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.     

Anaplasma marginale, Eperythrozoon wenyoni: lectin reactions with bovine erythrocytes

Normal bovine erythrocytes were agglutinated with four of five lectins specific for different oligosaccharides. The order of reactivity was wheat germ greater than ricin greater than soybean greater than peanut. Concanavalin A did not agglutinate normal bovine erythrocytes. After neuraminidase treatment of normal bovine erythrocytes, each lectin agglutinated the cells with decreased concentrations of lectin, verifying that partial removal of sialic acid exposes more of each lectin's binding sites or alters the binding site such that fewer molecules of lectin are required to initiate agglutination. A change in agglutination of erythrocytes using soybean agglutinin and peanut agglutinin occurred when cells were obtained from cattle infected with Eperythrozoon wenyoni. The results suggested that an alteration in erythrocyte membranes occurred as a result of this infection as manifested by the increased recognition of both the soybean agglutinin and peanut agglutinin receptor carbohydrates. A similar effect was indicated with erythrocytes obtained during an acute Anaplasma marginale infection; however, an ensuing reticulocytosis masked the effect, requiring the use of fluoresceinated lectins to verify that increased binding of each lectin occurred with infected cells when compared to normal cells.     

Effect of metabolic state on phytohemagglutinin-P agglutination of normal human erythrocytes

A reproducible quantitative assay for the lectin-mediated agglutination of human erythrocytes, depending on different rates of settling of agglutinated and non-agglutinated erythrocytes, was developed. This assay was used to study the aggregation of human erythrocytes by phytohemagglutinin-P. The aggregation of human erythrocytes by phytohemagglutinin-P was found to depend upon the metabolic state of the cells. Metabolically depleted erythrocytes agglutinated much less readily than did similar cells supplied with adenosine. this was not due to swelling and rigidity of the cells, since erythrocytes in hypotonic solution did not exhibit significantly altered phytohemagglutinin-P agglutination.Metabolically depleted erythrocytes, or erythrocytes from blood stored 8 weeks, lysed and resealed in the presence of ATP, were agglutinated by phytohemagglutinin-P to a much greater extent than control samples without ATP. The presence of Mg²⁺, either alone or with ATP, had little effect on the agglutinability of the resealed membranes. Low concentrations of Ca²⁺ (0.2 mM) had little effect on agglutinability, although high Ca²⁺ (5 mM) inhibited agglutinability of the resealed membranes somewhat.Both metabolically depleted erythrocytes and depleted erythrocytes, previously treated with adenosine, when treated with trypsin released similar amounts of sialic acid. The agglutinability of the trypsinized adenosine-supplemented cells increased more readily than did that of trypsinized metabolically depleted cells.The agglutination of erythrocytes was not affected by cytochalasin B (40 μg/ml). Vinblastine (0.2 mM) caused depleted erythrocytes to agglutinate similarly to adenosine-supplemented erythrocytes, but had no effect on the agglutination of adenosine-supplemented erythrocytes.It is concluded that ATP in the human erythrocyte probably participates in the modulation of phytohemagglutinin-P agglutinability. This is not a consequence of the more rigid membrane known to accompany ATP depletion in the erythrocyte, or of the effect of ATP levels on Ca²⁺ or Mg²⁺ content. It appears likely that ATP modulates human erythrocyte phytohemagglutinin-P agglutinability through interaction, direct or indirect, with a membrane-associated component, which might also be sensitive to vinblastine.     

Bean lectins

Summary Single seeds of over 100 bean cultivars were analyzed by two-dimensional electrophoresis. The cultivars could be classified into eight groups by virtue of their G2/albumin electrophoretic patterns: T_G2, S_G2, V_G2, Pr_G2, B_G2, M_G2, P_G2, and Pi_G2, The polypeptide compositions of these types were largely inter-related having particular polypeptides in common. It was possible to correlate the G2/albumin patterns with agglutinating activity of cow and rabbit blood cells as measured by the agglutination ratio (minimum concentration of extract required to agglutinate cow blood cells: minimum concentration of extract required to agglutinate rabbit blood cells). The active lectin polypeptides were identified by extracting lectins from agglutinated erythrocytes and by comparing the qualitative similarities and differences of the G2/albumin patterns and their agglutination activities. A reference catalogue of over 100 bean cultivars giving their phaseolin and G2/albumin electrophoretic patterns, and agglutination ratios is presented.     

