Flow cytometry, fluorescent probes, and flashing bacteria

C.J. Bunthof

Research output: Thesisinternal PhD, WU


<font size="3"><p> </p><p><hr/></p><p>Key words: fluorescent probes, flow cytometry, CSLM, viability, survival, microbial physiology, lactic acid bacteria, <em>Lactococcus lactis</em> , <em>Lactobacillus plantarum</em> , cheese, milk, probiotic</p><p> </p><p>In food industry there is a perceived need for rapid methods for detection and viability assessment of microbes. Fluorescent staining and flow cytometry provide excellent tools for microbial analysis. This thesis describes fluorescent techniques for assessment of the physiological state of lactic acid bacteria.</p><p>Lysis of lactic acid bacteria plays a crucial role in cheese manufacturing. It is generally considered that lysis results in leakage of intracellular enzymes in the cheese curd and, thus, plays an important role in ripening and flavor formation. <em>Bac</em> Light (Molecular Probes) was applied for monitoring the lysis process of <em>Lactococcus lactis</em> MG1363 in a buffered suspension with high osmolarity to mimic cheese conditions. The <em>Bac</em> Light kit combines the nucleic acid dyes propidium iodide (PI) and SYTO 9. PI is commonly used to determine membrane integrity based on dye exclusion. When used in combination with the permeant SYTO 9, membrane-damaged cells are stained by PI (red) while the intact cells are stained by SYTO 9 (green). Lysis was induced with mutanolysin and followed in time using fluorescence microscopy and flow cytometry. Also, enzyme assays and plate counts were performed. The results demonstrated a transient permeable cell status that has a significant role in the lysis process. Furthermore, permeable cells were demonstrated in ripening cheese with confocal scanning laser microcopy and <em>Bac</em> Light.</p><p>Viability assessment by conventional plate counting requires long incubation times and provides limited information. Flow cytometric assessment of the viability of lactic acid bacteria was investigated and compared with plate counts. The esterase substrate carboxyfluorescein diacetate (cFDA) and the impermeant nucleic acid dyes PI and TOTO-1 were tested using exponential phase at 70°C heat-killed cultures of a <em>Lactococcus</em> , a <em>Streptococcus</em> , three <em>Lactobacillus</em> , two <em>Leuconostoc</em> , an <em>Enterococcus</em> , and a <em>Pediococcus</em> species. The combination of cFDA and TOTO-1 gave the best results. The intact and membrane-damaged subpopulations were distinguished well. Sorting and plating showed that cFDA stained the culturable and TOTO-1 the nonculturable cells. The assay was applied to cultures exposed to deconjugated bile salts or to hydrochloric acid and results corresponded well with plate counts.</p><p>Subsequently, flow cytometry with cFDA and TOTO-1 staining was applied to <em>Lactobacillus plantarum</em> WCFS 1 suspended in milk. To facilitate flow cytometry clearing of the milk was required. A procedure based on a milk clearing solution was optimized to increase the signal-to-noise-ratio and flow cytometry enumerations were accurate to a lower limit of 10 <sup>5</SUP>cells/ml.</p><p>Finally, the novel assay was applied to starter cultures for cheese and yogurt and to the probiotic products Yakult, Mona Vifit, and Orthiflorplus. Flow cytometry in combination with plate counts revealed three populations: culturable cells, cells that are intact and metabolically active but not culturable, and permeabilized cells. The proportions of the populations differed between the tested products.</p><p>In conclusion, the development of flow cytometry for bacteria is an important asset for microbiological research. The rapid novel methods described in this thesis provide possibilities for examination of fermentation processes and food products.
Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • Wageningen University
  • Rombouts, F.M., Promotor, External person
  • Abee, Tjakko, Promotor
  • Breeuwer, P., Promotor
Award date13 May 2002
Place of PublicationS.l.
Print ISBNs9789058086327
Publication statusPublished - 2002


  • flow cytometry
  • fluorescence microscopy
  • lactic acid bacteria
  • food microbiology
  • lysis
  • dairy research

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