STUDIES ON PENETRATION OF DYES WITH GLASS ELECTRODE : IV. PENETRATION OF BRILLIANT CRESYL BLUE INTO NITELLA FLEXILIS

Glass electrode measurements of the pH value of the sap of Nitella show that cresyl blue in form of free base penetrates the vacuoles and raises pH value of the sap to about the same degree as the free base of the dye added to the sap in vitro, while the dye salt dissolved in the sap does not alter its pH value. It is proved conclusively that the increase in the pH value of the sap is due only to the presence of the dye and not to some other alkaline substance. Spectrophotometric measurements show that the dye which penetrates the vacuole is chiefly cresyl blue. When the protoplasm is squeezed into the sap, the pH value of the sap is higher than that of the pure sap. Such a mixture behaves very much like the sap in respect to the dye.     

SPECTROPHOTOMETRIC STUDIES OF PENETRATION : V. RESEMBLANCES BETWEEN THE LIVING CELL AND AN ARTIFICIAL SYSTEM IN ABSORBING METHYLENE BLUE AND TRIMETHYL THIONINE.

The rate of diffusion through the non-aqueous layer of the protoplasm depends largely on the partition coefficients mentioned above. Since these cannot be determined we have employed an artificial system in which chloroform is used in place of the non-aqueous layer of the protoplasm. The partition coefficients may be roughly determined by shaking up the aqueous solutions with chloroform and analyzing with the spectrophotometer (which is necessary with methylene blue because we are dealing with mixtures). This will show what dyes may be expected to pass through the protoplasm into the vacuole in case it behaves like the artificial system. From these results we may conclude that the artificial system and the living cell act almost alike toward methylene blue and azure B, which supports the notion of non-aqueous layers in the protoplasm. There is a close resemblance between Valonia and the artificial system in their behavior toward these dyes at pH 9.5. In the case of Nitella, on the other hand, with methylene blue solution at pH 9.2 the sap in the artificial system takes up relatively more azure B (absorption maximum at 650 mµ) than the vacuole of the living cell (655 mµ). But both take up azure B much more rapidly than methylene blue. A comparison cannot be made between the behavior of the artificial system and that of the living cell at pH 5.5 since in the latter case there arises a question of injury to cells before enough dye is collected in the sap for analysis.     

CERTAIN EFFECTS OF SALTS ON THE PENETRATION OF BRILLIANT CRESYL BLUE INTO NITELLA

The effect of various substances on living cells may be advantageously studied by exposing them to such substances and observing their subsequent behavior in solutions of a basic dye, brilliant cresyl blue. The rate of penetration of the basic dye, brilliant cresyl blue, is decreased when cells are exposed to salts with monovalent cations before they are placed in the dye solution (made up with borate buffer mixture). This inhibiting effect is assumed to be due to the effect of the salts on the protoplasm. This effect is not readily reversible when cells are transferred to distilled water, but it is removed by salts with bivalent or trivalent cations. In some cases it disappears in dye made up with phosphate buffer mixture, or with borate buffer mixture at the pH value in which the borax predominates, and in the case of NaCl it disappears in dye containing NaCl. No inhibiting effect is seen when cells are exposed to NaCl solution containing MgCl₂ before they are placed in the dye solution. The rate of penetration of dye is not decreased when cells are previously exposed to salts with bivalent and trivalent cations. The rate is slightly increased when cells are placed in the dye solution containing a salt with monovalent cation and probably with bivalent or trivalent cations. In the case of the bivalent and trivalent salts the increase is so slight that it may be negligible.     

THE KINETICS OF PENETRATION : XIV. THE PENETRATION OF IODIDE INTO VALONIA

When 0.1 M NaI is added to the sea water surrounding Valonia iodide appears in the sap, presumably entering as NaI, KI, and HI. As the rate of entrance is not affected by changes in the external pH we conclude that the rate of entrance of HI is negligible in comparison with that of NaI, whose concentration is about 10⁷ times that of HI (the entrance of KI may be neglected for reasons stated). This is in marked contrast with the behavior of sulfide which enters chiefly as H₂S. It would seem that permeability to H₂S is enormously greater than to Na₂S. Similar considerations apply to CO₂. In this respect the situation differs greatly from that found with iodide. NaI enters because its activity is greater outside than inside so that no energy need be supplied by the cell. The rate of entrance (i.e. the amount of iodide entering the sap in a given time) is proportional to the external concentration of iodide, or to the external product [N⁺]_o [I^-l_o, after a certain external concentration of iodide has been reached. At lower concentrations the rate is relatively rapid. The reasons for this are discussed. The rate of passage of NaI through protoplasm is about a million times slower than through water. As the protoplasm is mostly water we may suppose that the delay is due chiefly to the non-aqueous protoplasmic surface layers. It would seem that these must be more than one molecule thick to bring this about. There is no great difference between the rate of entrance in the dark and in the light.     

