CHANGES IN THE STABILITY AND POTENTIAL OF CELL SUSPENSIONS : II. THE POTENTIAL OF ERYTHROCYTES.

1. Human and sheep erythrocytes, when placed in 0.01 N buffer solutions at reactions more acid than pH 5.2, undergo a progressive change in potential, becoming less electronegative or more electropositive. This change usually occurs within 2 hours at ordinary room temperatures. It did not occur when rabbit erythrocytes were used. 2. This change is due primarily to the liberation of hemoglobin from some of the cells. 3. Hemoglobin, even in very low concentrations, markedly alters the potential of erythrocytes in the more acid reactions. This is due to a combination between the electropositive hemoglobin and the erythrocytes. The effect of the hemoglobin is most marked in the more acid solutions; it occurs only on the acid side of the isoelectric point of the hemoglobin. 4. The isoelectric point of erythrocytes in the absence of salt, or in the presence of salts having both ions monovalent, occurs at pH 4.7. This confirms the observations of Coulter (1920–21). Divalent anions shift the isoelectric point to the acid side. 5. The effect of salts on the potential of erythrocytes is due to the ions of the salts, and is analogous in every way to the effect of salts on albumin-coated collodion particles, as discussed by Loeb (1922–23).     

THE EFFECT OF OXIDANTS AND REDUCTANTS UPON THE BIOELECTRIC POTENTIAL OF NITELLA

Nitella cells were exposed to various oxidants and reductants, to determine their effect upon the bioelectric potential. These included five systems, with an E_h range from +0.454 v. to –0.288 v., a total range of 0.742 v. When proper regard was given to buffering against acidity changes, and concentration changes of Na or K ions in the oxidized and reduced forms, no significant effect upon the bioelectric potential was found: 1. When an oxidant or reductant (K ferri- or ferrocyanide) was applied instead of an equivalent normality of an "indifferent" salt (KCl). 2. In changing from a given oxidant to its corresponding reductant (ferri- to ferrocyanide; oxidized to leuco-dye, etc.). 3. When a mixture of 2 dyes, (indophenol with positive E''₀, and safranin with negative E''₀) was oxidized and reduced, to give better poising at the extremes. It is conduded that the outer surface of this cell is not influenced by the state of oxidation or reduction of the systems employed; at least it does not respond with a manifest change of bioelectric potential to changes in oxidation-reduction intensity of the medium. The cells continued to show, however, at all times their usual response to concentration changes of KCl, NaCl, etc., and to electrical stimulation.     

STUDIES ON SALT ACTION : X. THE INFLUENCE OF ELECTROLYTES UPON THE VIABILITY AND ELECTROPHORETIC MIGRATION OF BACTERIUM COLI.

1. The strain of Bacterium coli used in these experiments multiplies in distilled water at pH 6.0 and pH 8.0 and in Ringer-Locke solution at pH 6.0. Under all the other conditions studied the numbers decrease with the passage of time. 2. The electrophoretic charge of the cells is highest in distilled water at pH 6.0 and pH 8.0. Under all other conditions studied the velocity of migration is decreased, but the decrease is immediate and is not affected by more prolonged exposure. 3. A strongly acid solution (pH 2.0) causes a rapid death of the cells and a sharp decrease in electrophoretic charge, sometimes leading to complete reversal. 4. A strongly alkaline solution (pH 11.0) is almost as toxic as a strongly acid one, although in distilled water the organisms survive fairly well at this reaction. Electrophoretic charge, on the other hand, is only slightly reduced in such an alkaline medium. 5. In distilled water, reactions near the neutral point are about equally favorable to both viability and electrophoretic charge, pH 8.0 showing slightly greater multiplication and a slightly higher charge than pH 11.0. In the presence of salts, however, pH 8.0 is much less favorable to viability and somewhat more favorable to electrophoretic charge than is pH 6.0. 6. Sodium chloride solutions, in the concentrations studied, all proved somewhat toxic and all tended to depress electrophoretic charge. Very marked toxicity was, however, exhibited only in a concentration of .725 M strength or over and at pH 8.0, while electrophoretic migration velocity was only slightly decreased at a concentration of .0145 M strength. 7. Calcium chloride was more toxic than NaCl, showing very marked effects in .145 M strength at pH 8.0 and in 1.45 M strength at pH 6.0. It greatly depressed electrophoretic charge even in .0145 M concentration. 8. Ringer-Locke solution proved markedly stimulating to the growth of the bacteria at pH 6.0 while at pH 8.0 it was somewhat toxic, though less so than the solutions of pure salts. It depressed migration velocity at all pH values, being more effective than NaCl in this respect, but less effective than CaCl₂. 9. It would appear from these experiments that a balanced salt solution (Ringer-Locke''s) may be distinctly favorable to bacterial viability in water at an optimum reaction while distinctly unfavorable in a slightly more alkaline solution. 10. Finally, while there is a certain parallelism between the influence of electrolytes upon viability and upon electrophoretic charge, the parallelism is not a close one and the two effects seem on the whole to follow entirely different laws.     

