Rapid fluorescence assessment of the viability of stressed Lactococcus lactis

Abstract

The aim of this study was to establish the use of the fluorescent probes carboxyfluorescein (cF) and propidium iodide (PI) for rapid assessment of viability, using Lactococcus lactis subsp. lactis ML3 exposed to different stress treatments. The cF labeling indicated the reproductive capacity of mixtures of nontreated cells and cells killed at 70 degrees C very well. However, after treatment up to 60 degrees C the fraction of cF-labeled cells remained high, whereas the survival decreased for cells treated at above 50 degrees C and was completely lost for those treated at 60 degrees C. In an extended series of experiments, cell suspensions were exposed to heating, freezing, low pH, or bile salts, after which the colony counts, acidification capacity, glycolytic activity, PI exclusion, cF labeling, and cF efflux were measured and compared. The acidification capacity corresponded with the number of CFU. The glycolytic activity, which is an indicator of vitality, was more sensitive to the stress conditions than the reproduction, acidification, and fluorescence parameters. The cF labeling depended on membrane integrity, as was confirmed by PI exclusion. The fraction of cF-labeled cells was not a general indicator of reproduction or acidification, nor was PI exclusion or cF labeling capacity (the internal cF concentration). When the cells were labeled by cF, a subsequent lactose-energized efflux assay was needed for decisive viability assessment. This novel assay proved to be a good and rapid indicator of the reproduction and acidification capacities of stressed L. lactis and has potential for physiological research and dairy applications related to lactic acid bacteria.

FIG. 1

Labeling of L. lactis by cF and subsequent active extrusion. A nontreated cell suspension was labeled with cF, by incubation with 50 μM cFDA at 30°C and pH 7.0 for 10 min, and washed once (A). This labeled cell suspension was incubated with 20 mM lactose at 30°C for 2 min (B) and for 15 min (C). Cell suspensions were photographed with simultaneous light and epifluorescence microscopy (excitation wavelength, 450 to 490 nm; emission wavelength, >520 nm) to visualize both stained and nonstained cells. Bar represents 10 μm for all micrographs.

FIG. 2

Retention of cF by L. lactis. Cells were loaded with cF and resuspended in 50 mM KP_i buffer (pH 7.0). (A) Retention of cF in the absence of an energy source when cells were kept on ice (∗), at 30°C (■), and at 30°C with the addition of 2 μM valinomycin and nigericin (▴). (B) Retention of cF in cells with the addition of 20 mM lactose (■) and with the addition of 20 mM lactose after dissipation of the PMF by adding 2 μM valinomycin and nigericin (▴). The internal ATP levels in cells with the addition of lactose (□) and with the addition of lactose after dissipation of the PMF (▵) were also measured.

FIG. 3

Fluorescence microscopy of L. lactis cells double labeled with cF and PI. Suspensions containing 100% nontreated cells (A), 50% nontreated cells mixed with 50% heat-killed cells (B), and 100% heat-killed cells (C) were incubated with 50 μM cFDA and 44 μM PI. Cell suspensions were photographed with simultaneous light and epifluorescence microscopy (excitation wavelength, 450 to 490 nm; emission wavelength, >520 nm) to visualize both stained and nonstained cells. Bar represents 10 μm for all micrographs.

FIG. 4

Experimental discrimination of viable and nonviable bacteria. An L. lactis cell suspension (10¹⁰ CFU/ml) was divided into two portions. One was not treated and the other was exposed for 10 min to 70°C. The nontreated and the heat-treated portions were mixed in various proportions and plated on M17 agar supplemented with 0.5% (wt/vol) lactose or labeled for 10 min with 50 μM cFDA, washed, and analyzed by fluorescence microscopy and spectrofluorimetry. The experiment was performed with two batches of cells. Plate counts (A), fraction cF-labeled cells (B), and average intracellular cF concentration (C) are all plotted against the known fraction of nontreated cells.

FIG. 5

Effects of temperature treatment on L. lactis survival and cF labeling. L. lactis cell suspensions (10¹⁰ CFU/ml) were subjected to elevated temperatures for 90 s. After the treatment the suspensions were plated on M17 agar supplemented with 0.5% (wt/vol) lactose and tested for cF labeling. Both the fraction cF-labeled cells and the cF labeling capacity were determined. Data are the means and standard deviations of three experiments and are expressed as values relative to that for nontreated cells (taken as 100). RT, room temperature.

FIG. 6

Effect of stresses on the cF efflux by lactose-energized L. lactis. Cell suspensions were pretreated under different stress conditions, loaded with cF, and resuspended in 50 mM KP_i buffer (pH 7.0). Five minutes after the start of the incubation at 30°C, lactose was added to a final concentration of 20 mM. Results of representative experiments with a non-treated-cell suspension and five of the stressed cell suspensions are given. No treatment, 100% = 364 μM (■); exposure to 60°C for 90 s, 100% = 149 μM (□); exposure to −20°C for 24 h, 100% = 36 μM (∗); exposure to pH 5.0, 100% = 137 μM (); exposure to 1.0% CBS, 100% = 105 μM (●); and exposure to 0.02% DBS, 100% = 136 μM (◊).

FIG. 7

Comparisons of fluorescence parameters, plate counts, acidification capacity, and glycolytic activity of L. lactis after exposure to different types of stress. Comparisons are shown for the fraction cF-labeled cells with plate counts (A), the cF labeling capacity with plate counts (B), the cFDA hydrolysis activity with plate counts (C), the fraction PI-excluding cells with plate counts (D), the product of cF labeling and efflux with plate counts (E), the plate counts with acidification capacity (F), the product of cF labeling and efflux with acidification capacity (G), and the glycolytic activity with acidification capacity (H). For each panel the data are extracted from Table 1. The symbols indicate no treatment (■), exposure to 70°C for 10 min (), exposure to 60°C for 90 s (□), exposure to −20°C for 24 h (∗), exposure to −20°C and 30% glycerol for 24 h (+), exposure to pH 7.0 (▵), exposure to pH 5.0 (), exposure to pH 2.0 (▴), exposure to 0.2% CBS (○), exposure to 1.0% CBS (●), exposure to 0.02% DBS (◊), exposure to 0.06% DBS (), and exposure to 1.0% DBS (⧫). The error bars indicate the standard deviations. The bold lines indicate the optimal situation, that is, linear regression through the origin and the point for the non-treated-cell suspensions.