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It is widely recognized that fluorescent labeling of microorganisms is an effective means to determine total cell numbers or how many viable cells exist in a sample. Flow cytometry combined with fluorescent staining is a powerful tool to analyze heterogeneous microbial populations (8, 13). Fluorescein diacetate and its derivatives are nonfluorescent molecules that diffuse into cells and are hydrolyzed by intracellular nonspecific esterases to give fluorescent products. The fluorescent products can be accumulated only in those cells that have intact cell membranes; therefore, dead cells with leaky membranes are not stained. Breeuwer et al. reported the precise kinetics of membrane transport and intracellular hydrolysis of fluorescein diacetate and carboxyfluorescein (CF) diacetate (CFDA) as determined in studies aimed to optimize fluorescent staining for detection of yeasts in food materials by flow cytometry (6, 7). However, the fluorescence intensity of labeled cells varies considerably among strains, probably because of differences in intracellular esterase activity. This may cause inaccurate detection of yeast cells in heterogeneous populations by flow cytometry. Recently, I found, by chance, that accumulation of CF or carboxydichlorofluorescein (CDCF) is facilitated by nonlethal levels of hydrostatic pressure. Hydrostatic pressure is a thermodynamic variable that acts to decrease the total volume of a system at equilibrium in the case of liquids and solutions. Although the physicochemical basis of the effect of hydrostatic pressure is well established (3, 9), the pressure-induced phenomena that occur in living microorganisms have not been fully defined.

In this study, the effect of hydrostatic pressure on the fluorescent staining of living yeasts with CFDA and CDCF diacetate (CDCFDA) was analyzed, which may contribute to the efficient detection of microorganisms by flow cytometry.

Cell culture and application of hydrostatic pressure. Cells of S. cerevisiae were grown in YPD (1% yeast extract, 2% Bacto Peptone, 2% D-glucose) broth at 24°C. Cells from a log-phase culture (2 × 10⁷ to 4 × 10⁷/ml) were collected by centrifugation, resuspended in fresh YPD containing 50 mM citric acid (pH 3.0 or 5.0), and then placed in plastic tubes (Cryotubes; Nunc) at 2 × 10⁷ to 3 × 10⁷/ml. After being sealed with Parafilm, the tubes were put into titanium pressure vessels and subjected to hydrostatic pressure. The required hydrostatic pressures were reached in 2 min by using a hand pump (Rigo-sha). To obtain samples, the pressure was released in approximately 15 s. It is known that adiabatic compression from 0.1 to 10 MPa (0.1 MPa = 1 bar = 0.9869 atm = 1.0197 kg of force cm²) raises water temperature by 0.3°C. When hydrostatic pressure was slowly applied to water at 1.7 MPa s¹, the increase in temperature was estimated to be approximately 0.2°C (10). Assuming that the compressibility of YPD and Good's buffers is close to that of water, the increase in temperature was estimated to be less than 1.2°C when a pressure of 60 MPa was applied in 2 min. In a preliminary experiment, adiabatic decompression from 60 to 0.1 MPa reduced the temperature of MB buffer (100 mM morpholinoethanesulfonic acid [MES]-bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane [Bis-Tris], pH 5.0) by approximately 0.5°C. These small changes in temperature are negligible. In fact, no significant difference was observed in either cell viability or accumulation of fluorescent dyes, even when compression and decompression were rapid (data not shown).

Labeling of cells with fluorescent dyes. Cells from a log-phase culture (2 × 10⁷ to 4 × 10⁷/ml) were incubated with 10 µM CFDA (Sigma catalog no. C-5041) in YPD containing 50 mM citric acid (pH 3.0) or with 10 µM CDCFDA (Molecular Probe catalog no. C-369) in YPD containing 50 mM citric acid (pH 5.0) under hydrostatic pressures of up to 60 MPa. To analyze the glucose dependence of pressure-induced dye accumulation, vegetative cells were collected, washed twice in distilled water, and starved for 30 min on ice. The cells were then incubated with CDCFDA in MBA buffer (100 mM MES-Bis-Tris, 100 mM ammonium sulfate, pH 5.0) containing various concentrations of D-glucose under hydrostatic pressures of up to 60 MPa for 1 h. To analyze the effect of 2-deoxyglucose, cells were incubated with CDCFDA in MBA buffer containing 100 mM D-glucose. Unless otherwise specified, the final concentration of the fluorescent dyes was 10 µM. After application of hydrostatic pressure for 1 h, the cells were washed twice with 10 mM MES-Bis-Tris (pH 5.0) and suspended in MB buffer. Fluorescence of the labeled cells emitted at 535 nm with excitation at 485 nm was detected by using a CytoFluor 2350 plate reader (Millipore) or an RF5300PC spectrofluorometer (Shimazu). Fluorescence intensity (arbitrary units [AU]) was recorded as the fluorescence of labeled cells minus the fluorescence of non-labeled cells for 10⁷ cells.

