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.     

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.     

STUDIES OF THE INNER AND OUTER PROTOPLASMIC SURFACES OF LARGE PLANT CELLS : II. MECHANICAL PROPERTIES OF THE VACUOLAR SURFACE

The vacuolar surface of Nitella is covered with a non-aqueous film too thin to be visible as a separate membrane. The motion of the protoplasm may subject this film to a good deal of mechanical disturbance. Apparently this does not rupture the film for no dye escapes into the protoplasm as the result of such disturbance when the vacuolar sap is deeply stained with neutral red or brilliant cresyl blue. When the deeply stained central vacuole breaks up into several smaller vacuoles, leaving the outer protoplasmic surface in its normal position, there is no evidence of the escape of dye into the protoplasm through the film surrounding the vacuole.     

SPECTROPHOTOMETRIC STUDIES OF PENETRATION : IV. PENETRATION OF TRIMETHYL THIONIN INTO NITELLA AND VALONIA FROM METHYLENE BLUE.

Spectrophotometric measurements show that it is chiefly the trimethyl thionin that is present in the sap extracted from the vacuoles of uninjured cells of Nitella or Valonia which have been placed in methylene blue solution at a little above pH 9. Whether these measurements were made immediately or several hours later the same results were obtained. Methylene blue is detected in the sap (1) when the cells are injured or (2) when the contamination of the sap from the stained cell wall occurs at the time of extraction. The sap is found to be incapable of demethylating methylene blue dissolved in it even on standing for several hours. It is somewhat uncertain as to whether the trimethyl thionin penetrated as such from the external methylene blue solution which generally contains this dye as impurity (in too small concentration for detection by spectrophotometer but detectable by extraction with chloroform), or whether it has formed from methylene blue in the protoplasm. The evidences described in the text tend to favor the former explanation. Theory is discussed on basis of more rapid penetration of trimethyl thionin (in form of free base) than of methylene blue, or of trimethyl thionin in form of salt.     

The purification of methylene blue and azure B by solvent extraction and crystallization.

Detailed schemes are described for the preparation of purified methylene blue and azure B from commercial samples of methylene blue. Purified methylene blue is obtained by extracting a solution of the commercial product in an aqueous buffer (pH 9.5) with carbon tetrachloride. Methylene blue remains in the aqueous layer but contaminating dyes pass into the carbon tetrachloride. Metal salt contaminants are removed when the dye is crystallized by the addition of hydrochloric acid at a final concentration of 0.25 N. Purified azure B is obtained by extracting a solution of commercial methylene blue in dilute aqueous sodium hydroxide (pH 11-11.5) with carbon tetrachloride. In this pH range, methylene blue is unstable and yields azure B. The latter passes into the carbon tetrachloride layer as it is formed. Metal salt contaminants remain in the aqueous layer. A concentrated solution oa azure B is obtained by extracting the carbon tetrachloride layer with 4.5 X 10(-4)N hydrobromic acid. The dye is then crystallized by increasing the hydrobromic acid concentration to 0.23 N. Thin-layer chromatography of the purified dyes shows that contamination with related thiazine dyes is absent or negligible. Ash analyses reveal that metal salt contamination is also negligible (sulphated ash less than 0.2%).     

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.     

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.     

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.     

PROTOPLASMIC POTENTIALS IN HALICYSTIS : IV. VACUOLAR PERFUSION WITH ARTIFICIAL SAP AND SEA WATER

Perfusion of the vacuole of living cells of Halicystis is described, the method employing two longitudinally fused capillaries as entrance and exit tubes. Natural sap, artificial sap, and sea water have been successfully perfused, with various additions and deficiencies, within the limits of physiological balance. In H. ovalis the P.D. remains positive and scarcely reduced in value when normal sea water, at pH 8.1, is perfused in the vacuole. In H. Osterhoutii the P.D. reverses in sign when the perfused solution has a higher pH than 6.5. In both cases a large P.D. persists when the solutions are the same on both sides of the protoplasm. In the absence of external gradients, there must be some internal gradient or asymmetry of the protoplasm itself to account for the P.D. Since appreciable currents are produced, there must be some metabolic activity as a source of energy. The higher normal P.D. in H. ovalis is not due to the higher KCl content of its sap (as earlier suggested by the author) since it persists nearly unchanged when sea water is substituted for sap.     

