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Cell Viability of Japanese Radish Cylinders Immersed in Hypertonic Solutions
3rd IFAC/CIGR Workshop on Control Applications in post-Harvest and Processing Technology, october 3-5,2001, Tokyo, Japan CELL VIABILITY OF JAPANESE RADISH CYLINDERS IMMERSED IN HYPERTONIC SOLUTIONS
Sboji Koide·, Kazuyuki Sbibuya ., Yosbio Nishiyama·, Matsuo Uemura
b
a Defartment
ofEnvironmental Sciences. lwate University. Morioka 020-8550 Japan Cryohiosystem Research Center, lwate University. Morioka 020-8550 Japan
Abstract: To determine the effect of immersion time and osmolality of solution on cell viability, the triphenyl tetrazolium chloride test was performed using cylindrically-shaped Japanese radish after immersion in several hypertonic solutions (NaCl, KCI, NaNOJ and CaCh solutions at 20 0 q. There was a significant decrease in cell viability with immersion time. The immersion time at which cell viability decreased to 50% (CT so ) was calculated using an empirical equation. It was indicated that increase of osmolality resulted in a decrease of the CT so . Copyright © 20011FAC Keywords: Tissues, Evaluation, Stress, Control, Pressure, Physiology
1. INTRODUCTION Vegetable deteriorates and ultimately decreases its life vitality during postharvest period. The viability of plant has been reported quantitatively by survival and/or cell viability index (Steponkus and Lanphear, 1967; Li and Sakai, 1982; Kartha, et al., 1985; Uemura and Steponkus, 1989) using methods such as electrolyte leakage from cells, plasmolysis, vital staining, mitochondrial enzyme activity (TIC reduction), and photochemical fluorescence. However, very few studies have been reported on cell viability of harvested vegetable. In the present study, observations have been focused on the osmolality when vegetable tissue is subjected to hypertonic solutions. Commonly, the fresh vegetable tissue has been dehydrated and shrunk resulting from water flow from the tissue to the hypertonic solution, and penetration of the hypertonic solution into the tissue sample (Barat et al., 1998; Yao and Le Maguer, 1998). This permeation is mainly caused by mass transfer, which is a very complex phenomenon involving multiple factors. It depends upon the diffusivity of solute, temperature, the space occupied by the individual cells, and the size and geometry of the tissue (Sakai and Miki, 1982; Biswal and Le Maguer, 1998; Yao and Le Maguer, 1998; Rastogi et al., 1999; 2000). Living cell tends to be injured by high osmolality (osmotic pressure), and if cell is dead the plasma membrane loses its semipermeability, resulting in the free exchange between hypertonic solution and cell contents. Thus, whether cells in tissue are alive or dead would change the diffusivity of solute, and simultaneously produce physical and chemical changes (Rastogi et al., 2000).
It is well known that osmotic treatment by the use of the hypertonic solution decreases the vitality of vegetable instead of improving its chemical and textural quality. Therefore, quantitative data between cell viability of vegetable tissue and osmolality when immersed in the hypertonic solution can give important information to food engineers for storage, circulation, and quality keeping of vegetable from a physiological point of view. The objective of the present study is to determine the effect of immersion time and osmolality on the cell viability using tissue samples.
2. MATERIALS & METHODS
2.1 Sample preparation Japanese radish (Raphanus sativus L.) was purchased from a commercial market in Morioka, Japan. Japanese radish was peeled, and then hollowed out in the stem-base section with a cork borer. Each tissue sample was sliced (1.5 mm thick and 17.5 mm diameter), and weighed by an electric balance (A&D HA-180M, Japan). Initial weight of tissue sample used in this measurement was between 0.371 and 0.423 g (average 0.396 g).