THE RELATION OF THE STABILITY OF PROTOPLASMIC FILMS IN NOCTILUCA TO THE DURATION AND INTENSITY OF AN APPLIED ELECTRIC POTENTIAL

1. The experiments demonstrate that when a constant electric potential of sufficient intensity is applied to Noctiluca, the protoplasmic films which represent a part of the visible continuous phase of the cytoplasm and plasma membrane at the surface of the cell, become unstable and break down, thus releasing the acid contents of one of the internal discontinuous phases present in the cytoplasm of Noctiluca. This process which occurs first at anode then at the cathode side of the cell, appears to be a selective deemulsification or coalescence similar to that at the surface of an emulsion having a viscous continuous phase. 2. The experiments demonstrate that Nernst''s equation See PDF for Equation which expresses approximately the relation of duration and intensity of a constant electric current to threshold stimulation of striated muscle, applies equally well to the process of anodal coalescence in Noctiluca. 3. Anodal and cathodal coalescence have different thresholds, due to the fact that the semipermeable plasma film at the surface of the cell is asymmetric with respect to the direction of the applied current. Attention is called to the possible relation between this phenomenon and the conditions occurring at the synapse between neurons. 4. The stability of the protoplasmic films in relation to the applied electric potential is greater in young cells than in old cells, or in other words the threshold intensity of the stimulus is higher for young than for old cells. 5. Attention is called to the occurrence in the same cell of different receptor-affector mechanisms having a corresponding difference in intensity threshold when an electric current is acting as a stimulus.     

THE EFFECTS OF CURRENT FLOW ON BIOELECTRIC POTENTIAL : I. VALONIA

The effect of direct current flow upon the potential difference across the protoplasm of impaled Valonia cells was studied. Current density and direction were controlled in a bridge which balanced the ohmic resistances, leaving the changes (increase, decrease, or reversal) of the small, normally negative, bioelectric potential to be recorded continuously, before, during, and after current flow, with a string galvanometer connected into a vacuum tube detector circuit. Two chief states of response were distinguished: State A.—Regular polarization, which begins to build up the instant current starts to flow, the counter E.M.F. increasing most rapidly at that moment, then more and more slowly, and finally reaching a constant value within 1 second or less. The magnitude of counter E.M.F. is proportional to the current density with small currents flowing in either direction across the protoplasm, but falls off at higher density, giving a cusp with recession to lower values; this recession occurs with slightly lower currents outward than inward. Otherwise the curves are much the same for inward and outward currents, for different densities, for charge and discharge, and for successive current flows. There is a slight tendency for the bioelectric potential to become temporarily positive following these current flows. Records in the regular state (State A) show very little effect of increased series resistance on the time constant of counter E.M.F. This seems to indicate that a polarization rather than a static capacity is involved. State B.—Delayed and non-proportional polarization, in which there is no counter E.M.F. developed with small currents in either direction across the protoplasm, nor with very large outward currents. But with inward currents a threshold density is reached at which a counter E.M.F. rather suddenly develops, with a sigmoid curve rising to high positive values (200 mv. or more). There is sometimes a cusp, after which the P.D. remains strongly positive as long as the current flows. It falls off again to negative values on cessation of current flow, more rapidly after short flows, more slowly after longer ones. The curves of charge are usually quite different in shape from those of discharge. Successive current flows of threshold density in rapid succession produce quicker and quicker polarizations, the inflection of the curve often becoming smoothed away. After long interruptions, however, the sigmoid curve reappears. Larger inward currents produce relatively little additional positive P.D.; smaller ones on the other hand, if following soon after, have a greatly increased effectiveness, the threshold for polarization falling considerably. The effect dies away, however, with very small inward currents, even as they continue to flow. Over a medium range of densities, small increments or decrements of continuing inward current produce almost as regular polarizations as in State A. Temporary polarization occurs with outward currents following soon after the threshold inward currents, but the very flow of outward current tends to destroy this, and to decondition the protoplasm, again raising the threshold, for succeeding inward flows. State A is characteristic of a few freshly gathered cells and of most of those which have recovered from injuries of collecting, cleaning, and separating. It persists a short time after such cells are impaled, but usually changes over to State B for a considerable period thereafter. Eventually there is a reappearance of regular polarization; in the transition there is a marked tendency for positive P.D. to be produced after current flow, and during this the polarizations to outward currents may become much larger than those to inward currents. In this it resembles the effects of acidified sea water, and of certain phenolic compounds, e.g. p-cresol, which produce State A in cells previously in State B. Ammonia on the other hand counteracts these effects, producing delayed polarization to an exaggerated extent. Large polarizations persist when the cells are exposed to potassium-rich solutions, showing it is not the motion of potassium ions (e.g. from the sap) which accounts for the loss or restoration of polarization. It is suggested that inward currents restore a protoplasmic surface responsible for polarization by increasing acidity, while outward currents alter it by increasing alkalinity. Possibly this is by esterification or saponification respectively of a fatty film. For comparison, records of delayed polarization in silver-silver chloride electrodes are included.     