THE KINETICS OF PENETRATION : IX. MODELS OF MATURE CELLS

To imitate cells which have ceased to grow we have made models in which artificial sap is separated from the external solution by a non-aqueous layer (representing the protoplasm). A stream of CO₂ is bubbled through the artificial sap to imitate its production by the living cell. Potassium passes from the external solution through the non-aqueous layer into the artificial sap and there reacts with CO₂ to form KHCO₃: its rate of entrance depends on the supply of CO₂. Hence the increase of volume depends on the supply of CO₂ (as is probably true of the living cell). By regulating the supply of CO₂ and the osmotic pressure we are able to keep the volume and composition of the artificial sap approximately constant while maintaining a higher concentration of potassium than in the external solution. In these respects the model resembles certain mature cells which have ceased to grow.     

Zinc Chloride Methylene Blue. I. Biological Stain History, Physical Characteristics and Approximation of Azure B Content of Commercial Samples

Zinc chloride methylene blue appeared on the market almost contemporaneously with the zinc-free medicinal form. The former has rarely been reported as being used in blood stains. Recent suspension of manufacture of medicinal methylene blue by it. principal American producer has excited interest in the use of the zinc chloride form for the preparation of blood stains. According to Lillie (1944a,b) the azure B content of zinc chloride methylene blue may have varied from 5 to 30% in the samples studied. Taking the Merck Index (1968, 1976) figures for the spectroscopic absorption maximum (λmax) of 667.8 and 668 nm as standard, recent samples of zinc chloride methylene blue are calculated to contain 6-8% azure B. These figures are baaed on 1) the shift of λmax after exhaustive pH 9.5 chloroform extraction, 2) evaluation of the actual ratio of the observed TiCl₂ dye content to the theoretical for pure zinc chloride methylene blue, 3) comparison of spectroscopic and staining effects of graded hot dichromate oxidation products with those of highly purified azure B-methylene blue mixtures of known proportions.

As far as can be found, medicinal methylene blue is almost the exclusive source of cosin polychrome methylene blue blood stains. Lillie (1944c) included a short series comparing 5 zinc chloride methylene blues with a dozen medicinal methylene blue samples; all were oxidized with hot dichromate to produce successful Wright stains. No effort was made to remove the zinc Exhaustive pH 9.5 chloroform extraction of zinc chloride methylene blue (lot MCB 12-H-29) yielded a small amount of red dye which when extracted into 0.1 N HCI gave λmax = 650. The extraction moved the absorption peak of the zinc chloride methylene blue from 667 to 668 nm and the midpoint of the 90% maximum absorption band, 18 nm wide, from 666.5 to 667.5 nm.     

