Assessment of viability of microorganisms employing fluorescence techniques

P. Breeuwer

Research output: Thesisinternal PhD, WU


<br/>Viability assessment of microorganisms is relevant for a wide variety of applications in industry, including evaluation of inactivation treatments and quality assessment of starter cultures for beer, wine, and yoghurt production.<p>Usually, the ability of microbial cells to reproduce is considered as the benchmark method for determination of cell viability, and this is most commonly determined by the plate count method. The time needed to form visible colonies, however, is relatively long. Therefore, there is an increasing interest in rapid methods which exploit criteria other than reproduction. The advantages of fluorescent methods are a high sensitivity, a high time resolution and the potential to analyze individual cells.<p>Fluorescent probes such as fluorescein, carboxyfluorescein (cF), and BCECF may be incorporated in microorganisms as (non-fluorescent) acetoxymethyl or diacetyl esters. These esters are membrane permeable and are cleaved in the cytoplasm by esterases, which results in accumulation of the fluorescent form. In <em>Saccharomyces</em><em>cerevisiae</em> it was found that the carrier- mediated efflux of cF was coupled to the energy metabolism. Subsequently, a twostep procedure was developed, consisting of loading the cells with cF, followed by incubation in the presence of glucose. The efflux experiments showed an excellent correlation between the viability of <em>S.</em><em>cerevisiae</em> cells (determined by plate count) and the ability to translocate cF.<p>The intracellular pH (pH <sub><font size="-2">in</font></sub> ) is critical for the control of many cellular processes, such as DNA transcription, protein synthesis, and enzyme activities. To determine the intracellular pH of bacteria a novel method was developed based on the intracellular conjugation of the fluorescent probe 5 (and 6-)-carboxyfluorescein succinimidyl ester (cFSE). This cFSE method significantly reduced problems due to efflux of fluorescent probe from the cells. Moreover, the method was successfully used to determine the intracellular pH in bacteria under stress conditions, such as elevated temperatures and the presence of detergents.<p>The viability of <em>R. oligosporus</em> sporangiospores was determined microscopically and with flow cytometry. Swelling of the sporangiospores was accompanied by an increase of the pH <sub><font size="-2">in</font></sub> . In the presence of nonanoic acid, a self-inhibitor produced by various fungi, an increase of the pH <sub><font size="-2">in</font></sub> , was prevented and swelling inhibited, suggesting a crucial role of the pHin in the germination of the spores.<p>The effects of detergents on the viability of <em>Lactobacillus lactis</em> and <em>Bacillus subtilis</em> were investigated. Triton X-100 and Lauryl sulfobetaine efficiently inhibited growth at concentrations below the critical micelle concentration. Surprisingly, the application of fluorescence techniques showed that Triton X-100 did not permeabilize the cytoplasmic membrane of <em>L. lactis</em> .<p>In conclusion, fluorescence techniques offer the possibility to study viability of a cell population employing microscopy, flow cytometry and image analysis. This may contribute to a better understanding of the mechanisms involved in selective survival of microorganisms under different stress conditions.
Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • Rombouts, F.M., Promotor, External person
  • Abee, Tjakko, Promotor
Award date18 Oct 1996
Place of PublicationS.l.
Print ISBNs9789054855859
Publication statusPublished - 1996


  • staining
  • fluorescent dyes
  • microbiology
  • methodology
  • techniques

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