellular pH as a viability indicator of Clavibacter michiganensis subsp. michiganensis. J Appl Microbiol 88:809–816

    CAS  Article  PubMed  Google Scholar 

  13. Smigic N, Rajkovic A, Nielsen DS et al (2009) Intracellular pH as an indicator of viability and resuscitation of Campylobacter jejuni after decontamination with lactic acid. Int J Food Microbiol 135:136–143. https://doi.org/10.1016/j.ijfoodmicro.2009.07.023

    CAS  Article  PubMed  Google Scholar 

  14. Breeuwer P, Drocourt JL, Rombouts FM, Abee T (1996) A novel method for continuous determination of the intracellular pH in bacteria with the internally conjugated fluorescent probe 5 (and 6-)—carboxyfluorescein succinimidyl ester. Appl Env Microbiol 62:178–183

    CAS  Google Scholar 

  15. Glaasker E, Konings WN, Poolman B (1996) The application of pH-sensitive fluorescent dyes in lactic acid bacteria reveals distinct extrusion systems for unmodified and conjugated dyes. Mol Membr Biol 13:173–181

    CAS  Article  PubMed  Google Scholar 

  16. Hache C, Cachon R, Wache Y et al (1999) Influence of lactose-citrate co-metabolism on the differences of growth and energetics in Leuconostoc lactis, Leuconostoc mesenteroides ssp. mesenteroides and Leuconostoc mesenteroides ssp. cremoris. Syst Appl Microbiol 22:507–513

    CAS  Article  PubMed  Google Scholar 

  17. Espariz M, Repizo G, Blancato V et al (2011) Identification of malic and soluble oxaloacetate decarboxylase enzymes in Enterococcus faecalis. FEBS J 278:2140–2151. https://doi.org/10.1111/j.1742-4658.2011.08131.x

    CAS  Article  PubMed  Google Scholar 

  18. Zuljan FA, Repizo GD, Alarcon SH, Magni C (2014) α-acetolactate synthase of Lactococcus lactis contributes to pH homeostasis in acid stress conditions. Int J Food Microbiol 188:99–107. https://doi.org/10.1016/j.ijfoodmicro.2014.07.017

    CAS  Article  PubMed  Google Scholar 

  19. Zhang J, Wu C, Du G, Chen J (2012) Enhanced acid tolerance in Lactobacillus casei by adaptive evolution and compared stress response during acid stress. Biotechnol Bioprocess Eng 17:283–289

    CAS  Article  Google Scholar 

  20. Cachon R, Antérieux P, Diviès C (1998) The comparative behavior of Lactococcus lactis in free and immobilized culture processes. J Biotechnol 63:211–218

    CAS  Article  Google Scholar 

  21. Cook GM, Russell JB (1994) The effect of extracellular pH and lactic acid on pH homeostasis in Lactococcus lactis and Streptococcus bovis. Curr Microbiol 28:165–168

    CAS  Article  Google Scholar 

  22. Paulsen IT, Brown MH, Skurray RA (1996) Proton-dependent multidrug efflux systems. Microbiol Rev 60:575–608

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Lowry OH, Rosebrough NJ, Farr AL, Randell RJ (1951) Protein measurement with the folin phenol reagent. J Biol Chem 193:265–275

    CAS  PubMed  Google Scholar 

  24. Hellingwerf KJ, van Hoorn P (1985) A polyvinylchloride-membrane based anion selective electrode for continuous registration of delta pH (interior alkaline) with salicylate as the indicator probe. J Biochem Biophys Methods 11:83–93

    CAS  Article  PubMed  Google Scholar 

  25. Lolkema JS, Hellingwerf KJ, Konings WN (1982) The effect of “probe binding” on the quantitative determination of the proton-motive force in bacteria. Biochim Biophys Acta 681:85–94

    CAS  Article  Google Scholar 

  26. Siegumfeldt H, Rechinger KB, Jakobsen M (2000) Dynamic changes of intracellular pH in individual lactic acid bacterium cells in response to a rapid drop in extracellular pH. Appl Environ Microbiol 66:2330–2335. https://doi.org/10.1128/AEM.66.6.2330-2335.2000

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  27. Ramos CL, Thorsen L, Ryssel M et al (2014) Effect of the gastrointestinal environment on pH homeostasis of Lactobacillus plantarum and Lactobacillus brevis cells as measured by real-time fluorescence ratio-imaging microscopy. Res Microbiol 165:215–225. https://doi.org/10.1016/j.resmic.2014.02.005

    CAS  Article  PubMed  Google Scholar 

  28. Spilimbergo S, Quaranta A, Garcia-Gonzalez L et al (2010) Intracellular pH measurement during high-pressure CO2 pasteurization evaluated by cell fluorescent staining. J Supercrit Fluids 53:185–191

    CAS  Article  Google Scholar 

  29. Setty OH, Hendler RW, Shrager RI (1983) Simultaneous measurements of proton motive force, delta pH, membrane potential, and H+/O ratios in intact Escherichia coli. Biophys J 43:371–381

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  30. Molenaar D, Abee T, Konings WN (1991) Continuous measurement of the cytoplasmic pH in Lactococcus lactis with a fluorescent pH indicator. Biochim Biophys Acta 1115:75–83

    CAS  Article  PubMed  Google Scholar 

  31. Cachon R, Wache Y, Alwazeer D et al (2005) Cell for measuring biological activities and/or physiological parameters of micro-organisms. World Patent WO2005026714

  32. Allonneau C, Olmos E, Guyot S et al (2015) Hydrodynamic characterization of a new small-scale reactor mixed by a magnetic bar. Biochem Eng J 96:29–37. https://doi.org/10.1016/j.bej.2014.12.005

    CAS  Article  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Duried Alwazeer.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Alwazeer, D., Riondet, C. & Cachon, R. Comparison Between Fluorescent Probe and Ion-Selective Electrode Methods for Intracellular pH Determination in Leuconostoc mesenteroides. Curr Microbiol 75, 1493–1497 (2018). https://doi.org/10.1007/s00284-018-1550-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00284-018-1550-9