Fluorescence analysis under hydrostatic pressure. Fluorescence emission was examined under several hydrostatic pressures in a hydrostatic chamber with transparent windows which were made of sapphire (10 by 8 mm). Each sample in a transparent cuvette was placed in the chamber, which was set up in an RF5300PC spectrofluorometer. Fluorescence was emitted at 535 nm with excitation at 485 nm. Fluorescence intensity of labeled cells was strong enough to be detected through the sapphire windows, even though the emmision was reduced compared to the analysis without the chamber. Hydrostatic pressure was applied by using a hand pump (Teramecs, Co. Ltd.).

Flow cytometry. Cells were incubated with 10 µM CFDA or CDCFDA under several hydrostatic pressures for 1 h. After decompression, the labeled cells were washed twice with distilled water and resuspended in MB buffer. Cells were analyzed by using the Bryte-HS Flow Cytometry System (Bio-Rad) at atmospheric pressure.

Accumulation of CF and CDCF under elevated hydrostatic pressure. CFDA and CDCFDA are known to be hydrolyzed by intracellular nonspecific esterases, and the fluorescent products CF (pH sensitive) and CDCF (pH insensitive) are accumulated in acidic compartments such as vacuoles (11, 12). CDCFDA is useful for labeling of viable yeast cells because it is more stable than CFDA in less acidic medium (pH ~5.0), and the molar fluorescence intensity of the hydrolysis product CDCF emitted at 530 to 540 nm is greater than that of CF. Since the variability of staining with different strains is known, two strains, sake yeast strain IFO2347 (a strongly labeled strain) and strain IFO10159 (a weakly labeled strain which is less readily detectable by flow cytometry), were used in this study. Application of hydrostatic pressure markedly promoted the accumulation of CF and CDCF in strain IFO2347, which peaked at 40 and 30 MPa, respectively (Fig. 1A). The degree of pressure-induced accumulation of CDCF was greater than that of CF in the cells. Thus, CDCFDA was mainly used for labeling during the following experiments. The total fluorescence intensity of CF-labeled IFO10159 cells at atmospheric pressure (15.5 AU/10⁷ cells) was only 1.6-fold greater than that of nonlabeled cells (9.6 AU/10⁷ cells), and that of CDCF-labeled cells (24.8 AU/10⁷ cells) was 2.5-fold greater than that of nonlabeled cells (9.9 AU/10⁷ cells). A pressure of 50 MPa enhanced the accumulation of dyes 5- to 10-fold (Fig. 1B). No significant difference in the fluorescence of nonlabeled cells was observed at 50 MPa. Although there are no data to explain why the 5- to 10-fold enhancement would have a significant impact on detection limits in natural samples, application of moderate hydrostatic pressure could potentially be a new procedure for detection of living yeast cells.

View larger version (19K): [in this window] [in a new window]   FIG. 1.   Pressure-induced accumulation of CF or CDCF in strains IFO2347 (A) and IFO10159 (B). Cells were labeled with 10 µM CFDA or CDCFDA under several hydrostatic pressures for 1 h, and the labeled cells were analyzed by using CytoFluor 2350. Symbols: , CF-labeled cells; , CDCF-labeled cells.