THE KINETICS OF PENETRATION : VIII. TEMPORARY ACCUMULATION

A model is described which throws light on the mechanism of accumulation. In the model used an external aqueous phase A is separated by a non-aqueous phase B (representing the protoplasm) from the artificial sap in C. A contains KOH and C contains HCl: they tend to mix by passing through the non-aqueous layer but much more KOH moves so that most of the KCl is formed in C, where the concentration of potassium becomes much greater than in A. This accumulation is only temporary for as the system approaches equilibrium the composition of A approaches identity with that of C, since all the substances present can pass through the non-aqueous layer. Such an approach to equilibrium may be compared to the death of the cell as the result of which accumulation disappears. During the earlier stages of the experiment potassium tends to go in as KOH and at the same time to go out as KCl. These opposing tendencies do not balance until the concentration of potassium inside becomes much greater than outside (hence potassium accumulates). The reason is that KCl, although its driving force be great, moves very slowly in B because its partition coefficient is low and in consequence its concentration gradient in B is small. This illustrates the importance of partition coefficients for penetration in models and in living cells. It also indicates that accumulation depends on the fact that permeability is greater for the ingoing compound of the accumulating substance than for the outgoing compound. Other things being equal, accumulation is increased by maintaining a low pH in C. Hence we may infer that anything which checks the production of acid in the living cell may be expected to check accumulation and growth. This model recalls the situation in Valonia and in most living cells where potassium accumulates as KCl, perhaps because it enters as KOH and forms KA in the sap (where A is an organic anion). In some plants potassium accumulates as KA but when HCl exists in the external solution it will tend to enter and displace the weaker acid HA (if this be carbonic it can readily escape): hence potassium may accumulate to a greater or less extent as KCl. Injury of the cell may produce a twofold effect, (1) increase of permeability, (2) lessened accumulation. The total amount of electrolyte taken up in a given time will be influenced by these factors and may be greater than normal in the injured cell or less, depending somewhat on the length of the interval of time chosen.     

EXIT OF DYE FROM LIVING CELLS OF NITELLA AT DIFFERENT pH VALUES

Experiments on the exit of brilliant cresyl blue from the living cells of Nitella, in solutions of varying external pH values containing no dye, confirm the theory that the relation of the dye in the sap to that in the external solution depends on the fact that the dye exists in two forms, one of which (DB) can pass through the protoplasm while the other (DS) passes only slightly. DB increases (by transformation of DS to DB) with an increase in the pH value, and is soluble in substances like chloroform and benzene. DS increases with decrease in pH value and is insoluble (or nearly so) in chloroform and benzene. The rate of exit of the dye increases as the external pH value decreases. This may be explained on the ground that DB as it comes out of the cell is partly changed to DS, the amount transformed increasing as the pH value decreases. The rate of exit of the dye is increased when the pH value of the sap is increased by penetration of NH₃.     

THE KINETICS OF PENETRATION : VI. SOME FACTORS AFFECTING PENETRATION

Some of the factors affecting penetration in living cells may be advantageously studied in models in which the organic salts KG and NaG diffuse from an aqueous solution A, through a non-aqueous layer B (representing the protoplasmic surface) into an aqueous solution C (representing the sap and hence called artificial sap) where they react with CO₂ to form KHCO₃ and NaHCO₃. Their relative proportions in C depend chiefly on the partition coefficients and on the diffusion constants in the non-aqueous layer. But the ratio is also affected by other variables, among which are the following: 1. Temperature, affecting diffusion constants and partition coefficients and altering the thickness of the unstirred layers by changing viscosity. 2. Viscosity (especially in the non-aqueous layers) which depends on temperature and the presence of solutes. 3. Rate of stirring, which affects the thickness of the unstirred layers and the transport of electrolyte in those that are stirred. 4. Shape and surface area of the non-aqueous layer. 5. Surface forces. 6. Reactions occurring at the outer surface such as loss of water by the electrolyte or its molecular association in the non-aqueous phase. The reverse processes will occur at the inner surface and here also combinations with acids or other substances in the "artificial sap" may occur. 7. Outward diffusion from the artificial sap. The outward movement of KHCO₃ and NaHCO₃ is small compared with the inward movement of KG and NaG when the concentrations are equal. This is because the partition coefficients³ of the bicarbonates are very low as compared with those of NaG and KG. Since CO₂ and HCO₃ ^- diffuse into A and combine with KG and NaG the inward movement of potassium and sodium falls off in proportion as the concentration of KG and NaG is lessened. 8. Movement of water into the non-aqueous phase and into the artificial sap. This may have a higher temperature coefficient than the penetration of electrolytes. 9. Variation of the partition coefficients with concentration and pH. Many of these variables may occur in living cells. (It happens that the range of variation in the ratio of potassium to sodium in the models resembles that found in Valonia.)     