THE STABILITY OF BACTERIAL SUSPENSIONS : IV. THE COMBINATION OF ANTIGEN AND ANTIBODY AT DIFFERENT HYDROGEN ION CONCENTRATIONS.

1. The amount of immune body required to agglutinate a suspension of Bacillus typhosus increases in direct proportion to the concentration of the suspension. 2. The amount of immune body combined with the organisms is constant from pH 9 to pH 3.7. Below the latter value the amount in combination is decreased. 3. The addition of immune serum to a suspension of Bacillus typhosus at a pH of 2,5 increases the positive charge of the organisms. These results are contradictory to the idea that the combination is caused by a difference in the sign of the charge carried by the immune body and the organism. They agree with the assumption that the immune body forms a film on the surface of the organism and that the effect on the charge is the result of this film.     

THE STABILITY OF BACTERIAL SUSPENSIONS : III. AGGLUTINATION IN THE PRESENCE OF PROTEINS,NORMAL SERUM,AND IMMUNE SERUM.

1. The addition of proteins or serum to suspensions of bacteria, (Bacillus typhosus or rabbit septicemia) at different pH widens the acid agglutination zone and shifts the isoelectric point to that of the added substance. 2. The amount of serum required to agglutinate is much less near the acid agglutination point of the organisms. 3. The addition of immune serum prevents the salt from decreasing the cohesive force between the organisms, and agglutination therefore is determined solely by the potential, provided excess immune body is present. Whenever the potential is decreased below 15 millivolts the suspension agglutinates.     

THE ALKALINE ISOPOTENTIAL POINT OF THE BACTERIAL CELL

Irregularities in migration velocity of bacterial cells in the highly alkaline solutions are due to the buffering effect of the cells upon the immediately adjacent zone of menstruum. Consistent results can be obtained by shaking the suspension thoroughly before placing it in the electrophoretic cell. When observed in this way both Bacillus cereus and Bacterium coli show an isopotential point near pH 13.5, that for Bacillus cereus being slightly below, and that for Bacterium coli slightly above this point. At more alkaline reactions the cells acquire a positive charge which increases with further increase in pH to very high values.     

THE INFLUENCE OF ELECTROLYTES ON THE CATAPHORETIC CHARGE OF COLLOIDAL PARTICLES AND THE STABILITY OF THEIR SUSPENSIONS : II. EXPERIMENTS WITH PARTICLES OF GELATIN,CASEIN, AND DENATURED EGG ALBUMIN.

1. This paper gives measurements of the influence of various electrolytes on the cataphoretic P.D. of particles of collodion coated with gelatin, of particles of casein, and of particles of boiled egg albumin in water at different pH. The influence of the same electrolyte was about the same in all three proteins. 2. It was found that the salts can be divided into two groups according to their effect on the P.D. at the isoelectric point. The salts of the first group including salts of the type of NaCl, CaCl₂, and Na₂SO₄ affect the P.D. of proteins at the isoelectric point but little; the second group includes salts with a trivalent or tetravalent ion such as LaCl₃ or Na₄Fe(CN)₆. These latter salts produce a high P.D. on the isoelectric particles, LaCl₃ making them positively and Na₄Fe(CN)₆ making them negatively charged. This difference in the action of the two groups of salts agrees with the observations on the effect of the same salts on the anomalous osmosis through collodion membranes coated with gelatin. 3. At pH 4.0 the three proteins have a positive cataphoretic charge which is increased by LaCl₃ but not by NaCl or CaCl₂, and which is reversed by Na₄Fe(CN)₆, the latter salt making the cataphoretic charge of the particles strongly negative. 4. At pH 5.8 the protein particles have a negative cataphoretic charge which is strongly increased by Na₄Fe(CN)₆ but practically not at all by Na₂SO₄ or NaCl, and which is reversed by LaCl₃. the latter salt making the cataphoretic charge of the particles strongly positive. 5. The fact that electrolytes affect the cataphoretic P.D. of protein particles in the same way, no matter whether the protein is denatured egg albumin or a genuine protein like gelatin, furnishes proof that the solutions of genuine proteins such as crystalline egg albumin or gelatin are not diaphasic systems, since we shall show in a subsequent paper that proteins insoluble in water, e.g. denatured egg albumin, are precipitated when the cataphoretic P.D. falls below a certain critical value, while water-soluble proteins, e.g. genuine crystalline egg albumin or gelatin, stay in solution even if the P.D. of the particles falls below the critical P.D.     