THE PENETRATION OF BASIC DYE INTO NITELLA AND VALONIA IN THE PRESENCE OF CERTAIN ACIDS,BUFFER MIXTURES,AND SALTS

When living cells of Nitella are exposed to an acetate buffer solution until the pH value of the sap is decreased and subsequently placed in a solution of brilliant cresyl blue, the rate of penetration of dye into the vacuole is found to decrease in the majority of cases, and increase in other cases, as compared with the control cells which are transferred to the dye solution directly from tap water. This decrease in the rate is not due to the lowering of the pH value of the solution just outside the cell wall, as a result of diffusion of acetic acid from the cell when cells are removed from the buffer solution and placed in the dye solution, because the relative amount of decrease (as compared with the control) is the same whether the external solution is stirred or not. Such a decrease in the rate may be brought about without a change in the pH value of the sap if the cells are placed in the dye solution after exposure to a phosphate buffer solution in which the pH value of the sap remains normal. The rate of penetration of dye is then found to decrease. The extent of this decrease is the greater the lower the pH value of the solution. It is found that hydrochloric acid and boric acid have no effect while phosphoric acid has an inhibiting effect at pH 4.8 on stirring. Experiments with neutral salt solutions indicate that a direct effect on the cell (decreasing penetration) is due to monovalent base cations, while there is no such effect directly on the dye. It is assumed that the effect of the phosphate and acetate buffer solutions on the cell, decreasing the rate of penetration, is due (1) to the penetration of these acids into the protoplasm as undissociated molecules, which dissociate upon entrance and lower the pH value of the protoplasm or to their action on the surface of the protoplasm, (2) to the effect of base cations on the protoplasm (either at the surface or in the interior), and (3) possibly to the effect of certain anions. In this case the action of the buffer solution is not due to its hydrogen ions. In the case of living cells of Valonia under the same experimental conditions as Nitella it is found that the rate of penetration of dye decreases when the pH value of the sap increases in presence of NH₃, and also when the pH value of the sap is decreased in the presence of acetic acid. Such a decrease may be brought about even when the cells are previously exposed to sea water containing HCl, in which the pH value of the sap remains normal.     

ON THE NATURE OF THE DYE PENETRATING THE VACUOLE OF VALONIA FROM SOLUTIONS OF METHYLENE BLUE

When uninjured cells of Valonia are placed in methylene blue dissolved in sea water it is found, after 1 to 3 hours, that at pH 5.5 practically no dye penetrates, while at pH 9.5 more enters the vacuole. As the cells become injured more dye enters at pH 5.5, as well as at pH 9.5. No dye in reduced form is found in the sap of uninjured cells exposed from 1 to 3 hours to methylene blue in sea water at both pH values. When uninjured cells are placed in azure B solution, the rate of penetration of dye into the vacuole is found to increase with the rise in the pH value of the external dye solution. The partition coefficient of the dye between chloroform and sea water is higher at pH 9.5 than at pH 5.5 with both methylene blue and azure B. The color of the dye in chloroform absorbed from methylene blue or from azure B in sea water at pH 5.5 is blue, while it is reddish purple when absorbed from methylene blue and azure B at pH 9.5. Dry salt of methylene blue and azure B dissolved in chloroform appears blue. It is shown that chiefly azure B in form of free base is absorbed by chloroform from methylene blue or azure B dissolved in sea water at pH 9.5, but possibly a mixture of methylene blue and azure B in form of salt is absorbed from methylene blue at pH 5.5, and azure B in form of salt is absorbed from azure B in sea water at pH 5.5. Spectrophotometric analysis of the dye shows the following facts. 1. The dye which is absorbed by the cell wall from methylene blue solution is found to be chiefly methylene blue. 2. The dye which has penetrated from methylene blue solution into the vacuole of uninjured cells is found to be azure B or trimethyl thionine, a small amount of which may be present in a solution of methylene blue especially at a high pH value. 3. The dye which has penetrated from methylene blue solution into the vacuole of injured cells is either methylene blue or a mixture of methylene blue and azure B. 4. The dye which is absorbed by chloroform from methylene blue dissolved in sea water is also found to be azure B, when the pH value of the sea water is at 9.5, but it consists of azure B and to a less extent of methylene blue when the pH value is at 5.5. 5. Methylene blue employed for these experiments, when dissolved in sea water, in sap of Valonia, or in artificial sap, gives absorption maxima characteristic of methylene blue. Azure B found in the sap collected from the vacuole cannot be due to the transformation of methylene blue into this dye after methylene blue has penetrated into the vacuole from the external solution because no such transformation detectable by this method is found to take place within 3 hours after dissolving methylene blue in the sap of Valonia. These experiments indicate that the penetration of dye into the vacuole from methylene blue solution represents a diffusion of azure B in the form of free base. This result agrees with the theory that a basic dye penetrates the vacuole of living cells chiefly in the form of free base and only very slightly in the form of salt. But as soon as the cells are injured the methylene blue (in form of salt) enters the vacuole. It is suggested that these experiments do not show that methylene blue does not enter the protoplasm, but they point out the danger of basing any theoretical conclusion as to permeability on oxidation-reduction potential of living cells from experiments made or the penetration of dye from methylene blue solution into the vacuole, without determining the nature of the dye inside and outside the cell.     