2.2 Hypertonic solutions and treatments The hypertonic solutions used are shown in Table I. Concentration and molar concentration of each solution was obtained from the literature (Robert, C. W., 1988). KCI, NaNO J , and CaCh solutions at the osmolality of 0.478, 0.799, 1.638, and 3.529 Oslkg at
- 149--
20°C were equivalent to NaCl solutions with concentrations of 1.5, 2.5, 5.0, and 10.0 % (w/w) at 20 oC, respectively. In this measurement, ethanol and sucrose solutions at the osmolality of 1.638 Os/kg were used as solutions with nonelectrolytes. Furthermore, NaCI solution at the concentration of 5.0 % (w/w), at IOC, 10 °c, and 30 °c were used to test the temperature effect on the cell viability.. The tissue samples were immersed in a hypertonic solution for 0.5, I, 3, 5, 10, 30 and 60 min. The temperature of solution was maintained in a temperature-controlled incubator at constant temperature, as described previously (Koide et al., 2000). The solution was stabilized with a magnetic stirrer. The ratio of the weight of the tissue sample to that of the solution was less than I: 100. After immersion, the surface of the tissue sample was dried immediately with paper towels. The cell viability of the tissue sample was then determined, as described below. More than three tissue samples were used for each TTC test.
2.3 Triphenyl tetrazolium chloride (ITC) test
TIC test was performed according to Steponkus and Lanphear (1967). The absorbance of tissue sample at 530 nm was obtained by a spectrometer (Milton Loy Spectronic 301, USA). In this study, the cell viability was expressed as follows;
C= 00-000 OOF-OD o
(1)
where, C: cell viability (-) OD: value of optical density of the tissue sample per initial weight after immersion (g.1 initial) OD F : value of optical density of the fresh tissue sample per initial weight (g.1 initial) ODD: value of optical density of the inactivated tissue sample per initial weight (g'J initial) In this measurement, OD F was determined with the tissue sample without immersion. The value of ODF was taken on assumption that all cells in tissue sample are alive. On the other hand, the tissue sample after the termination of the process of immersion was placed in a test tube, heated in a boiling water bath for 20 minutes, and used to determine the ODD. The value of ODD was taken on assumption that all cells in the tissue sample are dead. In this study, it was assumed that the total number of cells in the tissue sample when subjected to the hypertonic solution remained constant even if the weight of the tissue sample changed due to shrinkage/swelling during immersion (Koide et al., 2000).
Table I Osmolality. concentration and molar concentration of each hypertonic solution at the temperature of 20°C Osmolality· Concentration Molar concentration (%, w/w) (Os/kg) (mol/L) 0.478 1.5 0.26 0.799 2.5 NaCl 0.44 1.638 5.0 0.89 3.529 10.0 1.83 0.478 1.9 0.26 0.799 3.2 KCI 0.44 1.638 6.5 0.91 3.529 13.2 1.92 0.478 2.25 0.27 0.799 3.8 0.46 NaN~ 1.638 7.9 0.98 3.529 17.0 2.24 0.478 2.0 0.18 0.799 3.3 0.30 CaCh 1.638 6.2 0.59 3.529 10.8 1.06 Ethanol 1.638 7.1 1.52 Sucrose 1.638 32.6 1.09 • Osmolality = (273.15-T)/1.86 (Os/kg), where T is a subfreezing temperature. Solutions having same osmolality (Os/kg) indicate solutions which have same osmotic pressure (Pa). Solute type
- 150-
3. Results & Discussion
that the total amount of solutes increases with immersion time until equilibrium (Biswal and Bozorgmehr, 1991; Biswal and Le Maguer, 1989; Koide et aI., 2000). Since, the increase of the osmotic pressure proceeds from the cell near the interface to the center of the tissue, and the cell begins to be plasmolyzed (Yao and Le Maguer, 1998), it can be said that the cell viability progressively decreases with immersion time. Fig. 3 shows the cell viability with immersion time in various electrolyte solutions (KCI, NaNO) and CaCI 2 solutions). It was found that the decrease in cell viability occurred with an increase in immersion time and the osmolality in all three solutions. These tendencies are similar to the result obtained with NaCl solutions (Fig. 2). In this study, to discuss the above phenomena quantitatively, the immersion time at which cell viability decreased to 50% (CT so) was determined using an empirical equation as follows;
3.1 Cell viability as affected by initial weight o/tissue sample The effect of initial weight of the fresh tissue sample on initial cell viability (ODr. g-I initial) is presented in Fig. I. It is indicated that the cell viability increased slightly with initial weight increase. However, within the limits of the considered range of initial weight (between 0.371 and 0.423 g), there is no significant difference in cell viability. Therefore, the effect of initial weight of the tissue sample on initial cell viability is considered to be negligible in this study.