Agglutination of African Primate and Rodent Erythrocytes by Adenoviruses, Reoviruses, and Enteroviruses

African nonhuman primate and rodent erythrocytes were tested for agglutination by adenoviruses, reoviruses, and enteroviruses. Squirrel erythrocytes were agglutinated by reovirus serotypes and adenovirus types 3, 11, 16, and 21. Adenoviruses also agglutinated brazza monkey erythrocytes to the same titers as those obtained with either rhesus or grey monkey cells. Prototype reovirus types 1 and 2 agglutinated grey monkey erythrocytes to much lower titers than either squirrel or human group O red cells. Among the enteroviruses tested, only echovirus types 7 and 12 agglutinated grey, red-tail, brazza, and rhesus monkey erythrocytes. The specificity of agglutination of squirrel, grey, and brazza monkey erythrocytes by reoviruses, echoviruses, and adenoviruses, respectively, was confirmed by hemagglutination-inhibition tests. The titers obtained were similar to those obtained with erythrocytes usually used in these tests. Erythrocytes of bush babies, potto unstriped grass mice, swamp rat, rusty-nosed rat, bush rat, harsh-furred mice, soft-furred rat, and giant rat were not agglutinated by adenoviruses, reoviruses, or enteroviruses.     

Protoplasmic streaming of an internodal cell of Nitella flexilis; its correlation with electric stimulus

The sudden cessation or sudden decrease in velocity of the protoplasmic streaming of Nitella flexilis is observed whenever an action potential is elicited. The action potential can be generated by an electric stimulus after its refractory period, whether the flow is at a complete standstill or on the way to recovery. The membrane potential is generally decreased more or less when the rate of flow is decreased on application of salts or other agents. There is, however, no parallelism between these two. The membrane potential decreases proportionally with applied voltage of subthreshold intensity, while the rate of flow does not change appreciably. Only on application of a superthreshold voltage does the flow stop suddenly. In one case the rate of flow decreased to half without appreciable decrease in membrane potential. In another case it continued flowing at about one-half rate, although the membrane potential was almost zero. The Q₁₀ of the rate of flow is about 2, while it is 1.1 to 1.5 for the membrane potential. The sudden cessation of the protoplasmic streaming is supposed to be caused by the temporary formation of certain interlinkages among contractile protein networks in the endoplasm during excitation at the cathodal half of Nitella.     

Effect of metabolic state on phytohemagglutinin-P agglutination of normal human erythrocytes.

A reproducible quantitative assay for the lectin-mediated agglutination of human erythrocytes, depending on different rates of settling of agglutinated and nonagglutinated erythrocytes, was developed. This assay was used to study the aggregation of human erythrocytes by phytohemagglutinin-P. The aggregation of human erythrocytes by phytohemagglutinin-P was found to depend upon the metabolic state of the cells. Metabolically depleted erythrocytes agglutinated much less readily than did similar cells supplied with adenosine. This was not due to swelling and rigidity of the cells, since erythrocytes in hypotonic solution did not exhibit significantly altered phytohemagglutinin-P agglutination. Metabolically depleted erythrocytes, or erythrocytes from blood stored 8 weeks, lysed and resealed in the presence of ATP, were agglutinated by phytohemagglutinin-P to a much greater extent than control samples without ATP. The presence of Mg2+, either alone or with ATP, had little effect on the agglutinability of the resealed membranes. Low concentrations of Ca2+ (0.2 mM) had little effect on agglutinability, although high Ca2+ (5 mM) inhibited agglutinability of the resealed membranes somewhat. Both metabolically depleted erythrocytes and depleted erythrocytes, previously treated with adenosine, when treated with trypsin released similar amounts of sialic acid. The agglutinability of the trypsinized adenosine-supplemented cells increased more readily than did that of trypsinized metabolically depleted cells. The agglutination of erythrocytes was not affected by cytochalasin B (40 mug/ml). Vinblastine (0.2 mM) caused depleted erythrocytes to agglutinate similarly to adenosine-supplemented erythrocytes, but had no effect on the agglutination of adenosine-supplemented erythrocytes. It is concluded that ATP in the human erythrocyte probably participates in the modulation of phytohemagglutinin-P agglutinability. This is not a consequence of the more rigid membrane known to accompany ATP depletion in the erythrocyte, or of the effect of ATP levels on Ca2+ or Mg2+ content. It appears likely that ATP modulates human erythrocyte phytohemagglutinin-P agglutinability through interaction, direct or indirect, with a membrane-associated component, which might also be sensitivie to vinblastine.     