THE INFLUENCE OF ELECTROLYTES UPON THE ELECTROPHORETIC MIGRATION OF BACTERIA AND OF YEAST CELLS

1. It seems first of all clear from our results that the effect of electrolytes upon electrophoretic charge is essentially the same, whether one is dealing with silica dust, bacteria, or yeast cells, although certain quantitative differences appear which will later be discussed. 2. The normal negative charge on the suspended particles appears to be slightly increased by very low concentrations of electrolytes, markedly so in the case of yeast cells. Increase in charge due to minimal concentrations of electrolytes has been recorded by Loeb (1922) for collodion particles. 3. Higher concentrations of electrolytes cause a marked and progressive decrease in negative charge, sometimes leading to an isopotential condition and sometimes to a complete reversal of charge with active migration toward the cathode. This effect is apparently due to the cation alone and increases with the valency of the cation, except that the H ion shows specially marked activity, between that of bivalent and trivalent ions. Since NaOH behaves like an ordinary univalent salt, increased alkalinity of a solution does not further depress the charge already depressed by salts; but, since the H ion is much more active than other univalent or bivalent ions, increased acidity does cause a further progressive depression of charge, even in salt solutions. Certain electrolytes appear to show individual peculiarities due to something else than their valency. Thus KCl for example is distinctly more effective than NaCl. Sodium chloride in general appears to exert less influence upon electrophoretic charge, either in low or high dilution, than do other compounds of univalent ions studied. This depressing effect of moderately high concentrations of electrolytes is much less marked with yeast cells than with Bacterium coli. Silica dust is still less affected by monovalent and bivalent ions than are the yeast cells but appears to be more affected than either yeast or Bacterium coli by AlCl₃. 4. Very high concentrations of AlCl₃ (above 10^–2 M) show a third effect, a decrease of the positive charge produced by concentrations of moderate molar strength. This is analogous to phenomena observed for trivalent salts by Northrop and De Kruif (1921–22) and for acid by Winslow, Falk, and Caulfield (1923–24). 5. Organic substances, such as glucose, glycerol, and saponin produce no effect on electrophoretic velocity until they reach a concentration at which viscosity changes are involved. 6. The first two results observed,—(a) the increase in charge as a result of slight additions of electrolytes, and (b) the marked decrease in charge with further concentration of electrolytes, depending on the valency of the cation, so far as vegetable cells are concerned, are entirely in accord with the theory of the Donnan equilibrium as worked out by Loeb (1922). We might assume in explaining such phenomena that the plant cell contains a certain proportion of unbound protein material and that the first modicum of cation which enters the cell is bound by the protein, leading to an increase in the relative negative charge of the cell as compared with its menstruum, while subsequent increments of cation remain unbound in the cell and thus lower its charge. When we find, however, that the same phenomena are apparent with collodion particles, as shown by Loeb, and with silica dust, it seems difficult to apply such a theory, involving the conceptions of a permeable membrane and unbound organic compounds. Loeb (1923–24) suggests that the primary increase may be due to an aggregation of anions in the part of the electrical double layer adjacent to the suspended particles; but why there should be first an aggregation of anions and later (with increasing concentration) an aggregation of cations, is not easy to conceive. The third result,—the reversion to a more negative charge in the presence of a marked excess of trivalent ions,—is again difficult to explain. Loeb, in this connection, postulates the existence of complex ion-protein compounds, which can scarcely be assumed in the case of the silica particles.     