SEPARATION OF POTASSIUM ISOTOPES IN VALONIA AND NITELLA

The ratio of K³⁹ ÷ K⁴¹ appears to be lower in the sap of Valonia and Nitella than in the environment, indicating that the living cell can separate these isotopes to some extent. Experiments with a mixture of guaiacol and p-cresol suggest that a similar separation may occur here but further experiments are needed.     

USE OF BLUE LAKE AS AN INDICATOR OF BACTERIAL PENETRATION INTO EGGS

Blue Lake, an insoluble dye, was evaluated as an indicator of potential bacterial penetration into eggs. Various groups of eggs (fresh-laid; commercial; water-washed) were dipped in 0.25% (w/v) Blue Lake in 0.1% Triton X-100 solution for 2 min and incubated at room temperature up to 1 ± 24 h. Penetration was detected by counting the blue dots on shell membranes after breaking the eggs. Commercial eggs allowed the easiest penetration. All commercial eggs showed blue dots even at 2 min incubation and the average count reached 111/egg at 1 h. Water-washed eggs allowed much less penetration than commercial eggs and the counts of blue dots on those eggs were 10/egg at 2 min and 22 at 1 h. Fresh-laid eggs did not allow any penetration up to 24 h. Above results corresponded very well with the penetration study with Salmonella enteritidis and also with the morphological study of eggshell surfaces using electron microscopy where fresh-laid eggs had intact cuticle layers, but commercial eggs did not. Thus, Blue Lake dye might be used as a rapid indicator of bacterial penetration through eggshells.     

STUDIES ON PENETRATION OF DYES WITH GLASS ELECTRODE : V. WHY DOES AZURE B PENETRATE MORE READILY THAN METHYLENE BLUE OR CRYSTAL VIOLET?

Glass electrode measurements of the pH value of the sap of cells of Nitella show that azure B in the form of free base penetrates the vacuoles and raises the pH value of the sap to about the same degree as the free base of the dye added to the sap in vitro, but the dye salt dissolved in the sap does not alter the pH value of the sap. It is concluded that the dye penetrates the vacuoles chiefly in the form of free base and not as salt. The dye from methylene blue solution containing azure B free base as impurity penetrates and accumulates in the vacuole. This dye must be azure B in the form of free base, since it raises the pH value of the sap to about the same extent as the free base of azure B dissolved in the sap in vitro. The dye absorbed by the chloroform from methylene blue solution behaves like the dye penetrating the vacuole. These results confirm those of spectrophotometric analysis previously published. Crystal violet exists only in one form between pH 5 and pH 9.2, and does not alter the pH value of the sap at the concentrations used. It does not penetrate readily unless cells are injured. A theory of "multiple partition coefficients" is described which explains the mechanism of the behavior of living cells to these dyes. When the protoplasm is squeezed into the sap, the pH value of the mixture is higher than that of the pure sap. The behavior of such a mixture to the dye is very much like that of the sap except that with azure B and methylene blue the rise in the pH value of such a mixture is not so pronounced as with sap when the dye penetrates into the vacuoles. Spectrophotometric measurements show that the dye which penetrates from methylene blue solution has a primary absorption maximum at 653 to 655 mµ (i.e., is a mixture of azure B and methylene blue, with preponderance of azure B) whether we take the sap alone or the sap plus protoplasm. These results confirm those previously obtained with spectrophotometric measurements.     

THE EFFECT OF ACETATE BUFFER MIXTURES,ACETIC ACID,AND SODIUM ACETATE,ON THE PROTOPLASM,AS INFLUENCING THE RATE OF PENETRATION OF CRESYL BLUE INTO THE VACUOLE OF NITELLA

When living cells of Nitella are exposed to a solution of sodium acetate and are then placed in a solution of brilliant cresyl blue made up with a borate buffer mixture at pH 7.85, a decrease in the rate of penetration of dye is found, without any change in the pH value of the sap. It is assumed that this inhibiting effect is caused by the action of sodium on the protoplasm. This effect is not manifest if the dye solution is made up with phosphate buffer mixture at pH 7.85. It is assumed that this is due to the presence of a greater concentration of base cations in the phosphate buffer mixture. In the case of cells previously exposed to solutions of acetic acid the rate of penetration of dye decreases with the lowering of the pH value of the sap. This inhibiting effect is assumed to be due chiefly to the action of acetic acid on the protoplasm, provided the pH value of the external acetic acid is not so low as to involve an inhibiting effect on the protoplasm by hydrogen ions as well. It is assumed that the acetic acid either has a specific effect on the protoplasm or enters as undissociated molecules and by subsequent dissociation lowers the pH value of the protoplasm. With acetate buffer mixture the inhibiting effect is due to the action of sodium and acetic acid on the protoplasm. The inhibiting effect of acetic acid and acetate buffer mixture is manifested whether the dye solution is made up with borate or phosphate buffer mixture at pH 7.85. It is assumed that acetic acid in the vacuole serves as a reservoir so that during the experiment the inhibiting effect still persists.     