Microspectrophotometric Analysis of Accumulation of the Fluorones K-Fluorescein, Rose Bengal and Phloxine Red in Living Plant Cells

Spectrophotometry investigations of dye solutions in different media and of living stained cells from the upper epidermis of the scaleleaf of Allium cepa were carried out with the dyes K-fluorescein, rose Bengal and phloxine red to elucidate the mechanism of the accumulation of these dyes in the cytoplasm, the nucleus and the cell sap. Thin layer chromatography and paper electrophoresis indicate that the K-fluorescein used here contains no detectable contaminants. Besides the main component, rose Bengal contains two components in small quantities with Rf values of 0.64 and 0.57, plus three more components in traces. Besides the two main components (Rf values of 0.83 and 0.73), phloxine red also contains five more components in traces. Electrophoretic investigations reveal that in aqueous solution the fluorones rose Bengal and phloxine red from pH 2.0-11 show a migration toward the anode. K-fluorescein from pH 2.9-10.4 shows a migration toward the anode, but at pH 1.9 a migration toward the cathode. By shaking aqueous solutions of K-fluorescein, rose Bengal and phloxine red at different pH values with different organic solvents, the above used stainings show different spectral absorption curves according to the polarity of the solvent. The position of the absorption maxima and the shape of the absorption curves of these three anionic dyes lead to the conclusion that the staining of the living cytoplasm and nucleus is due to ion accumulation by means of the “ion trap mechanism” within the aqueous phase of the cytoplasm (cytosol) and the nucleus. Adsorption of dye particles in the protein phase of the cytoplasm cannot be excluded. There seems to be a fundamental difference in the vital staining of the protoplasm by anionic and cationic dyes, the latter apparently accumulating as neutral dye molecules in the lipid phase of the protoplasm. The concentration of the dyes used in the living cytoplasm (cytosol) is approximately 0.2-0.05%. During natural and artificial displacement of K-fluorescein from the cytoplasm to the vacuole, it appears that accumulation of the dye within the vacuole is performed through an ion trap mechanism in the form of bivalent ions. Along with natural displacement, it is possible that ion accumulation also occurs in metabolic products.     

Metal contaminants in commercial thiazine dyes.

The iron, potassium, sodium and zinc content of commercial samples of the thiazine dyes azure A (C.I. 52005), azure B (C.I. 52010), azure C (C.I. 52002), methylene blue (C.I. 52015), new methylene blue (C.I. 52030), polychrome methylene blue, thionine (C.I. 52000) and toluidine blue (C.I. 52040) have been determined by atomic absorption spectrophotometry. The metal concentration varied widely in the 38 samples examined--iron, potassium, sodium and zinc together comprised between 0.02% and 25.35% of individual samples.     

Topo-optical reactions on the membrane of the human red cell ghost (author's transl)]

Previous studies have proved that the thiazin dyes toluidine blue, azure A, azure B, 1.9-dimethyl methylene blue and the quinolin dyes N,N'-diethylpseudoisocyanine chloride, N,N'-6,6'-dichlorpseudoisocyanine chloride are suitable for topo-optical reaction on the membrane of the red blood cells. In the present study the applicability of the thiazin and quinolin dyes on the membrane of the human red cell ghost was examined. Optical analysis revealed that the thiazin dyes are bound in radial position to the membrane, while the quinolin dyes are bound parallel to the membrane's plane.     

The azure dyes: their purification and physicochemical properties. II. Purification of azure B.

A method is described for the purification of the dye azure B in quantities sufficient for biological staining experiments on a larger scale. The method is based on the use of column chromatography. Two columns are employed. In column A with silica gel as adsorbent the azure B fraction is isolated from a suitable substrate ('technical' azure B gained by a modification of Bernthsen's synthesis of methylene blue, or polychrome methylene blue) using an acetate-formate mixture as eluent. In column B, on an Amberlite polymeric adsorbent (XAD-2) the acetate-formate anions are exchanged in chloride. Regeneration of both columns is possible: KMnO4, Na2S2O4 and water are run through column A; 5% NaOH, methanol and water through column B. Purification of azure B on economic terms is thus attained. The opinion is expressed that this method is also applicable to the purification of other cationic dyes.     

