Abstract
The analysis of growing or resting bacterial populations by flow cytometry offers several advantages over traditional methods for determining mean-value parameters. This method has been applied here to measure both the distribution of single-cell fluorescence intensity and the light-scatter behaviour of the methylotrophical strains of Methylobacterium rhodesianum MB126 and Methylocystis GB25 as well as Pseudomonas fluorescens and a strain isolated from the soil. The four different bacterial populations were analysed concerning the DNA and the poly-3-hydroxybutyrate (PHB) content. A new cell-preservation method is presented. Optimized staining methods for each strain were developed in detail, in two cases DNA had to be dehybridized before staining with a mixture of mithramycin/ethidium bromide. Nile red is used for detecting PHB. Both stains were excited by an argonion laser at 488 nm; fluorescence emission for mithramycin/ethidium bromide was measured from 520 nm and for Nile red from 600 nm onwards. It is shown that changes in the DNA content and in the forward-light-scattering behaviour of the bacterial strains chosen were measurable. These changes could be related to different cultivation conditions and correlated, in the case of strains that accumulate PHB, with alterations of that biopolymer content. In addition it was found that these methods provide a contribution to the differentiation of mixed bacterial populations.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Babel W (1992) Peculiarities of methylotrophs concerning overflow metabolism, especially the synthesis of polyhydroxyalkanoates, FEMS Microbiol Rev 103:141–148
Button DK, Robertson BR (1989) Kinetics of bacterial processes in natural aquatic systems based on biomass as determined by high-resolution flow cytometry. Cytometry 10:558–563
Degelau A (1993) Durchflußzytometrie als Analysenmethode in der Biotechnologie. Thesis A, Universität Hannover
Degelau A, Freitag R, Linz F, Middendorf C, Scheper T, Bley T, Müller S, Stoll P, Reardon KF (1992) Immuno- and flow cytometric analytical methods for biotechnological research and process monitoring. J Biotechnol 25:115–144
Grattarola M, Carlo P, Gianetti G, Finollo R, Viviani R, Chiabrera A (1985) Laser flow measurements of scattering and fluorescence from cell nuclei in the presence of increasing Mg+ + concentrations. Biophys J 47:461–468
Green PN, Bousfield IJ, Hood D (1988) Three new Methylobacterium species: M. rhodesianum sp. nov., M. zatmanii sp. nov., M. fugusawaense sp. nov. Int J Syst Bacteriol 38:124–127
Hagemann R (1990) Gentechnologische Arbeitsmethoden. Akademie-Verlag, Berlin, pp 160–163
Linz F (1989) Durchflußzytometrie zur Prozeßbeobachtung in der Biotechnologie. Thesis A. Universität Hannover
Lloyd D (1993) Flow cytometry in microbiology. Springer-Verlag, London Berlin Heidelberg New York
Lösche A, Stoll P, Müller S, Bley T (1991) Durchflußzytometrie — eine physikalische Meßmethode in der Biotechnologie. Exp Tech Phys 39:97–102
Lösche A, Müller S, Bley T (1994) Flow cytometry — Charakterisierung von Bakterien. Bioforum 17:56–60
Molenaar D, Bolhuis H, Abbe T, Poolman B, Konings WN (1992) The efflux of a fluorescent probe is catalyzed by an ATP-driven extrusion system in Lactococcus lactis. J Bacteriol 174:3118–3124
Müller S (1992) Darstellung der Populationsdynamik von Saccharomyces cerevisiae anhand durchflußzytometrischer Messung des 3β-Hydroxysterol- und DNS-Gehaltes. Thesis A, Universität Halle-Wittenberg
Müller S, Lösche A, Bley T (1993) Staining procedures for flow cytometric monitoring of bacterial populations. Acta Biotechnol 13:289–297
Paulhuhn I, Naujok A (1980) Quantitative fluorometric determination of cellular DNA by ethidium bromide and the constancy of the quantum yield of the DNA-ethidium complex in the biological environment. Z Naturforsch 35:7–8
Sanders CA, Yajko DM, Hyun W, Langlois RG, Nassos PS, Fulwyler MJ, Hadley WK (1990) Determination of guanine-pluscytosine content of bacterial DNA by dual-laser flow cytometry. J Gen Microbiol 136:359–365
Scheper T, Hitzmann B, Rinas U, Schügerl K (1987) Flow cytometry of Escherichia coli for process monitoring. J Biotechnol 5:139–148
Skarstadt K, Steen HB, Boye E (1983) Cell cycle parameters of slowly growing Escherichia coli B/r studied by flow cytometry. J Bacteriol 154:656–662
Steen HB, Boye E (1980) Bacterial growth studied by flow cytometry. Cytometry 1:32–36
Steinbüchel A (1991) Recent advances in the knowledge of bacterial poly(hydroxyalkanoic acid) metabolism and potential impacts on the production of biodegradable thermoplasts. Acta Biotechnol 11:13–21
Steinbüchel A and Pieper U (1992) Production of a copolyester of 3-hydroxy butyric acid and 3-hydroxy valeric acid from single unrelated carbon sources by a mutant of Alcaligenes eutrophus. Appl Microbiol Biotechnol 37:1–6
Van Dilla MA, Langlois RG, Pinkel D, Yaiko D, Hadley WK (1983) Bacterial characterization by flow cytometry. Science 220:620–622
Zimmermann HW (1986) Physikochemische und cytochemische Untersuchungen zur Bindung von Ethidium- und Acridinfarbstoffen an DNA und Organellen in lebenden Zellen. Angew Chem 98:115–131
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Müller, S., Lösche, A., Bley, T. et al. A flow cytometric approach for characterization and differentiation of bacteria during microbial processes. Appl Microbiol Biotechnol 43, 93–101 (1995). https://doi.org/10.1007/BF00170629
Received:
Revised:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00170629