CORRELATION OF OXIDATION AND PHOSPHORYLATION IN HEMOLYZED BLOOD IN PRESENCE OF METHYLENE BLUE AND PYOCYANINE

1. The system:hemolyzed blood + glucose never exhibits glycolysis or, in the air, oxidation of glucose. When glucose is replaced by hexosephosphate ester, addition of methylene blue causes oxidation in air. 2. When cozymase is added also, the oxidation is increased, and a synthesis of hexosephosphate esters takes place. 3. When pyocyanine is used instead of methylene blue, the rate of oxidation is the same as with methylene blue, but a synthesis of phosphate esters takes place without addition of cozymase. 4. There is never a phosphate ester synthesis without oxidation going on, but oxidation does not necessarily go hand in hand with phosphate synthesis. 5. In order to couple the oxidation process with phosphate synthesis, two methods are available: either to start oxidation by methylene blue and to add coenzyme from yeast cells; or to start oxidation by pyocyanine, in which case coenzyme is unnecessary, though it improves the effect. 6. Iodoacetate always suppresses synthesis, but only under certain conditions decreases oxidation. Cyanide has no effect upon either process.     

THE KINETICS OF PENETRATION : XIX. ENTRANCE OF ELECTROLYTES AND OF WATER INTO IMPALED HALICYSTIS

When cells of Halicystis are impaled on a capillary so that space is provided into which the sap can migrate, the rate of entrance of water and of electrolyte is increased about 10-fold. In impaled Valonia cells the rate is increased about 15-fold. After a relatively rapid non-linear rate of increase of sap volume immediately after impalement (which may possibly represent the partial dissipation of the difference of the osmotic energy between intact and impaled cells) the volume increases at a linear rate, apparently indefinitely. Since the halide concentration of the sap at the end of the experiment is (within the limits of natural variation) the same as in the intact cell, we conclude that electrolyte also enters the sap about 10 times as fast as in the intact cell. As in the case of Valonia we conclude that there is a mechanism whereby in the intact cell the osmotic concentration of the sap is prevented from greatly exceeding that of the sea water. This may be associated with the state of hydration of the non-aqueous protoplasmic surfaces.     

CHEMICAL RESTORATION IN NITELLA : IV. EFFECTS OF GUANIDINE

Leaching in distilled water may remove irritability and the potassium effect in Nitella but both of these may be restored by appropriate treatment with guanidine.     

THE ACCUMULATION OF ELECTROLYTES : I. THE ENTRANCE OF AMMONIA INTO VALONIA MACROPHYSA

When 0.005 M NH₄Cl is added to sea water containing cells of Valonia macrophysa ammonia soon appears in the sap and may reach a concentration inside over 40 times as great as outside. It appears to enter as undissociated NH₃ (or NH₄OH) and tends to reach a pseudoequilibrium in which the activity of undissociated NH₃ (or NH₄OH) is the same inside and outside. When ammonia first enters, the pH value of the sap rapidly rises but it soon reaches a maximum and subsequently falls off. At the same time there is an increase of halide in the sap which, however, does not run a parallel course to the ammonia accumulation, but it comes to a new equilibrium value and remains constant. The increase in NH₃ in the sap is accompanied by a decrease in the concentration of K. As NH₃ enters the specific gravity of the sap decreases and the cells rise to the surface and continue to grow as floating organisms. The growth of the cells is increased.     

CYTOTAXONOMY OF THE CHARACEAE: KARYOTYPE ANALYSIS OF NITELLA OPACA AND NITELLA FLEXILIS

The haploid chromosome complement of Nitella flexilis (n = 12) is composed of two quite different basic karyotypes. One of these is symmetrical and appears to be identical to the karyotype of N. opaca (n = 6), and the other is quite asymmetrical and seems to be identical to that of an apparently undescribed dioecious Nitella from Kansas (n = 6). This may indicate that the monoecious N. flexilis has arisen through hybridization between two dioecious species. Although heteromorphic sex chromosomes were not observed in either species, it appears that female and male potentialities are confined to separate basic chromosome complements and mechanisms determining the monoecious and dioecious states are in close relation to ploidy.