TRANSPLANTATION AND POTENTIAL IMMORTALITY OF MAMMALIAN TISSUES

1. Serial transplantation of tumors made it possible in 1901 and following years to draw the conclusion that various mammalian tissues have potential immortality. Serial transplantations of normal tissues did not succeed at first, because the homoioreaction on the part of the lymphocytes and connective tissue of the host injures the transplant. 2. In continuation of these experiments we found that cartilage of the rat can be transplanted serially to other rats at least for a period of 3 years. At the end of that time great parts of the transplanted cartilage and perichondrium are alive. 3. Not only the cartilage of young rats can be homoiotransplanted, but also the cartilage of very old rats which are nearing the end of life. By using such animals we have been able to obtain cartilage and perichondrium approaching an age of 6 years which is almost double the average age of a rat. 4. We found that cartilage can be homoiotransplanted more readily than other tissues for the following reasons: (a) While in principle the homoioreaction towards cartilage is the same as against other tissues, cartilage elicits this reaction with less intensity; (b) cartilage is better able to resist the invasion of lymphocytes and connective tissue than the majority of other tissues; (c) a gradual adaptation between transplant and host seems to take place in the case of cartilage transplantation, as a result of which the lymphocytic reaction on the part of the host tissue decreases progressively the longer the cartilage is kept in the strange host. 5. At time of examination we not only found living transplanted cartilage tissue, but also perichondrial tissue, which in response to a stimulus apparently originating in the necrotic central cartilage, had been proliferating and replacing it. These results suggest that it may perhaps be possible under favorable conditions to keep cartilage alive indefinitely through serial transplantations. 6. At the same time these experiments permit the analysis of the factors which are favorable or unfavorable to the continued life of the transplants. Favorable factors are: (a) Well preserved perichondrium around transplant; (b) cellular newly formed perichondrial cartilage—though it is doubtful whether such young cartilage cells allow a state of stable equilibrium. Host connective tissue does not invade transplant under these conditions. Unfavorable factors are: (a) Cartilage differentiation and the production of paraplastic substances (hyaline capsules in parts of transplant far removed from vessels and sources of oxygen and food; (b) cartilage necrosis when a still greater distance from nourishment exists; (c) disturbance of equilibrium between host connective tissue and transplant due to above conditions, resulting in (d) attack by host connective tissue on transplanted cartilage, which is the chief danger in the preservation of the life of the whole transplant 7. It is pointed out that also in old age there exist similar problems of disturbances of tissue equilibria, due to degenerative changes in certain parenchymatous structures and to proliferative processes on the part of connective tissue and glia elements together with increase in paraplastic structures.     

ACTION POTENTIAL OF NITELLA INTERNODES

The ionic current during a non-propagating action potentialis analysed from the voltage clamp experiments. The shape ofthe action potential of the Nitella internode can be reconstructedfrom the data of the voltage clamp experiments. The N-shapedcurrent-voltage characteristics (I-V curve) of the Nitella membraneis not constant with time as it is in the tunnel diode, butdecays with time, converging finally into a delayed rectificationcurve. The temporal locus of the potential at which each I-Vcurve crosses the voltage axis coincides almost exactly withthe action potential. The membrane resistance which is calculatedfrom the slope of the I-V curve at each intersection with thevoltage axis also changes in parallel to the action potential.Such correlations are found in the Nitella not only in the pondwater, but also in high Na, high Ca or high Mg medium, wherethe shape of the action potential is modified in various ways.It is highly probable that the action potential is a locus ofthe change of the membrane potential so that the net membranecurrent may be maintained at zero after the transient modificationof the membrane structure by stimulation. (Received June 30, 1966; )     

ON THE GROWTH AND RESPIRATION OF SULFUR-OXIDIZING BACTERIA

1. It is shown that Sulfomonas thiooxidans oxidizes elementary sulfur completely to sulfuric acid. Sodium thiosulfate is oxidized by this organism completely to sulfate. Sulfomonas thiooxidans differs, in this respect, from various other sulfur-oxidizing bacilli which either produce elementary sulfur, from the thiosulfate, or convert it into sulfates and persulfates. 2. The organism derives its carbon from the CO₂ of the atmosphere, but is incapable of deriving the carbon from carbonates or organic matter. 3. The S:C, or ratio between the amount of sulfur oxidized to sulfate and amount of carbon assimilated chemosynthetically from the CO₂ of the atmosphere, is, with elementary sulfur as a source of energy, 31.8, and with thiosulfate 64.2. The higher ratio in the case of the thiosulfate is due to the smaller amount of energy liberated in the oxidation of sulfur compound than in the elementary form. 4. Of the total energy made available in the oxidation of the sulfur to sulfuric acid, only 6.65 per cent is used by the organism for the reduction of atmospheric CO₂ and assimilation of carbon. 5. Sulfates do not exert any injurious effect upon sulfur oxidation by Sulfomonas thiooxidans. Any effect obtained is due to the cation rather than the sulfate radical. Nitrates exert a distinctly injurious action both on the growth and respiration of the organism. 6. There is a definite correlation between the amount of sulfur present and velocity of oxidation, very similar to that found in the growth of yeasts and nitrifying bacteria. Oxidation reaches a maximum with about 25 gm. of sulfur added to 100 cc. of medium. However, larger amounts of sulfur have no injurious effect. 7. Dextrose does not exert any appreciable injurious effect in concentrations less than 5 per cent. The injurious effect of peptone sets in at 0.1 per cent concentration and brings sulfur oxidation almost to a standstill in 1 per cent concentration. Dextrose does not exert any appreciable influence upon sulfur oxidation and carbon assimilation from the carbon dioxide of the atmosphere. 8. Sulfomonas thiooxidans can withstand large concentrations of sulfuric acid. The oxidation of sulfur is affected only to a small extent even by 0.25 molar initial concentration of the acid. In 0.5 molar solutions, the injurious effect becomes marked. The organism may produce as much as 1.5 molar acid, without being destroyed. 9. Growth is at an optimum at a hydrogen ion concentration equivalent to pH 2.0 to 5.5, dropping down rapidly on the alkaline side, but not to such an extent on the acid, particularly when a pure culture is employed. 10. Respiration of the sulfur-oxidizing bacteria can be studied by using the filtrate of a vigorously growing culture, to which a definite amount of sulfur is added, and incubating for 12 to 24 hours.     

