RESULTS AND DISCUSSION

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1 ASSESSMENT WITH A COMPACT FLOW CYTOMETER OF LIVE, INJURED AND DEAD PROBIOTIC LACTOBACILLI AND BIFIDOBACTERIA AFTER SUBJECTING TO OSMOTIC, FREEZE AND HEAT STRESS Thien Trung Le,4, Tony Ruyssen 2, Mieke Uyttendaele, Paul Van Assche 3 and Koen Dewettinck Ghent University, Department of Food Safety and Food Quality, Belgium 2 University College Ghent, Department Food Science and Technology, Belgium 3 Institute for Agricultural and Fisheries Research, Technology and Food Unit, Belgium 4 Ho Chi Minh City Nong Lam University, Faculty of Food Science and Technology, Vietnam Abstract A single light-source flow cytometer with the aid of two fluorescent probes SYTO 8 and PI was used to examine the viability of three commercial probiotic strains Lactobacillus acidophilus, Lactobacillus paracasei ssp. tolerans and Bifidobacterium animalis ssp. lactis after they were subjected to elevated temperatures, high salt concentrations and freezing. The flow cytometry was validated to be a rapid viability assay by comparing its results with those of the conventional plate counting method. Key words: flow cytometry, SYTO 8 PI, viability, probiotics, INTRODUCTION A quick evaluation of the viability of bacteria is of great importance not only for scientific research but also in many industrial applications such as food & drinks, cosmetics, biotechnology, water industry, and pharmaceutics. In this research, cell suspensions of three commercial probiotic strains Lactobacillus acidophilus (L), Lactobacillus paracasei ssp. tolerans () and Bifidobacterium animalis ssp. lactis (B42) were subjected to stress conditions namely elevated temperatures, high salt concentrations and freezing. The capacity of a compact, single light-source flow cytometer was used to examine the viability of the stressed cells, based on membrane integrity with the aid of a combination of two fluorescent stains, namely cell membrane impermeant propidium iodine (PI) and cell-permeant SYTO 8. The staining mechanism of the combination of these two dyes is explained elsewhere (Haugland, 26). Results of flow cytometry (FCM) counting were compared with those of plate counting to validate the use of flow cytometers coupled with the two fluorescent probes as a rapid viability assay that provides a generally valid indicator for reproduction. MATERIAL AND METHODS Bacterial strains and culture conditions L (Food Specialties, Australia), (Rhodia Food, Dangé St. Romain, France) and B42 (Danisco Cultor/Wisby, Niebüll, Germany) were stored as frozen stocks at - C on cryobeads. Media for the activation and plate counting of the first two strains were MRS broth and MRS agar. For B42, the media supplemented with.5%w/v L-cystein hydrochloride monohydrate were used. The incubation was always carried under anaerobic condition at 37 o C. Prior to the assays, the strains were activated and serially transferred at least twice. One percent of each culture was then inoculated in the fresh medium and incubated for 2 hours before being subjected to experiments. 78

2 Experimental treatments Suspensions of % inoculum ( 6-7 cells/ml) of activated cultures were made in autoclaved MRD (maximum recovery diluent), MRD supplemented with NaCl at different concentrations and MRD supplemented with the cryoprotectants (see section results and dicussion) for the experiments of the exposure to heat, osmotic shock and freezing, respectively. Untreated samples were also measured and served as control samples. Each experiment was carried out in at least three replicates with each performed on a different day from a new inoculum. FCM measurement Bacterial samples were stained with 5 μm for SYTO 8 (Molecular Probes) and. mg/ml (or 4.97 μm) for PI (P47, Sigma) in the dark at 37 o C for 5 minutes. The FCM analyses were performed with a compact flow cytometer, "BACTIFLOW GL" (Partec-Chemunex), equipped with a mw green Nd:YAG solid state laser, operating at 532 nm. FSC (forward scatter), SSC (side scatter), orange (fluorescence ) and red of each single cell were recorded. FSC was selected as the trigger parameter. Orange fluorescence emitted from SYTO 8 stained cells was collected with bandpass filter 5-59 nm, whereas red fluorescence emitted from PI stained cells was collected with bandpass filter nm. The detection gain and thresholds were set to have maximum collection of signals from the bacteria. Dilution of bacterial suspensions for FCM measuring was done in phosphate-buffered saline which had been filtered through.2μm Supor Acrodisc syringe filter (Pall Gelman). All measurements were carried out in duplicate at dilution factor of -2 after the experimental treatments. Speed of the flow was adjusted to obtain flow rate of 5-5 cells/second. Blank samples (for fluorescence background counting) with the same constituents but without bacteria were also counted on the flow cytometer. Counts of bacterial samples were subtracted to those of blank ones before doing further calculation. Data analysis Data acquisition and analysis were performed with the Partec Flomax software. Two parameter dot plots FSC-SSC and - were primarily used to resolve the data. First, bacterium gate on FSC- SSC plots was defined and applied to discriminate bacteria from noise (background). Second, compensate crosstalk was applied on - plots to disperse the overlap between the two fluorescent parameters. Finally, counts of the separated subpopulations on - plots (Figure ) were obtained by setting quadrants (sometimes polygon gates) on these plots. RESULTS AND DISCUSSION Effect of temperatures In untreated samples, almost all cells were only stained with SYTO 8 and gated in Q4 (live cell) (fig. ). Under a certain intensity of the treatment depending on the strains, part of the population was not only stained with SYTO 8 but also with PI and was acquired in gate Q2 (sublethal or injured cell). The membranes of all cells were ruptured at above 67.5 o C for L, above 65 o C for and above o C for B42, hence the cells were solely stained by PI throughout the population and were therefore gated in Q (dead cell) (Fig. & 2a). The number of injured cells (Q2) was highest at around 62.5 o C (Fig. 2a) and they were probably not resurrected on MRS agar plates since the line of only Q4 fraction was already located above that of the plate counts (Fig. 2b). Use of SYTO 8-PI combination to assess the viability of B42 (can be genera, species of this strain) under thermal stress is not suitable (Fig. 2b). Total (Q+Q2+Q4) detected cell numbers of L and B42 decreased when treatment temperatures exceeded certain values. The decrease in total cells of L could be due to the disintegration (lyse) of cells or intense loss of the components caused by their leakage through the damaged membrane. On the other hand, the decrease in total B42 detected cells was due to a part of heat treated cells 79

