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.     

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 EFFECT OF HYDROGEN ION CONCENTRATION ON THE PRODUCTION OF CARBON DIOXIDE BY BACILLUS BUTYRICUS AND BACILLUS SUBTILIS

1. The maximum rate of CO₂ production of Bacillus butyricus was found to be at a pH value of 7; of Bacillus subtilis at pH 6.8. If the pH value be raised or lowered there is a progressive decrease in the rate of production of CO₂. 2. Spontaneous recovery follows the addition of alkali to either organism, while addition of acid is followed by recovery only upon addition of an equivalent amount of alkali, and is not complete except when the amount of acid is very small.     

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 STABILITY OF BACTERIAL SUSPENSIONS : VI. THE INFLUENCE OF THE CONCENTRATION OF THE SUSPENSION ON THE CONCENTRATION OF SALT REQUIRED TO CAUSE COMPLETE AGGLUTINATION.

1. The concentrations of various salts required to agglutinate different concentrations for a suspension of typhoid bacilli sensitized with immune serum have been determined. 2. The electrolytes may be divided into two classes; (1) those with which the concentration required to agglutinate is independent of the concentration of the suspension; and (2) those with which the agglutinating concentration increases in proportion to the concentration of the suspension. 3. The salts comprised under (1) do not reverse the sign of the charge of the suspension. 4. The salts of Class (2) (with the exception of ZnSO₄) do reverse the sign of the charge.     

ELECTROKINETIC PHENOMENA. III : THE "ISOELECTRIC POINT" OF NORMAL AND SENSITIZED MAMMALIAN ERYTHROCYTES

A survey of the published electrophoretic mobilities of certain mammalian red cells reveals that the isoelectric points accorded to these cells are the result of equilibria incidental to red cell destruction. The electrophoretic mobilities of normal washed sheep and human cells have now been studied in 0.85 per cent NaCl solutions from about pH 3.6 to 7.4. All measurements were made within 2 minutes of the preparation of the suspension of red cells. In no case was reversal of sign of charge observed under these conditions. Reversal of sign of charge occurred only after sufficient time had elapsed to permit sufficient adsorption of the products of red cell destruction. There is little change in mobility as the pH of the medium is decreased. Reversal of sign of charge does occur in the presence of normal and immune (anti-sheep) rabbit sera. The isoelectric point determined under these conditions does not appear to be connected specifically with the immune body but is perhaps associated with phenomena incidental to red cell destruction and the presence of serum. The characteristic lowering of mobility by amboceptor occurs, however, from pH 4.0 to pH 7.4. The curves of mobility plotted against pH for normal and for immune sera support the viewpoint that the identity of the isoelectric points for normal and sensitized sheep cells is not primarily concerned with the immune reaction. It is most unlikely that an "albumin" or a "globulin" surface covers red cells with a complete protein film. Although serum protein reacts with red cells in acid solutions, this is not demonstrable for gelatin. The lowering of mobility usually ascribed to anti-sheep rabbit serum may also occur, but to a lesser degree, in normal rabbit serum. This diminution of mobility is not, in the first place, associated with sensitization to hemolysis induced by complement. This supports the view that only a very small part of the red cell surface need be changed in order to obtain complete hemolysis in the presence of complement.     

CHANGES IN THE STABILITY AND POTENTIAL OF CELL SUSPENSIONS : I. THE STABILITY AND POTENTIAL OF BACTERIUM COLI.

1. Stability and potential of Bacterium coli suspensions depend, not only on the strain of the organism and the medium in which it is suspended, but also on the previous treatment of the suspension, and the length of time it has been in the medium. 2. When treated at acid reactions, the negative charge on the bacteria is diminished; with some strains, a positive charge is acquired. Changes in stability accompany the changes in potential. 3. Washing acid-treated bacteria at neutral or slightly alkaline reactions does not restore the original potential; the zone of flocculation is moved toward the alkaline side. 4. These changes are due to two factors: the extraction of a soluble protein which combines with the surfaces of the cells, and a further irreversible change of the cell or its membrane.     

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.     

Surface electrical properties of <Emphasis Type="Italic">bacillus subtilis</Emphasis> cells and the effect of interaction with silicon dioxide particles

Depending on the amount of phosphate in the growth medium, the surface layer of the Bacillus subtilis cell wall may be composed mainly of either teichoic or teichuronic acids. Only in the former case the negative charge of the cell surface increases upon addition of silicon dioxide particles into the cell suspension.     