The role of water in protoplasmic permeability and in antagonism

The behavior of the cell depends to a large extent on the permeability of the outer non-aqueous surface layer of the protoplasm. This layer is immiscible with water but may be quite permeable to it. It seems possible that a reversible increase or decrease in permeability may be due to a corresponding increase or decrease in the water content of the non-aqueous surface layer. Irreversible increase in permeability need not be due primarily to increase in the water content of the surface layer but may be caused chiefly by changes in the protoplasm on which the surface layer rests. It may include desiccation, precipitation, and other alterations. An artificial cell is described in which the outer protoplasmic surface layer is represented by a layer of guaiacol on one side of which is a solution of KOH + KCl representing the external medium and on the other side is a solution of CO₂ representing the protoplasm. The K⁺ unites with guaiacol and diffuses across to the artificial protoplasm where its concentration becomes higher than in the external solution. The guaiacol molecule thus acts as a carrier molecule which transports K⁺ from the external medium across the protoplasmic surface. The outer part of the protoplasm may contain relatively few potassium ions so that the outwardly directed potential at the outer protoplasmic surface may be small but the inner part of the protoplasm may contain more potassium ions. This may happen when potassium enters in combination with carrier molecules which do not completely dissociate until they reach the vacuole. Injury and recovery from injury may be studied by measuring the movements of water into and out of the cell. Metabolism by producing CO₂ and other acids may lower the pH and cause local shrinkage of the protoplasm which may lead to protoplasmic motion. Antagonism between Na⁺ and Ca⁺⁺ appears to be due to the fact that in solutions of NaCl the surface layer takes up an excessive amount of water and this may be prevented by the addition of suitable amounts of CaCl₂. In Nitella the outer non-aqueous surface layer may be rendered irreversibly permeable by sharply bending the cell without permanent damage to the inner non-aqueous surface layer surrounding the vacuole. The formation of contractile vacuoles may be imitated in non-living systems. An extract of the sperm of the marine worm Nereis which contains a highly surface-active substance can cause the egg to divide. It seems possible that this substance may affect the surface layer of the egg and cause it to take up water. A surface-active substance has been found in all the seminal fluids examined including those of trout, rooster, bull, and man. Duponol which is highly surface-active causes the protoplasm of Spirogyra to take up water and finally dissolve but it can be restored to the gel state by treatment with Lugol solution (KI + I). The transition from gel to sol and back again can be repeated many times in succession. The behavior of water in the surface layer of the protoplasm presents important problems which deserve careful examination.     

THE KINETICS OF PENETRATION : XV. THE RESTRICTION OF THE CELLULOSE WALL

When Valonia cells are impaled on capillaries, it is in some ways equivalent to removing the comparatively inelastic cellulose wall. Under these conditions sap can migrate into a free space and it is found that on the average the rate of increase of volume of the sap is 15 times what it is in intact cells kept under comparable conditions. The rate of increase of volume is a little faster during the first few hours of the experiment, but it soon becomes approximately linear and remains so as long as the experiment is continued. The slightly faster rate at first may mean that the osmotic pressure of the sap is approaching that of the sea water (in the intact cell the sap osmotic pressure is always slightly above that of the sea water). This might result from a more rapid entrance of water than of electrolyte, as would be expected when the restriction of the cellulose wall was removed. During the linear part of the curve the osmotic concentration and the composition of the sap suffer no change, so that entrance of electrolyte must be 15 times as fast in the impaled cells as it is in the intact cells. The explanation which best accords with the facts is that in the intact cell the entrance of electrolyte tends to increase the osmotic pressure. As a consequence the protoplasm is partially dehydrated temporarily and it cannot take up more water until the cellulose wall grows so that it can enclose more volume. The dehydration of the protoplasm may have the effect of making the non-aqueous protoplasm less permeable to electrolytes by reducing the diffusion and partition coefficients on which the rate of entrance depends. In this way the cell is protected against great fluctuations in the osmotic concentration of the sap.