3 not being stained with PI or SYTO 8 since the number of bacterial cells recorded on bacteria FSC- SSC gates did not decrease (figure not shown). Gate: R Q:.53% 565 / ml Q2:.56% / ml Gate: R Q:.26% 92 / ml Q2:.7% 245 / ml Gate: R Q: 7.4% 48 / ml SSC Q2:.7% 76 / ml Q3: 5.5% 5875 / ml Q4: 93.4% 997 / ml Q3: 8.94% 655 / ml Q4: 88.% / ml Q3: 9.2% 632 / ml Q4: 72.59% / ml.. Gate: R Q: 86.% 473 / ml.... Ctrl o C 62.5 o C SSC Q2:.8% 985 / ml Gate: R Q: 87.52% / ml Q2:.2% / ml Gate: R Q:.9% / ml Q2:.9% / ml Q3:.% 35 / ml Q4:.47% 255 / ml Q3: 2.2% 59 / ml Q4:.7% 35 / ml Q3: 96.64% 5325 / ml Q4:.8% 65 / ml o C 67.5 o C Blank sample Figure. Fluorescence properties of Lactobacillus acidophilus (L) under the treatment with different temperatures for 5 mins. Cells were stained with SYTO8 and PI. Q-cells stained with PI (dead cell), Q2 cells stained with both SYTO8 and PI (sublethal cell), Q3-background and Q4-cell stained with SYTO8 (live cell). PI stained (Q) SYTO8 stained (Q4) total cells detected (Q+Q2+Q4) (a) SYTO8 and PI stained (Q2) plate counts (b) Percentage of cells 2 L Ctrl L Ctrl Ctrl Ctrl

4 2 Ctrl B Ctrl B42 75 Temperature ( o C) Figure 2. Comparison of FCM using SYTO8-PI staining with plate counts under thermal treatments on Lactobacillus acidophilus (L), Lactobacillus paracasei ssp. paracasei () and Bifidobacterium animalis ssp. lactis (B42). Graphs show the relative changes of PI ( ), SYTO8 & PI ( ), SYTO8 ( ) stained fractions, total cells detected ( ) on - dot plots and plate counts ( L) Effect of osmotic shock Total recorded cells of L decreased from % to ± 6.54 % and 73.6 ± 2.59 % with 5% and 25% salt treatments respectively at 37 o C (Figure 3a). These high salt concentrations caused cells to shrink and some were not included in the bacterial clusters which were gated on the FSC- SSC plot. Increase of total detected cells of and B42 after the treatment could be due to the disintegration of cell aggregates (Figure 3a). In relation to cell aggregation, plate counts and FCM lead to an underestimation of bacterial numbers. Plate counts of L coincided more with SYTO 8 stained fraction rather than total SYTO 8 stained fraction (Q2+Q4). While for and B42, plate counts was in better correlation with Q2+Q4 rather than Q4 only (Figure 3b). Therefore, the former rather than the latter could be an indicator for culturability of these strains under the treatment. This may point out that (part of) injured cells and B42 gated in Q2 could multiply in the plates. Application of sorting technique will determine the percentage of cell recovery on plates of the subpopulation. 8