3.2 Cell viability as affected by immersion time osmolality. solution, and temperature During immersion of the tissue sample in the NaCI solution at 200 C, there was an exponential decrease in cell viability with immersion time (Fig. 2). It was also found that the increase of osmolality of the NaCl solution resulted in a decrease of the cell viability. These results are mainly due to the osmotic pressure caused by diffusion of Na" cr, and nonelectrolyte NaCl of the solution into the tissue sample, indicating
C=(Co-C.}exp(-k.t)+C e
where, Co: cell viability at initial (-) Ce: equilibrium cell viability (-) k: parameter (min- I) t: immersion time (min) Equation (2) is analogous to Newton's law of cooling. The equation could be applied in the case of slow adsorption or desorption (Murata et aI., 1996). The calculated parameters Ce and k, and CTso for each condition are shown in Table 2. It was observed that the increase of osmolality resulted in a decrease of the CT so with electrolyte solutions (NaCl, KCI, NaNO) and CaCh solutions). The CT 50 for ethanol and sucrose solutions were longer than those of the electrolyte solutions. On the other hand, cell viability of tissue samples immersed in the NaCI solution at four temperatures of I, 10,20, and 30 0 C indicated that significant influence could not be seen in temperature in this study (Table 2).
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Fig. 2 Change of cell viability with immersed time. 0: NaCl (l.5%w/w), 6; NaCl (2.5%w/w), D: NaCI(5.00low/w), 0: NaCl (IO.OOlow/w). Each point represents a mean of three trials.
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of cell viability with immersion time in various electrolyte solutions (KCI, NaN03 and solutions) at the temperature of 20°C. 0: 0.478 Os/kg, 6: 0.799 Os/kg, 0: 1.638 Os/kg, ::>: 3.529 Osfkg were equivalent to NaCl solutions with concentrations of 1.5, 2.5, 5.0, and % (w/w) at the temperature of 20 °C, respectively. Each point represents a mean of three trials. rimental details are given in the text.
1
- 152-
Table 2 Parameters in egyation (2) and calculated immersion time at which cell viability decreased to 50% R1 k C. (min'l) (g'l initial) type (-) 0.994 20DC 0.1114 0.2345 NaCI 20DC 0.992 0.1618 0.0952 IDC -* 0.995 0.4793 0.1638 -* 0.4266 0.984 10DC 0.1523 1.638 0.0958 0.989 20DC 0.3491 D -* 0.5798 0.994 30 C 0.1222 3.529 20DC 1.0853 0.994 0.1380 0.478 20DC 0.0902 0.2919 0.998 KCI 0.799 20DC 0.2413 0.2228 0.978 1.638 20DC 0.2484 0.1173 0.997 3.529 20DC 1.3872 0.1302 0.994 0.478 20DC 0.0967 0.2597 0.981 NaN03 0.799 20DC 0.2435 0.1765 0.993 1.638 20DC 0.3601 0.0903 0.990 3.529 0.998 20DC 0.8285 0.0617 0.478 20DC 0.4764 0.1665 0.956 0.799 20DC 0.1225 0.0998 0.991 CaCh D 1.638 0.1351 0.995 20 C 0.3323 3.529 20DC 0.2200 1.8049 0.983 Ethanol 1.638 20DC 0.2081 0.6821 0.961 Sucrose 1.638 20DC 0.0966 0.3294 0.971 CT so: The immersion time at which cell viability decreased to 50% determined parameters by the use of equation (2). ND: CT so is not determined in the period of immersion time (60 min). *: 5.0 % (w/w) NaCl solution. Osmolality ofNaCI solution is 1.638 Os/kg at 20De. Osmolality = (273.15-T)/1.86 (Os/kg), where T is a subfreezing temperature. Solute
Osmolality (Oslkg) 0.478 0.799
temperature
eC)
4. Conclusions The TIC test was performed to determine the cell viability of cylindrically-shaped Japanese radish during immersion in several hypertonic solutions (NaCl, KCI, NaNO), and CaCI2 solutions) at 20°e. There was an exponential decrease in cell viability with immersion time. It was also found that the decrease in cell viability occurred as the osmolality of the solution increased. These phenomena vary with the diffusivity of solute, and the size and geometry of the tissue. However, the influence of immersion time and osmolality on cell viability observed in this measurement would support a physiological understanding of living vegetable such as freshness when subjected to the hypertonic solution. To effectively evaluate the cell viability nondestructively, it is necessary to determine the relationship between the cell viability and some physical properties.