THE ISOELECTRIC POINT OF RED BLOOD CELLS AND ITS RELATION TO AGGLUTINATION

1. The movement of normal and sensitized red blood cells in the electric field is a function of the hydrogen ion concentration. The isoelectric point, at which no movement occurs, corresponds with pH 4.6. 2. On the alkaline side of the isoelectric point the charge carried is negative and increases with the alkalinity. On the acid side the charge is positive and increases with the acidity. 3. On the alkaline side at least the charge carried by sensitized cells is smaller and increases less rapidly with the alkalinity than the charge of normal cells. 4. Both normal and sensitized cells combine chemically with inorganic ions, and the isoelectric point is a turning point for this chemical behavior. On the acid side the cells combine with the hydrogen and chlorine ions, and in much larger amount than on the alkaline side; on the alkaline side the cells combine with a cation (Ba), and in larger amount than on the acid side. This behavior corresponds with that found by Loeb for gelatin. 5. The optimum for agglutination of normal cells is at pH 4.75, so that at this point the cells exist most nearly pure, or least combined with anion and cation. 6. The optimum for agglutination of sensitized cells is at pH 5.3. This point is probably connected with the optimum for flocculation of the immune serum body.     

MECHANISM OF HEMOLYSIS BY COMPLEMENT : I. COMPLEMENT FIXATION AS AN ESSENTIAL PRELIMINARY TO HEMOLYSIS.

1. Sensitization confers upon the red cell the property of adsorbing complement from solution. The submicroscopic film of immune serum protein deposited upon the cell surface during sensitization, and completely analogous to the precipitate formed in a soluble antigen-antibody reaction (e.g., sheep serum vs. rabbit anti-sheep serum) acts as absorbent, the degree of sensitization (size of the film) determining the amount of complement "fixed" (adsorbed). 2. This adsorption of complement by the sensitized cell is an essential preliminary to hemolysis, and when inhibited, even large quantities of demonstrably active complement have no hemolytic action. The marked influence of electrolytes and of the hydrogen ion concentration upon hemolysis is due primarily to corresponding effects upon the fixation of complement by the sensitized cell. In the case of salts with monovalent cations, complement fixation (and hemolysis) is completely inhibited at any concentration < 0.02 M or > 0.35 M. Electrolytes with bivalent cations are much more inhibitory, and in low as concentration 0.07 M completely prevent fixation (and hemolysis). The optimal reaction for complement fixation (and hemolysis) is pH 6.5 to 8.0. In slightly more acid range both are inhibited. But at a reaction pH 5.3, and in the alkaline range, there is an irreversible inactivation of complement, complete at pH 4.8 and 8.8 respectively. It is perhaps more than a coincidence that complement fixation, and therefore, hemolysis, are prevented by just those factors which suppress the ionization of serum proteins, and lead to an increased aggregation state. Between a suspension of macroscopically visible particles of euglobulin in distilled water, and a solution is physiological saline, there is certainly a gradual transition, manifested at low electrolyte concentrations by the opacity of the solution. At pH 7.4, globulin would ionize as a Na-salt, an ionization inhibited as the isoelectric point (5.3) is approached, with a coincident greater tendency of the globulin to separate from solution. And the cataphoretic velocity of particles of globulin, as well as all the other properties which are a function of its ionization (viscosity, osmotic pressure, etc.), are suppressed by electrolytes, the degree of suppression being determined by the concentration and valence of the cation (on the alkaline side of the isoelectric point). The analogy with complement fixation is too complete to be dismissed as fortuitous. 3. The fact that the degree of complement "fixation" increases with the degree of sensitization explains one of the most puzzling phenomena in hemolysis,—that immune serum and complement are, to a certain extent, interchangeable, a decrease in either factor being compensated by an increase in the other (8), (20), (22). The explanation is evident from Figs. 1,2, and 3. The exact quantitative relationships involved will be developed in a later paper. With increasing sensitization there is an enormously more complete and more rapid fixation of complement, and correspondingly more rapid hemolysis, exactly the effect produced by increasing the quantity of complement instead of amboceptor (Fig. 3). All other variables being constant, the velocity of hemolysis is determined by the amount of complement adsorbed. With more amboceptor, a greater proportion is "fixed" by the cell; with more complement, a smaller proportion, but a larger absolute amount. The result is the same: more complement adsorbed, and a corresponding acceleration of hemolysis. If this mobilization of complement is the sole function of immuneserum (and there is as yet no reason to assume any other), then the accepted terminology, in which amboceptor, immune body, and hemolysin are used synonymously, is erroneous. The immune body would function only as an "amboceptor," mobilizing the effective hemolysin, complement, upon the surface of the cell. Nothing has been said of the multiple components into which complement may be split. A priori, it would be expected that the adsorption demonstrated is of the so called midpiece fraction.     