Flow cytometry analysis. Figure 2 shows the histograms of populations of IFO2347 cells labeled with CDCFDA under several hydrostatic pressures. When the cells were labeled at atmospheric pressure, the peak and mean fluorescences of labeled cells were 23 and 26 AU, respectively (Fig. 2A, 0.1 MPa). When the cells were subjected to a pressure of 30 MPa for 1 h, the accumulation of CDCF increased four- to sixfold. The peak and mean fluorescences were 92 and 162 AU, respectively (Fig. 2A, 30 MPa). However, staining was not enhanced at a pressure of 60 MPa (Fig. 2A, 60 MPa). Similar results were obtained with strain IFO10159 (Fig. 2B). Almost identical results were obtained when the cells were incubated with CFDA (data not shown). These results of flow cytometry analysis are mostly consistent with the results obtained by ordinary fluorescence analysis shown in Fig. 1.

View larger version (19K): [in this window] [in a new window]   FIG. 2.   Histograms of populations of labeled cells. Labeled cells (approximately 100,000) were analyzed by using the Bryte-HS flow cytometry system. (A) Cells of strain IFO2347 subjected to hydrostatic pressures of 0.1, 30, and 60 MPa in the presence of 10 µM CDCFDA for 1 h. (B) Cells of strain IFO10159 subjected to hydrostatic pressures of 0.1, 40, and 60 MPa in the presence of 10 µM CDCFDA for 1 h.

Effects of hydrostatic pressure on hydrolysis of CDCFDA. Fluorescence of strain IFO2347 was measured in a hydrostatic chamber after addition of 50 µM CDCFDA. The fluorescence intensity increased to 300 and 130 AU when hydrostatic pressures of 0.1 and 60 MPa were applied, respectively (Fig. 3A). In addition, fluorescence increased to 770 AU when a pressure of 30 MPa was applied. Cells were preincubated at several hydrostatic pressures for 1 h in YPD (pH 5.0), and the hydrolysis activity was subsequently measured at atmospheric pressure. Figure 3B shows the changes in fluorescence emission after addition of 50 µM CDCFDA to the pressure-adapted cells. The cells adapted to 30 MPa hydrolyzed CDCFDA at a rate approximately three times greater than that of cells adapted to 0.1 or 60 MPa. These results indicate that preincubation of the cells at 30 MPa induced CDCFDA hydrolysis activity, and it was maintained after decompression. Almost identical results were obtained by using CFDA (data not shown).

View larger version (16K): [in this window] [in a new window]   FIG. 3.   Effect of hydrostatic pressure on the hydrolysis of CDCFDA in strain IFO2347. Fluorescence was emitted in a hydrostatic chamber with transparent windows and analyzed by using an RF5300PC spectrofluorometer. (A) Hydrolysis of CDCFDA under elevated hydrostatic pressures. A cuvette containing the cells was placed in the chamber, and hydrostatic pressure was applied at time P after addition of 50 µM CDCFDA. (B) Hydrolysis of CDCFDA at atmospheric pressure after preincubation at several hydrostatic pressures. A cuvette containing pressure-adapted cells was placed in the chamber, and then 50 µM CDCFDA was added.

Dependence on glucose metabolism. Glucose was found to be required for the pressure-induced accumulation of CDCF. The glucose concentration required for half-maximal CDCF accumulation was approximately 13 mM (Fig. 4A). 2-Deoxyglucose significantly inhibited the pressure-induced accumulation of CDCF (Fig. 4B). The concentration at which half-maximal inhibition occurred was approximately 12 mM, very close to the concentration of glucose required for half-maximal CDCF accumulation. Although both values were slightly lower than the K_m of the low-affinity site of the hexose transporter (20 mM) (4, 5) and much greater than the K_m for sugar kinases (1 mM), the results suggest that glucose metabolism or ATP production has a role in the process of CDCF accumulation.

View larger version (18K): [in this window] [in a new window]   FIG. 4.   Effects of D-glucose and 2-deoxyglucose concentrations on the pressure-induced accumulation of CDCF in strain IFO2347. (A) Cells incubated with 10 µM CDCFDA at 0.1 MPa () or 30 MPa () in the presence of various concentrations of D-glucose in MBA buffer for 1 h. (B) Cells incubated with 10 µM CDCFDA at 0.1 () or 30 () MPa in the presence of 100 mM D-glucose and various concentrations of 2-deoxyglucose in MBA buffer for 1 h. Labeled cells were analyzed by using CytoFluor 2350.