References Atkins, P. W. (1998). The elements of physical chemistry, Oxford University Press, pp. 199-229. Barat, J.M.E., A. Chiralt, and P. Fito (1998). Equilibrium in cellular food osmotic solution systems as related to structure. J. Food Sci., 63(5),
CT so (min)
9.5 5.0 1.9 2.1 2.3 1.5
0.8 13.6 4.3 3.4 0.6 11.6 3.8 2.2 0.9 19.9 8.4 2.6 0.6 14.0 ND from data and
836-840. Biswal, R.N. and K. Bozorgmehr (1991): Equilibrium data for osmotic concentration of potato in NaCI solution, J. Food Process Engineering, 14, 237-245. Biswal, R.N. and M. Le Maguer (1989). Mass Transfer in Plant Materials in Contact with Aqueous Solutions of Ethanol and Sodium Chloride: Equilibrium Data, J. Food Process Engineering, 11, 159-176. Kartha, K.K. (1985). Cryopreservation ofplant cells and organs, CRC Press, pp243-267. Koide, S., Y. Nishiyama, H. Fukuda, and M. Uemura (2000). Change in density of Japanese radish cylinder immersed in NaCl solution, J. Jpn. Food Sci. Technol., 47(6), 439-444. Li, P.H. and A. Sakai (1982). Plant cold hardiness andjreezing stress, Academic Press (U.S.A.). Murata, S., K.S.P. Amaratunga, F. Tanaka, K. Shibuya, S. Koide, and T. Uchino (1996). Simulation of moisture adsorption by polished rice in deep-bed Food Sci. Technol., 1nt., 2(2), 86-91. Rastogi, N.K., A. Angersbach, and D. Knorr (2000). Evaluation of mass transfer mechanisms during osmotic treatment ofplant materials. J. Food Sci., 65(6),1016-1019. Rastogi, N.K., M.N. Eshtiaghi, and D. Knorr (1999).
- 153-
Accelerated mass transfer during osmotic dehydration of high intensity electrical field pulse pretreated carrots. 1. Food Sci., 64(6), 1020-1023. Robert, c.w. (1988), CRC handbook of chemistry and physics, CRC Press, Inc, pp.D220.269. Sakai, M. and M. Moo (1982). Transfer rates of salts in fish flesh, J. Jpn. Food &i. Technol., 29(8), 490-495. Steponkus, P.L. and F.O. Lanphear (1967). Refmement of the triphenyl tetrazolium chloride method of determining cold injury, Plant Physiol., 42(10), 1427-1426. Uemura, M. and P.L. Steponkus (1989). Effect of cold acclimation on the incidence of two forms of freezing injury in protoplasts isolated from rye leaves, Plant Physiol., 91, 1131-1 137. Yao, Y. and M. Le Maguer (1998). Possibility of using pseudo-diffusion approach to model mass transfer in osmotic dehydration, Trans. ASAE, 41(2),409-414.