THE BACTERICIDAL EFFECT OF ULTRAVIOLET RADIATION ON ESCHERICHIA COLI IN LIQUID SUSPENSIONS

1. The irradiation of bacteria in liquid suspension has been made possible through: (a) the use of a specially balanced physiological salt solution which is practically non-absorbing for the wave lengths used, and which is of such composition that subsequent dilution of the bacterial suspension gives the proper number of organisms; (b) special design of the exposure cell and a very thorough method of stirring which subjects each organism equally to the radiation; (c) practically complete absorption of the incident radiation, through the use of very dense suspensions, thus eliminating the necessity for a separate determination of the absorption coefficients of the bacteria for the wave lengths used. 2. The method also provides a means for determining the effects of sub-lethal doses. 3. A formula is given for calculating from observed survival ratios the energy required to inactivate bacteria with ultraviolet radiation. The formula corrects for the protective action of non-viable organisms. 4. Data are given for the inactivation of 15 hour and 240 hour cultures of E. coli, washed and unwashed) and for 6–7 hour cultures, unwashed. These data are compared with those of other investigators. 5. A possible explanation for the differences in energy required to inactivate old, young, and standard cultures of bacteria is suggested. 6. The possible mechanism of the action of ultraviolet radiation on microorganisms is discussed.     

THE REVERSAL OF THE SIGN OF THE CHARGE OF MEMBRANES BY HYDROGEN IONS

1. It had been shown in previous papers that when a collodion membrane has been treated with a protein the membrane assumes a positive charge when the hydrogen ion concentration of the solution with which it is in contact exceeds a certain limit. It is pointed out in this paper that by treating the collodion membrane with a protein (e.g. oxyhemoglobin) a thin film of protein adheres to the membrane and that the positive charge of the membrane must therefore be localized in this protein film. 2. It is further shown in this paper that the hydrogen ion concentration, at which the reversal in the sign of the charge of a collodion membrane treated with a protein occurs, varies in the same sense as the isoelectric point of the protein, with which the membrane has been treated, and is always slightly higher than that of the isoelectric point of the protein used. 3. The critical hydrogen ion concentration required for the reversal seems to be, therefore, that concentration where enough of the protein lining of the membrane is converted into a protein-acid salt (e.g. gelatin nitrate) capable of ionizing into a positive protein ion (e.g. gelatin) and the anion of the acid used (e.g. NO₃).     

High pH,ammonia toxicity,and the search for life on the Jovian planets

The Jovian planets have environments apparently suitable for the evolution of life, but, nevertheless, present severe challenges to organisms. One such challenge arises from the presence of ammonia. Ammonia is an efficient biocide, its effect being dependent on pH as well as on concentration. The effects of pH and ammonia concentration were studied separately, where possible, on a variety of organisms, including some isolated from natural environments of high pH and/or ammonia concentration. Escherichia coli andBacillus subtilis are both extremely sensitive to ammonia. An aerobic organism (growth up to pH 11.4) from an alkaline spring is more resistant, but exhibits a toxic response to ammonia at a pH much lower than its maximum for growth. The greatest ammonia resistance has been found in an unidentified organism growing at near neutral pH. Even in this case, however, survival at ammonia concentrations reasonably expected on the Jovian planets is measured in hours. This is, nevertheless, two to three orders of magnitude longer than forE. coli. Our data support the tentative conclusion that contamination of the Jovian planets with terrestrial organisms that can grow is unlikely. However, the range of toxic response noted, coupled with the observation that terrestrial life has not been exposed to high ammonia concentrations for millions of years, suggests that adaptation to greater ammonia tolerance may be possible.     

