Osmotic Adaptation of Nitella translucens Agardh. Application of Barger’s Method for determining the Osmotic Value to Vacuole Sap

Method. Nitella translucens was cultivated in solutions of various substances, the object of the investigation being to determine the rise in the osmotic value which the cells undergo in these solutions, the cause of this rise, and the factors which influence it. The well-known methods for the determination of the osmotic value of plants are discussed and the reasons are stated for which they were inapplicable for my purpose. The method used by me was that of Barger, originally intended for the determination of the molecular weight of substances; for the principle on which it is based see p. 238. The degree of accuracy which I considered necessary was about 0.01 n (n = mol p. liter). It is possible to carry out a determination with one cell, as for a determination according to the Barger method a minimum of 1.1 cub. mm. of cell-sap is required, and from one cell of average size about 20 cub. mm. of cell-sap can be obtained. The variability of the osmotic value of cells cultivated under the same conditions is usually no greater than 8 %; in view of this there is no use in working with greater accuracy than is possible with the Barger method as applied by me. In order to find the value of the cell-sap at a particular moment, I generally took the mean of the value of from 2 to 6 cells, and sometimes also mixed the liquid from several cells and made a determination with this. All the factors which play a part with the method are discussed in great detail, as are also biological and chemical applications made by others and improvements introduced by others but which were unnecessary for me. Physiological investigations. An annual periodicity of the osmotic value was found to exist; in the summer this is fairly constant, both in 1927, '28, '29, '30, and in 1931 the value was about 0.26 n; in the winter the value is higher, but is not the same every year; in January 1929 0.39 n, in the winter of '29/’30 0.60 n, in the winter of '30/'31 0.33 n. These periodical changes were found to occur independently of temperature and light, and they also occur in sugar solutions. The osmotic concentration of cells cultivated in the dark is little less than those cultivated in the light; low temperatures were found in all the experiments to give a somewhat higher value than high temperatures but to nothing like to such an extent as to account for the periodical increase in the winter. As the cause of the high winter values I therefore am obliged to assume anatonosis due to unknown autonomous factors, moreover as the periodic rise occurred outward and in the laboratory and either in water or in sugar. Nitella was cultivated in saccharose solutions of 0.10 n and 0.20 n; a practically complete osmotic adaptation was found to occur. In glucose solution 0.10 n the value was increased by 0.07 n, in 0.20 solution a complete adaptation came about; the value became 0.46 n. Nitella cannot live in 0.30 n solutions. With winter-cells which had a high value little or no rise was found to take place. It seems, furthermore, that no rise occurs in the dark either in the case of winter-cells or in that of summer-cells. In the dark Nitella cells can be freed from starch, in glucose and saccharose solutions in the dark this is again formed. With the micro-fermentation method of Van Lutsenburg Maas—Van Iterson sugar tests of the vacuole sap were made; for this method the amounts of sugar fermentable must be between 3.5 mgr. and 0.1 mgr., a drop of 0.010 cc. solution sufficing for one test. Cells cultivated in water were found to contain no sugar. My experiments show that the increase in osmotic value of the cells cultivated in 0.10 saccharose and glucose is entirely due to anatonosis; no sugar whatever was to be demonstrated in the cell-sap. About 13 % of the increase which the Nitella cells undergo in 0.20 n saccharose is to be accounted for by sugar, the presence of which could be demonstrated with the above-mentioned method; for the rest it is also an anatonosis. Probably either part of the starch (via sugars) is decomposed into osmotically active substances, or the saccharose (or glucose) is converted into such substances, in the protoplasm, these then permeating through the tonoplast. In 0.20 glucose, when the osmotic concentration of the cell-sap has reached 0.44 n, more than half the rise is to be accounted for by sugar; 10 days later, when the value is 0.47 n, almost the entire rise. In these cells, therefore, anatonosis occurs; as later on, when the osmotic value has reached its maximum, a great deal of sugar can be shown to be present in the cell-sap, it is probable that permeation of the sugar then gets the upper-hand of the anatonosis. It cannot, however, be considered impossible that the starch is then decomposed, which is also an anatonosis. The experiments with urea gave the following result. Nitella was cultivated in solutions of four concentrations; the smaller the concentration of the solution was, the more complete was the osmotic adaptation. By means of micro-chemical reactions it was possible to demonstrate that this increase was due to permeated urea. After having reached a maximum value the value then fell again, until it was about the same as that of the environment, so that the urea evidently had a toxic action. With the object of investigating this more closely, cells were put into water, after having been for a long time in urea of different concentrations; at that moment they were evidently still in a healthy condition, but it was found that the osmotic value in water fell after a longer or shorter period and finally approached zero — the cells died. It is therefore not impossible that, after being in urea, and before being transferred to water, the cells were already no longer healthy. This shows how cautious one must be in assuming the permeability for urea of intact protoplasm. I was able to show that — as was, indeed, to be anticipated — the toxic action of urea on Nitella increases as the concentration of the solution rises. Comparative determinations were further made of fresh green cells, such as were used for the other experiments, with other cells which were darker, coated with Algae and dirt, and which had a much more brittle cellwall; it was found that the course of the changes in osmotic value in urea varied (table 37). Cultivated in glycerol (0.10 n) the osmotic value after 13 days was still equal to the initial value, 0.26 n to 0.27 n. I have concluded from this that the protoplasm of Nitella is scarcely, if at all, permeable for glycerol. In 0.15 and 0.30 n solutions the cells quickly died. The investigations here published were carried out in the Laboratory for Plant Physiology of the State University at Groningen. I wish to tender my hearty thanks to Prof. Dr. W. H. Arisz for all his advice and constructive criticism.