- 154-
Sboji Koide·, Kazuyuki Sbibuya ., Yosbio Nishiyama·, Matsuo Uemura
b
a Defartment
ofEnvironmental Sciences. lwate University. Morioka 020-8550 Japan Cryohiosystem Research Center, lwate University. Morioka 020-8550 Japan
Abstract: To determine the effect of immersion time and osmolality of solution on cell viability, the triphenyl tetrazolium chloride test was performed using cylindrically-shaped Japanese radish after immersion in several hypertonic solutions (NaCl, KCI, NaNOJ and CaCh solutions at 20 0 q. There was a significant decrease in cell viability with immersion time. The immersion time at which cell viability decreased to 50% (CT so ) was calculated using an empirical equation. It was indicated that increase of osmolality resulted in a decrease of the CT so . Copyright © 20011FAC Keywords: Tissues, Evaluation, Stress, Control, Pressure, Physiology
1. INTRODUCTION Vegetable deteriorates and ultimately decreases its life vitality during postharvest period. The viability of plant has been reported quantitatively by survival and/or cell viability index (Steponkus and Lanphear, 1967; Li and Sakai, 1982; Kartha, et al., 1985; Uemura and Steponkus, 1989) using methods such as electrolyte leakage from cells, plasmolysis, vital staining, mitochondrial enzyme activity (TIC reduction), and photochemical fluorescence. However, very few studies have been reported on cell viability of harvested vegetable. In the present study, observations have been focused on the osmolality when vegetable tissue is subjected to hypertonic solutions. Commonly, the fresh vegetable tissue has been dehydrated and shrunk resulting from water flow from the tissue to the hypertonic solution, and penetration of the hypertonic solution into the tissue sample (Barat et al., 1998; Yao and Le Maguer, 1998). This permeation is mainly caused by mass transfer, which is a very complex phenomenon involving multiple factors. It depends upon the diffusivity of solute, temperature, the space occupied by the individual cells, and the size and geometry of the tissue (Sakai and Miki, 1982; Biswal and Le Maguer, 1998; Yao and Le Maguer, 1998; Rastogi et al., 1999; 2000). Living cell tends to be injured by high osmolality (osmotic pressure), and if cell is dead the plasma membrane loses its semipermeability, resulting in the free exchange between hypertonic solution and cell contents. Thus, whether cells in tissue are alive or dead would change the diffusivity of solute, and simultaneously produce physical and chemical changes (Rastogi et al., 2000).
It is well known that osmotic treatment by the use of the hypertonic solution decreases the vitality of vegetable instead of improving its chemical and textural quality. Therefore, quantitative data between cell viability of vegetable tissue and osmolality when immersed in the hypertonic solution can give important information to food engineers for storage, circulation, and quality keeping of vegetable from a physiological point of view. The objective of the present study is to determine the effect of immersion time and osmolality on the cell viability using tissue samples.
2. MATERIALS & METHODS
2.1 Sample preparation Japanese radish (Raphanus sativus L.) was purchased from a commercial market in Morioka, Japan. Japanese radish was peeled, and then hollowed out in the stem-base section with a cork borer. Each tissue sample was sliced (1.5 mm thick and 17.5 mm diameter), and weighed by an electric balance (A&D HA-180M, Japan). Initial weight of tissue sample used in this measurement was between 0.371 and 0.423 g (average 0.396 g).
2.2 Hypertonic solutions and treatments The hypertonic solutions used are shown in Table I. Concentration and molar concentration of each solution was obtained from the literature (Robert, C. W., 1988). KCI, NaNO J , and CaCh solutions at the osmolality of 0.478, 0.799, 1.638, and 3.529 Oslkg at
- 149--
20°C were equivalent to NaCl solutions with concentrations of 1.5, 2.5, 5.0, and 10.0 % (w/w) at 20 oC, respectively. In this measurement, ethanol and sucrose solutions at the osmolality of 1.638 Os/kg were used as solutions with nonelectrolytes. Furthermore, NaCI solution at the concentration of 5.0 % (w/w), at IOC, 10 °c, and 30 °c were used to test the temperature effect on the cell viability.. The tissue samples were immersed in a hypertonic solution for 0.5, I, 3, 5, 10, 30 and 60 min. The temperature of solution was maintained in a temperature-controlled incubator at constant temperature, as described previously (Koide et al., 2000). The solution was stabilized with a magnetic stirrer. The ratio of the weight of the tissue sample to that of the solution was less than I: 100. After immersion, the surface of the tissue sample was dried immediately with paper towels. The cell viability of the tissue sample was then determined, as described below. More than three tissue samples were used for each TTC test.