5 (a) (b) PI stained SYTO8 and PI stained SYTO8 stained plate counts total SYTO8 stained 2 L Ctrl 25%-4C 5% 25% L Ctrl 25%-4C 5% 25% Percentage of cells 2 2 Ctrl 25%-4C 5% 25% 2 2 Ctrl 25%-4C 5% 25% 2 B42 2 B Ctrl 25%-4C 5% 25% Ctrl 25%-4C 5% 25% NaCl concentration (%w/v) Figure 3. Comparison of FCM using SYTO8-PI staining with plate counts under high osmotic pressure treatmenton Lactobacillus acidophilus (L), Lactobacillus paracasei ssp. paracasei () and Bifidobacterium animalis ssp. lactis (B42). The treatment was performed for 2h at 37 o C, except 25%-4C at 4 o C. Graphs show the relative changes of PI ( ), SYTO8 & PI ( ), SYTO8 ( ), total SYTO8 ( ) (Q4+Q2) stained fractions, and plate counts ( ) Effect of freezing DMSO was less efficient than glycerin in the cryoprotective effect for L (Figure 4). When L is frozen in % dimethylsulfoxide and L and B42 in MRD without any cryoprotectants, the loss of culturability was seen primarily in the first 24h and the speed of loss decreased after that. Without the cryoprotectants, response to freezing of the three strains was not the same (fig. 4). For L, the percentage of SYTO 8 stained cells was significantly higher than that of plate counts (p<=.5) after 24h of freezing. The difference is insignificant after 7 days of freezing. For, the total SYTO 8 stained fraction (Q2+Q4) could be used to estimate plate counts after 24h of freezing. After 7 days- freezing, only Q4 can be used for the estimation. This proves that injured cells of this strain after 24h freezing could form colonies on plates but those after 7 days could not. It could be concluded that if the freezing time is long enough (e.g. week in this experiment) SYTO 8 stained fraction from FCM measurement can be used to estimate plate counts of L and. B42 seemed to be the most resistant to freezing-induced injury. Percentages of cells stained with SYTO 8 did not decrease (fig. 4). The difference between SYTO 8 fraction and plate counts was here statistically insignificant due to few replicates. The freezing of this strain should be 82

6 experimented in longer time to know whether SYTO8 stained fraction goes in agreement with the plate counts. SYTO8 stained plate counts total SYTO8 stained L_GLY 9 7 L_DMSO 9 7 L_MRD Percentage of cells _GLY _DMSO 2 _MRD B42_GLY B42_DMSO B42_MRD Freezing time Figure 4. Comparison of FCM using SYTO8-PI staining with plate counts under the effect of freezing on Lactobacillus acidophilus (L), Lactobacillus paracasei ssp. paracasei () and Bifidobacterium animalis ssp. lactis (B42). Graphs show the relative changes of SYTO8 ( ), total SYTO8 ( ) (Q4+Q2) stained fractions and plate counts ( L). Freezing was done in MRD with 3% glycerin (GLY), % dimethylsulfoxide (DMSO) and without the cryoprotectors (MRD) CONCLUSION Advantages of using FCM are the accuracy and speed of analysis. Single cell analysis using FCM gives the information on the heterogeneity of the bacterial populations, which can not be obtained with plate counts. Using a combination of SYTO 8 and PI, three subpopulations namely live, sublethal (injured) and dead cells could be distinguished. From the obtained graphs, FCM lines are generally located above plate counting lines. In the conditions provided for the plate counting method, probably some live cells did not multiply (Joux and Lebaron, 2). Under heat treatment, B42 cells gated in quadrant Q4 can not be called viable. In some cases, there were rather high percentages of sublethal cells (e.g. for ). Determining this subpopulation either or not grows on the plates is important to have accurate results in comparison with the culture method. 83

7 In summary, a combination of these two dyes provides a rapid and reliable method for discriminating and counting live, injured and dead (and total) bacteria. The ability to obtain results fast supports FCM to be applied for real-time monitoring of cell viability that is needed in versatile applications such as in the production of starter cultures and in other process practices. REFERENCES Haugland, P. 26. The Handbook: a Guide to Fluorescent Probes and Labeling Technologies, th ed. Molecular Probes, Inc., Eugene, Oreg. Joux, F., P. Lebaron, 2. Use of fluorescent probes to assess physiological functions of bacteria at singlecell level. Microbes and Infection 2, Mason, C.A., G. Hamer, and J.D. Brycrs, 986. The death and lysis of microorganisms in environmental processes. FEMS Microbiology Reviews 39,