THE PREPARATION OF RELATIVELY PURE BACTERIOPHAGE

The method described above, based on the electrophoretic migration of bacteriophage particles into an agar gel and their subsequent re-suspension in a suitable medium, has the following advantages: It is simple and can be readily carried out on a comparatively large scale by merely inserting additional units between the same electrode cups. It requires but one extraction and the resulting phage suspension is strongly lytic, an average sample being capable of completely lysing susceptible bacteria at a dilution of 10^–16. The suspension contains no proteins demonstrable by the biuret, alcohol, xanthoproteic, Millon or Hopkins-Cole reactions and yields but 0.044 mg. N/cc. directly attributable to the phage. Each corpuscle contains no more nitrogen than a single molecule of protein. In addition the method is applicable to determinations of the electric charge carried by biologically active substances of small dimensions, e.g., phage, toxins, and perhaps some viruses. It offers as well a possible means of purification of these substances. The purified bacteriophage obtained by such a procedure or similar ones is relatively unstable. Work now in progress indicates that it does not possess nearly the resistance to chemical agents, drying, etc., that non-purified phage displays. It is suggested that experiments designed to test the therapeutic value of bacteriophage be conducted, when possible, with purified suspensions thereby avoiding any possibility of obscure non-specific reactions due to other constituents of the lysates.     

A STUDY OF THE BACTERICIDAL ACTION OF ULTRA VIOLET LIGHT : II. THE EFFECT OF VARIOUS ENVIRONMENTAL FACTORS AND CONDITIONS

1. Wide differences in the intensity of incident ultra violet energy are not accurately compensated by corresponding changes in the exposure time, so that the Bunsen-Roscoe reciprocity law does not hold, strictly, especially for bactericidal action on young, metabolically and genetically active bacteria. In the present series of experiments, however, the energies used at various wave lengths did not differ by so much as to cause a significant error in the reported reactions. 2. The longer wave length limit of a direct bactericidal action on S. aureus was found to be between 302 and 313 mµ. The shorter limit was not determined because the long exposures required vitiate quantitative results. Bactericidal action was observed at λ225 mµ. 3. The temperature coefficient of the bactericidal reaction approaches 1 and thus furnishes empirical evidence that the direct action of ultra violet light on bacteria is essentially physical or photochemical in character. 4. The hydrogen ion concentration of the environment has no appreciable effect upon the bactericidal reaction between the limits of pH 4.5 and 7.5. At pH 9 and 10 evidence of a slight but definite increase in bacterial susceptibility was noted, but this difference may have been due to a less favorable environment for subsequent recovery and multiplication of injured organisms. 5. Plane polarization of incident ultra violet radiation has no demonstrable effect upon its bactericidal action. In a third paper of this group the ratios of incident to absorbed ultra violet energy at various wave lengths and the significance of these relations in an analysis of the bactericidal reaction will be discussed.     