The role of flagellar adhesion in sexual activation ofChlamydomonas eugametos

Summary Sex-specific agglutination in gametes ofChlamydomonas eugametos was carried out with dead partner cells (killed by glutaraldehyde, formaldehyde or OsO₄), with isolated flagella, flagellar appendages and isoagglutinins derived from cell-free culture medium. The activation of the plasma papilla in agglutinated cells was studied by scanning electron microscopy in relation to the agglutinative properties of the materials tested. The results reveal differences in agglutination and papilla activation between gametes of both mating types. They also indicate that papilla activation depends on the extent of agglutination, which is a function of sex-specific flagellar properties and the amount and nature of the agglutinative material used. A hypothesis is presented to explain the observed phenomena.     

Ontogeny of murine T lymphocytes: I. Maturation of thymocytes induced in vitro by tumor necrosis factor-positive serum (TNF+)

One question which is unresolved in developmental immunology is whether cortical thymocytes are the precursor cells which give rise to medullary thymocytes and peripheral T cells. Cortical thymocytes display a characteristic surface antigen phenotype (high TL and Thy-1, low H-2, no Qa-2, no Qa-3), are agglutinated by peanut agglutinin (PNA), and are unresponsive to concanavalin A (Con-A). The functionally more mature medullary thymocytes express a surface phenotype more closely resembling peripheral T cells (no TL, low Thy-1, high H-2, and some Qa-2), are not agglutinated by PNA, and are responsive to Con-A. An in vitro induction system has been devised in which mouse thymocytes undergo quantitative changes in surface antigens in less than 24 hr and increase their mitogen response to Con-A. The phenotypic changes are characterized by a decrease of TL and Thy-1 and an increase in H-2, Qa-2, and Qa-3. Studies in which thymocytes were fractionated on BSA gradients and by PNA agglutination demonstrate that the inducible cells have the properties of cortical thymocytes. Our data show that a subpopulation of cortical thymocytes can acquire phenotypic characteristics similar to medullary thymocytes and peripheral T cells.     

Lectin-induced lymphocyte agglutination : An active cellular process?

The agglutination of lymphocytes by lectins (Concanavalin A, phytohemagglutinin and pokeweed mitogen) is studied with fluorochrome-labelled lectins and by fluorescence microscopy. Both agglutinated and non-agglutinated cells pick up similar amounts of lectins as judged by their equivalence cell membrane brightness. At the level of resolution of optical microscopy no difference can be detected in the distribution of lectins bound by agglutinated or non-agglutinated cells. However a profound inhibition of agglutination by inhibitors of the capping process (sodium azide and cytochalasin B) suggests the involvement in the agglutination process of a step that requires active cell metabolism and microfilament function.     

Comparative lectin-binding and agglutination properties of the strain-specific,TA3-St,and the non-strain-specific,TA3-Ha,murine mammary carcinoma ascites sublines. Further studies of receptors in epiglycanin

The complex carbohydrates at the cell surfaces of two TA3, murine mammary carcinoma ascites sublines (the strain-specific, TA3-St subline and the nonstrain-specific, TA3-Ha line) were compared by binding studies with ¹²⁵I-labelled concanavalin A (con A), Ricinis communis agglutinin (RCA), and eel-serum agglutinin (ESA). The TA3-Ha cell bound equal amounts of con A, 1.5-fold more RCA, and 4-fold more ESA than the TA3-St cell. Binding-inhibition studies by these lectins and two others [wheat-germ agglutinin (WGA) and potato lectin (STA)] suggest complementary binding-sites between con A and both RCA and ESA. Quantitative agglutination studies with the five lectins, and inhibition determinations by both neuraminidase-treated and untreated epiglycanin revealed that TA3-St, but not TA3-Ha, cells were agglutinated by con A, and that epiglycanin inhibited this agglutination, as well as the agglutination of rabbit erythrocytes by con A. The presence of a con A receptor on epiglycanin was also suggested by the binding of epiglycanin to con A-Sepharose, and its specific elution with methyl α-d-manno-pyranoside. TA3-St cells were agglutinated at a 10-15-fold lower concentration of either STA or RCA than TA3-Ha cells, but both cells were agglutinated by the same concentration of WGA and ESA. Inhibition by epiglycanin of agglutination of TA3-St cells by either STA or ESA occurred at a concentration lower than that of TA3-Ha cells, but epiglycanin inhibited RCA agglutination of TA3-Ha cells at a concentration     