2.3 Triphenyl tetrazolium chloride (ITC) test
TIC test was performed according to Steponkus and Lanphear (1967). The absorbance of tissue sample at 530 nm was obtained by a spectrometer (Milton Loy Spectronic 301, USA). In this study, the cell viability was expressed as follows;
C= 00-000 OOF-OD o
(1)
where, C: cell viability (-) OD: value of optical density of the tissue sample per initial weight after immersion (g.1 initial) OD F : value of optical density of the fresh tissue sample per initial weight (g.1 initial) ODD: value of optical density of the inactivated tissue sample per initial weight (g'J initial) In this measurement, OD F was determined with the tissue sample without immersion. The value of ODF was taken on assumption that all cells in tissue sample are alive. On the other hand, the tissue sample after the termination of the process of immersion was placed in a test tube, heated in a boiling water bath for 20 minutes, and used to determine the ODD. The value of ODD was taken on assumption that all cells in the tissue sample are dead. In this study, it was assumed that the total number of cells in the tissue sample when subjected to the hypertonic solution remained constant even if the weight of the tissue sample changed due to shrinkage/swelling during immersion (Koide et al., 2000).
Table I Osmolality. concentration and molar concentration of each hypertonic solution at the temperature of 20°C Osmolality· Concentration Molar concentration (%, w/w) (Os/kg) (mol/L) 0.478 1.5 0.26 0.799 2.5 NaCl 0.44 1.638 5.0 0.89 3.529 10.0 1.83 0.478 1.9 0.26 0.799 3.2 KCI 0.44 1.638 6.5 0.91 3.529 13.2 1.92 0.478 2.25 0.27 0.799 3.8 0.46 NaN~ 1.638 7.9 0.98 3.529 17.0 2.24 0.478 2.0 0.18 0.799 3.3 0.30 CaCh 1.638 6.2 0.59 3.529 10.8 1.06 Ethanol 1.638 7.1 1.52 Sucrose 1.638 32.6 1.09 • Osmolality = (273.15-T)/1.86 (Os/kg), where T is a subfreezing temperature. Solutions having same osmolality (Os/kg) indicate solutions which have same osmotic pressure (Pa). Solute type
- 150-
3. Results & Discussion
that the total amount of solutes increases with immersion time until equilibrium (Biswal and Bozorgmehr, 1991; Biswal and Le Maguer, 1989; Koide et aI., 2000). Since, the increase of the osmotic pressure proceeds from the cell near the interface to the center of the tissue, and the cell begins to be plasmolyzed (Yao and Le Maguer, 1998), it can be said that the cell viability progressively decreases with immersion time. Fig. 3 shows the cell viability with immersion time in various electrolyte solutions (KCI, NaNO) and CaCI 2 solutions). It was found that the decrease in cell viability occurred with an increase in immersion time and the osmolality in all three solutions. These tendencies are similar to the result obtained with NaCl solutions (Fig. 2). In this study, to discuss the above phenomena quantitatively, the immersion time at which cell viability decreased to 50% (CT so) was determined using an empirical equation as follows;
3.1 Cell viability as affected by initial weight o/tissue sample The effect of initial weight of the fresh tissue sample on initial cell viability (ODr. g-I initial) is presented in Fig. I. It is indicated that the cell viability increased slightly with initial weight increase. However, within the limits of the considered range of initial weight (between 0.371 and 0.423 g), there is no significant difference in cell viability. Therefore, the effect of initial weight of the tissue sample on initial cell viability is considered to be negligible in this study.
3.2 Cell viability as affected by immersion time osmolality. solution, and temperature During immersion of the tissue sample in the NaCI solution at 200 C, there was an exponential decrease in cell viability with immersion time (Fig. 2). It was also found that the increase of osmolality of the NaCl solution resulted in a decrease of the cell viability. These results are mainly due to the osmotic pressure caused by diffusion of Na" cr, and nonelectrolyte NaCl of the solution into the tissue sample, indicating
C=(Co-C.}exp(-k.t)+C e
where, Co: cell viability at initial (-) Ce: equilibrium cell viability (-) k: parameter (min- I) t: immersion time (min) Equation (2) is analogous to Newton's law of cooling. The equation could be applied in the case of slow adsorption or desorption (Murata et aI., 1996). The calculated parameters Ce and k, and CTso for each condition are shown in Table 2. It was observed that the increase of osmolality resulted in a decrease of the CT so with electrolyte solutions (NaCl, KCI, NaNO) and CaCh solutions). The CT 50 for ethanol and sucrose solutions were longer than those of the electrolyte solutions. On the other hand, cell viability of tissue samples immersed in the NaCI solution at four temperatures of I, 10,20, and 30 0 C indicated that significant influence could not be seen in temperature in this study (Table 2).