THE EFFECTS OF CURRENT FLOW ON BIOELECTRIC POTENTIAL : II. HALICYSTIS

The effect of direct current, of controlled direction and density, across the protoplasm of impaled cells of Halicystis, is described. Inward currents slightly increase the already positive P.D. (70 to 80 mv.) in a regular polarization curve, which depolarizes equally smoothly when the current is stopped. Outward currents of low density produce similar curves in the opposite direction, decreasing the positive P.D. by some 10 or 20 mv. with recovery on cessation of flow. Above a critical density of outward current, however, a new effect becomes superimposed; an abrupt reversal of the P.D. which now becomes 30 to 60 mv. negative. The reversal curve has a characteristic shape: the original polarization passes into a sigmoid reversal curve, with an abrupt cusp usually following reversal, and an irregular negative value remaining as long as the current flows. Further increases of outward current each produce a small initial cusp, but do not greatly increase the negative P.D. If the current is decreased, there occurs a threshold current density at which the positive P.D. is again recovered, although the outward current continues to flow. This current density (giving positivity) is characteristically less than that required to produce reversal originally, giving the process a hysteretic character. The recovery is more rapid the smaller the current, and takes only a few seconds in the absence of current flow, its course being in a smooth curve, usually without an inflection, thus differing from the S-shaped reversal curve. The reversal produced by outward current flow is compared with that produced by treatment with ammonia. Many formal resemblances suggest that the same mechanism may be involved. Current flow was therefore studied in conjunction with ammonia treatment. Ammonia concentrations below the threshold for reversal were found to lower the threshold for outward currents. Subthreshold ammonia concentrations, just too low to produce reversal alone, produced permanent reversal when assisted by a short flow of very small outward currents, the P.D. remaining reversed when the current was stopped. Further increases of outward current, when the P.D. had been already reversed by ammonia, produced only small further increases of negativity. This shows that the two treatments are of equivalent effect, and mutually assist in producing a given effect, but are not additive in the sense of being superimposable to produce a greater effect than either could produce by itself. Since ammonia increases the alkalinity of the sap, and presumably of the protoplasm, when it penetrates, it is possible that the reversal of P.D. by current flow is also due to change of pH. The evidence for increased alkalinity or acidity due to current flow across phase boundaries or membranes is discussed. While an attractive hypothesis, it meets difficulties in H. ovalis where such pH changes are both theoretically questionable and practically ineffective in reversing the P.D. It seems best at the present time to assign the reversal of P.D. to the alteration or destruction of one surface layer of the protoplasm, with reduction or loss of its potential, leaving that at the other surface still intact and manifesting its oppositely directed potential more or less completely. The location of these surfaces is only conjectural, but some evidence indicates that it is the outer surface which is so altered, and reconstructed on recovery of positive P.D. This agrees with the essentially all-or-none character of the reversal. The various treatments which cause reversal may act in quite different ways upon the surface.     

THE STABILITY OF BACTERIAL SUSPENSIONS : II. THE AGGLUTINATION OF THE BACILLUS OF RABBIT SEPTICEMIA AND OF BACILLUS TYPHOSUS BY ELECTROLYTES.

1. Measurements have been made of the potential and of the cohesive force at the surface of Bacillus typhosus and the bacillus of rabbit septicemia in solutions of various salts and acids. 2. Electrolytes in low concentration (0.01 N) affect primarily the potential, and in high concentration decrease the cohesive force. 3. As long as the cohesive force is not affected, agglutination occurs whenever the potential is reduced below about 15 millivolts. 4. When the cohesive force is decreased the critical potential is also decreased, and in concentrated salt solution no agglutination occurs even though there is no measurable potential.     

THE EFFECTS OF CURRENT FLOW ON BIOELECTRIC POTENTIAL : III. NITELLA

String galvanometer records show the effect of current flow upon the bioelectric potential of Nitella cells. Three classes of effects are distinguished. 1. Counter E.M.F''S, due either to static or polarization capacity, probably the latter. These account for the high effective resistance of the cells. They record as symmetrical charge and discharge curves, which are similar for currents passing inward or outward across the protoplasm, and increase in magnitude with increasing current density. The normal positive bioelectric potential may be increased by inward currents some 100 or 200 mv., or to a total of 300 to 400 mv. The regular decrease with outward current flow is much less (40 to 50 mv.) since larger outward currents produce the next characteristic effect. 2. Stimulation. This occurs with outward currents of a density which varies somewhat from cell to cell, but is often between 1 and 2 µa/cm.² of cell surface. At this threshold a regular counter E.M.F. starts to develop but passes over with an inflection into a rapid decrease or even disappearance of positive P.D., in a sigmoid curve with a cusp near its apex. If the current is stopped early in the curve regular depolarization occurs, but if continued a little longer beyond the first inflection, stimulation goes on to completion even though the current is then stopped. This is the "action current" or negative variation which is self propagated down the cell. During the most profound depression of P.D. in stimulation, current flow produces little or no counter E.M.F., the resistance of the cell being purely ohmic and very low. Then as the P.D. begins to recover, after a second or two, counter E.M.F. also reappears, both becoming nearly normal in 10 or 15 seconds. The threshold for further stimulation remains enhanced for some time, successively larger current densities being needed to stimulate after each action current. The recovery process is also powerful enough to occur even though the original stimulating outward current continues to flow during the entire negative variation; recovery is slightly slower in this case however. Stimulation may be produced at the break of large inward currents, doubtless by discharge of the enhanced positive P.D. (polarization). 3. Restorative Effects.—The flow of inward current during a negative variation somewhat speeds up recovery. This effect is still more strikingly shown in cells exposed to KCl solutions, which may be regarded as causing "permanent stimulation" by inhibiting recovery from a negative variation. Small currents in either direction now produce no counter E.M.F., so that the effective resistance of the cells is very low. With inward currents at a threshold density of some 10 to 20 µa/cm.², however, there is a counter E.M.F. produced, which builds up in a sigmoid curve to some 100 to 200 mv. positive P.D. This usually shows a marked cusp and then fluctuates irregularly during current flow, falling off abruptly when the current is stopped. Further increases of current density produce this P.D. more rapidly, while decreased densities again cease to be effective below a certain threshold. The effects in Nitella are compared with those in Valonia and Halicystis, which display many of the same phenomena under proper conditions. It is suggested that the regular counter E.M.F.''S (polarizations) are due to the presence of an intact surface film or other structure offering differential hindrance to ionic passage. Small currents do not affect this structure, but it is possibly altered or destroyed by large outward currents, restored by large inward currents. Mechanisms which might accomplish the destruction and restoration are discussed. These include changes of acidity by differential migration of H ion (membrane "electrolysis"); movement of inorganic ions such as potassium; movement of organic ions, (such as Osterhout''s substance R), or the radicals (such as fatty acid) of the surface film itself. Although no decision can be yet made between these, much evidence indicates that inward currents increase acidity in some critical part of the protoplasm, while outward ones decrease acidity.     