ON THE LOCATION OF THE FORCES WHICH DETERMINE THE ELECTRICAL DOUBLE LAYER BETWEEN COLLODION PARTICLES AND WATER

1. The cataphoretic P.D. of suspended particles is assumed to be due to an excess in the concentration of one kind of a pair of oppositely charged ions in the film of water enveloping the particles and this excess is generally ascribed to a preferential adsorption of this kind of ions by the particle. The term adsorption fails, however, to distinguish between the two kinds of forces which can bring about such an unequal distribution of ions between the enveloping film and the opposite film of the electrical double layer, namely, forces inherent in the water itself and forces inherent in the particle (e.g. chemical attraction between particle and adsorbed ions). 2. It had been shown in a preceding paper that collodion particles suspended in an aqueous solution of an ordinary electrolyte like NaCl, Na₂SO₄, Na₄Fe(CN)₆, CaCl₂, HCl, H₂SO₄, or NaOH are always negatively charged, and that the addition of these electrolytes increases the negative charge as long as their concentration is below M/1,000 until a certain maximal P.D. is reached. Hence no matter whether acid, alkali, or a neutral salt is added, the concentration of anions must always be greater in the film enveloping the collodion particles than in the opposite film of the electrical double layer, and the reverse is true for the concentration of cations. This might suggest that the collodion particles, on account of their chemical constitution, attract anions with a greater force than cations, but such an assumption is rendered difficult in view of the following facts. 3. Experiments with dyes show that at pH 5.8 collodion particles are stained by basic dyes (i.e. dye cations) but not by acid dyes (i.e. dye anions), and that solutions of basic dyes are at pH 5.8 more readily decolorized by particles of collodion than acid dyes. It is also shown in this paper that crystalline egg albumin, gelatin, and Witte''s peptone form durable films on collodion only when the protein exists in the form of a cation or when it is isoelectric, but not when it exists in the form of an anion (i.e. on the alkaline side of its isoelectric point). Hence if any ions of dyes or proteins are permanently bound at the surface of collodion particles through forces inherent in the collodion they are cations but not anions. The fact that isoelectric proteins form durable films on collodion particles suggests, that the forces responsible for this combination are not ionic. 4. It is shown that salts of dyes or proteins, the cations of which are capable of forming durable films on the surface of the collodion, influence the cataphoretic P.D. of the collodion particles in a way entirely different from that of any other salts inasmuch as surprisingly low concentrations of salts, the cation of which is a dye or a protein, render the negatively charged collodion particles positive. Crystalline egg albumin and gelatin have such an effect even in concentrations of 1/130,000 or 1/65,000 of 1 per cent, i.e. in a probable molar concentration of about 10^–9. 5. Salts in which the dye or protein is an anion have no such effect but act like salts of the type of NaCl or Na₂SO₄ on the cataphoretic P.D. of collodion particles. 6. Amino-acids do not form durable films on the surface of collodion particles at any pH and the salts of amino-acids influence their cataphoretic P.D. in the same way as NaCl but not in the same way as proteins or dyes, regardless of whether the amino-acid ion is a cation or an anion. 7. Ordinary salts like LaCl₃ also fail to form a durable film on the surface of collodion particles. 8. Until evidence to the contrary is furnished, these facts seem to suggest that the increase of the negative charge of the collodion particles caused by the addition of low concentrations of ordinary electrolytes is chiefly if not entirely due to forces inherent in the aqueous solution but to a less extent, if at all, due to an attraction of the anions of the electrolyte by forces inherent in the collodion particles.     

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.     

STUDIES ON THE REGULATION OF OSMOTIC PRESSURE : I. THE EFFECT OF INCREASING CONCENTRATIONS OF GELATIN ON THE CONDUCTIVITY OF A SODIUM CHLORIDE SOLUTION.

1. In pure gelatin solutions the conductivity of the solution increases with increasing concentrations, regardless of the hydrogen ion concentration. The actual value of the specific conductivity is greater at that reaction where the degree of ionization is greater. 2. The addition of gelatin in increasing concentrations to a 0.6 per cent sodium chloride solution affects the conductivity of that solution in two ways: (a) At pH 3.3, (where gelatin is highly ionized) the conductivity increases with each added increment of gelatin. (b) At pH 5.1 and 7.4 (where gelatin is less highly ionized) the conductivity decreases with each added increment of gelatin. A similar study is being made of crystalline egg albumin.     

THE ANALYSIS OF THE DIVISION RATES OF CILIATES

1. Analysis of the division rates of Paramecium aurelia (mutant), Blepharisma undulans, and Histrio complanatus grown separately in pedigree isolation culture with the same culture medium, and in the same room at any given time, for a period of 3 years, discloses a secular trend and a seasonal rhythm for each organism. The seasonal rhythm is a yearly cycle with a maximum during July. 2. After removal of the effects of trend and seasonal rhythm, no correlation is found between the division rates of the several organisms. The distribution of the division rates is then one of chance order, except for large deviations known to be associated with changes in the culture technique. Each organism has a division rate varying independently of the others. 3. Consequently, seasonal rhythm alone has forced similar variations in the division rates of these three protozoans. The seasonal effect is gradually lost when the animals are raised for several years under laboratory conditions. Examination of the literature discloses other similar cases. 4. It is clear that unless all of the conditions of experiment are kept constant, one must analyze all protozoan division rate data in some such manner as that here presented before any conclusions may be drawn as to the existence of "cycles" or "rhythms."     

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.