3r-------------,
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o o (X) °n-eP CO 000 ( o ~ O-U 0 0
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o +-----.----.------,---4 0.3 0.35 0.4 0.45 0.5 Initial weight of sample tissue(g) Fig.1 Effect of initial weight of the fresh tissue sample on initial cell viability.
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Fig. 2 Change of cell viability with immersed time. 0: NaCl (l.5%w/w), 6; NaCl (2.5%w/w), D: NaCI(5.00low/w), 0: NaCl (IO.OOlow/w). Each point represents a mean of three trials.
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of cell viability with immersion time in various electrolyte solutions (KCI, NaN03 and solutions) at the temperature of 20°C. 0: 0.478 Os/kg, 6: 0.799 Os/kg, 0: 1.638 Os/kg, ::>: 3.529 Osfkg were equivalent to NaCl solutions with concentrations of 1.5, 2.5, 5.0, and % (w/w) at the temperature of 20 °C, respectively. Each point represents a mean of three trials. rimental details are given in the text.
1
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Table 2 Parameters in egyation (2) and calculated immersion time at which cell viability decreased to 50% R1 k C. (min'l) (g'l initial) type (-) 0.994 20DC 0.1114 0.2345 NaCI 20DC 0.992 0.1618 0.0952 IDC -* 0.995 0.4793 0.1638 -* 0.4266 0.984 10DC 0.1523 1.638 0.0958 0.989 20DC 0.3491 D -* 0.5798 0.994 30 C 0.1222 3.529 20DC 1.0853 0.994 0.1380 0.478 20DC 0.0902 0.2919 0.998 KCI 0.799 20DC 0.2413 0.2228 0.978 1.638 20DC 0.2484 0.1173 0.997 3.529 20DC 1.3872 0.1302 0.994 0.478 20DC 0.0967 0.2597 0.981 NaN03 0.799 20DC 0.2435 0.1765 0.993 1.638 20DC 0.3601 0.0903 0.990 3.529 0.998 20DC 0.8285 0.0617 0.478 20DC 0.4764 0.1665 0.956 0.799 20DC 0.1225 0.0998 0.991 CaCh D 1.638 0.1351 0.995 20 C 0.3323 3.529 20DC 0.2200 1.8049 0.983 Ethanol 1.638 20DC 0.2081 0.6821 0.961 Sucrose 1.638 20DC 0.0966 0.3294 0.971 CT so: The immersion time at which cell viability decreased to 50% determined parameters by the use of equation (2). ND: CT so is not determined in the period of immersion time (60 min). *: 5.0 % (w/w) NaCl solution. Osmolality ofNaCI solution is 1.638 Os/kg at 20De. Osmolality = (273.15-T)/1.86 (Os/kg), where T is a subfreezing temperature. Solute
Osmolality (Oslkg) 0.478 0.799
temperature
eC)
4. Conclusions The TIC test was performed to determine the cell viability of cylindrically-shaped Japanese radish during immersion in several hypertonic solutions (NaCl, KCI, NaNO), and CaCI2 solutions) at 20°e. There was an exponential decrease in cell viability with immersion time. It was also found that the decrease in cell viability occurred as the osmolality of the solution increased. These phenomena vary with the diffusivity of solute, and the size and geometry of the tissue. However, the influence of immersion time and osmolality on cell viability observed in this measurement would support a physiological understanding of living vegetable such as freshness when subjected to the hypertonic solution. To effectively evaluate the cell viability nondestructively, it is necessary to determine the relationship between the cell viability and some physical properties.
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CT so (min)
9.5 5.0 1.9 2.1 2.3 1.5
0.8 13.6 4.3 3.4 0.6 11.6 3.8 2.2 0.9 19.9 8.4 2.6 0.6 14.0 ND from data and
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