Utilization of physiological and taxonomic fluorescent probes to study Lactobacilli cells and response to pH challenge.
pH stress is recognized as an important feature for Lactobacillus in relation to lifestyle and commercial utility. Hence, this study aims to investigate the cell function of Lactobacilli cells subjected to pHs between 7.0 and 2.0. For this purpose, the Lactobacilli isolates of vegetable origin were first hybridized with fluorescent oligonucleotide rRNA probes for detecting Lactobacillus species. Then, cells were exposed to pH stress and labelled with fluorescent probes, carboxyfluorescein diacetate (CFDA) and propidium iodine (PI), which provided the insight into esterase activity and membrane integrity of cells. Among isolates, fluorescence in situ hybridization (FISH) enabled us to specifically detect L. plantarum and L. brevis. Interestingly, FCM analysis revealed that at pHs between 7.0 and 4.0 the cell membrane was intact, while after the exposure at pH 3.0, and 2.0 became perturbed or impaired. Finally, L. brevis and L. plantarum differed from each other in fluorescence labeling behaviour and culturability. However, the results showed that the same standard protocol for labeling enables discrimination of subpopulations of tested species. Depending on the species, the substantial culturability loss was observed at pH 3.0 and 2.0. These results suggest that the taxonomic and physiological fluorescent probes could be suitable for in situ detection of specific bacteria and rapid assessment of the physiological status of cells.
Chair of Industrial and Food Microbiology, Faculty of Food Science, University of Warmia and Mazury in Olsztyn, Plac Cieszyński 1, 10-726 Olsztyn, Poland. Electronic address: magdalena.olszewska@uwm.edu.pl.
Magdalena A
MA
Olszewska
Chair of Industrial and Food Microbiology, Faculty of Food Science, University of Warmia and Mazury in Olsztyn, Plac Cieszyński 1, 10-726 Olsztyn, Poland.
Aleksandra M
AM
Kocot
Research and Education Center, Laboratory of Molecular Diagnostics, University of Warmia and Mazury in Olsztyn, Prawocheńskiego 5, 10-720 Olsztyn, Poland.
Anna
A
Nynca
Chair of Industrial and Food Microbiology, Faculty of Food Science, University of Warmia and Mazury in Olsztyn, Plac Cieszyński 1, 10-726 Olsztyn, Poland.
Łucja
Ł
Łaniewska-Trokenheim
D018407:Bacterial Physiological Phenomena; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D006863:Hydrogen-Ion Concentration; D017404:In Situ Hybridization, Fluorescence; D007778:Lactobacillus / Q000145:classification* / Q000502:physiology*; D050296:Microbial Viability; D012336:RNA, Ribosomal, 16S / Q000235:genetics
D005456:Fluorescent Dyes; D012336:RNA, Ribosomal, 16S
Epifluorescence microscopy; Flow cytometry; Fluorescence in situ hybridization; Plate counts; Survival; pH