THE STABILITY OF BACTERIAL SUSPENSIONS : V. THE REMOVAL OF ANTIBODY FROM SENSITIZED ORGANISMS.

1. The removal of antibody from Bacillus typhosus is no more complete at pH 3 than at pH 7. 2. Approximately twelve agglutinating doses are firmly combined with the organisms. Immune body in excess of this amount is easily removable by distilled water. 3. A method of testing for the presence of immune body on the organism is described which depends on the difference in the acid agglutination of sensitized and unsensitized organisms. 4. Repeated washing in distilled water will serve to remove all the immune body from sensitized bacteria.     

ON THE CAUSE OF THE INFLUENCE OF IONS ON THE RATE OF DIFFUSION OF WATER THROUGH COLLODION MEMBRANES. I

1. In three previous publications it had been shown that electrolytes influence the rate of diffusion of pure water through a collodion membrane into a solution in three different ways, which can be understood on the assumption of an electrification of the water or the watery phase at the boundary of the membrane; namely, (a) While the watery phase in contact with collodion is generally positively electrified, it happens that, when the membrane has received a treatment with a protein, the presence of hydrogen ions and of simple cations with a valency of three or above (beyond a certain concentration) causes the watery phase of the double layer at the boundary of membrane and solution to be negatively charged. (b) When pure water is separated from a solution by a collodion membrane, the initial rate of diffusion of water into a solution is accelerated by the ion with the opposite sign of charge and retarded by the ion with the same sign of charge as that of the water, both effects increasing with the valency of the ion and a second constitutional quantity of the ion which is still to be defined. (c) The relative influence of the oppositely charged ions, mentioned in (b), is not the same for all concentrations of electrolytes. For lower concentrations the influence of that ion usually prevails which has the opposite sign of charge from that of the watery phase of the double layer; while in higher concentrations the influence of that ion begins to prevail which has the same sign of charge as that of the watery phase of the double layer. For a number of solutions the turning point lies at a molecular concentration of about M/256 or M/512. In concentrations of M/8 or above the influence of the electrical charges of ions mentioned in (b) or (c) seems to become less noticeable or to disappear entirely. 2. It is shown in this paper that in electrical endosmose through a collodion membrane the influence of electrolytes on the rate of transport of liquids is the same as in free osmosis. Since the influence of electrolytes on the rate of transport in electrical endosmose must be ascribed to their influence on the quantity of electrical charge on the unit area of the membrane, we must conclude that the same explanation holds for the influence of electrolytes on the rate of transport of water into a solution through a collodion membrane in the case of free osmosis. 3. We may, therefore, conclude, that when pure water is separated from a solution of an electrolyte by a collodion membrane, the rate of diffusion of water into the solution by free osmosis is accelerated by the ion with the opposite sign of charge as that of the watery phase of the double layer, because this ion increases the quantity of charge on the unit area on the solution side of the membrane; and that the rate of diffusion of water is retarded by the ion with the same sign of charge as that of the watery phase for the reason that this ion diminishes the charge on the solution side of the membrane. When, therefore, the ions of an electrolyte raise the charge on the unit area of the membrane on the solution side above that on the side of pure water, a flow of the oppositely charged liquid must occur through the interstices of the membrane from the side of the water to the side of the solution (positive osmosis). When, however, the ions of an electrolyte lower the charge on the unit area of the solution side of the membrane below that on the pure water side of the membrane, liquid will diffuse from the solution into the pure water (negative osmosis). 4. We must, furthermore, conclude that in lower concentrations of many electrolytes the density of electrification of the double layer increases with an increase in concentration, while in higher concentrations of the same electrolytes it decreases with an increase in concentration. The turning point lies for a number of electrolytes at a molecular concentration of about M/512 or M/256. This explains why in lower concentrations of electrolytes the rate of diffusion of water through a collodion membrane from pure water into solution rises at first rapidly with an increase in concentration while beyond a certain concentration (which in a number of electrolytes is M/512 or M/256) the rate of diffusion of water diminishes with a further increase in concentration.