Depolarization of Cell Membrane Potential during Trans-Plasma Membrane Electron Transfer to Extracellular Electron Acceptors in Iron-Deficient Roots of Phaseolus vulgaris L

Transfer of electrons from the cytosol of bean (Phaseolus vulgaris L.) root cells to extracellular acceptors such as ferricyanide and Fe^IIIEDTA causes a rapid depolarization of the membrane potential. This effect is most pronounced (30-40 millivolts) with root cells of Fe-deficient plants, which have an increased capacity to reduce extracellular ferric salts. Ferrocyanide has no effect. In the state of ferricyanide reduction, H⁺ (1H⁺/2 electrons) and K⁺ ions are excreted. The reduction of extracellular ferric salts by roots of Fe-deficient bean plants is driven by cellular NADPH (Sijmons, van den Briel, Bienfait 1984 Plant Physiol 75: 219-221). From this and from the membrane potential depolarization, we conclude that trans-plasma membrane electron transfer from NADPH is the primary process in the reduction of extracellular ferric salts.     

Lectin receptors on the cell surface membrane and the kinetics of lectin-induced cell agglutination.

Agglutination of malignant transformed hamster cells by concanavalin A (ConA) and the lectins from wheat germ (WGA) and soybean (SBA) has been automatically quantitated, by measuring the amount of light transmitted through a cell suspension. The transformed hamster cells were agglutinated by SBA only after treatment with neuraminidase. The initial rate of agglutination and the concentration of lectin (K_c) required for the half-maximum rate (V_m) has been determined. The initial rate and V_m were lower and more temperature-sensitive, and the K_c was higher, for ConA than for WGA and SBA. There was no detectable temperature-dependent phase transition for the initial rate of agglutination. The total number of receptors was lower for ConA than for WGA and SBA and the apparent association constant between lectin molecules and cell surface receptors was higher for ConA (10⁷M^?1) than for WGA and SBA (1.6 × 10⁶M^?1). The half V_m of agglutination required 75% saturation of the cell receptors for ConA, and only 13–17% saturation of the receptors for SBA and WGA. A 30% decrease in the number of SBA receptors present in agglutinable cells completely prevented their agglutination. The results indicate that there is heterogeneity of lectin receptors on the cell surface and that only a small proportion of the total number of WGA and SBA receptors have to be occupied for agglutination by these lectins.     

Surface Alterations of Cells Carrying RNA Tumour Virus Genetic Information

CELLS transformed by the DNA tumour viruses, polyoma virus and SV40, are agglutinated by lectins such as wheat germ agglutinin¹, concanavalin A (Con A)² and soybean agglutinin³. Agglutination in these cases presumably reflects changes in the cell surface related to the transformed properties of the cell; studies with a temperature-dependent mutant of polyoma virus has shown that cell surface changes are controlled by viral genes⁴. Here we describe experiments in which we investigated the agglutinability of cells transformed by RNA tumour viruses. One recent report had suggested that cells transformed by RNA tumour viruses were not specifically agglutinated⁵, whereas a second more recent report claimed the specific agglutination of cells transformed by RSV⁶. We find that transformed rat, mouse and cat cells that replicate the sarcoma-leukaemia virus complex of murine (MSV) and feline (FeSV) origin are strongly agglutinated by Con A, but mouse and human cells that replicate the murine and feline leukaemia virus components alone are not agglutinated. The ability to agglutinate is rapidly acquired by normal mouse cells on infection with the murine sarcoma virus at a rate that parallels virus replication. In contrast to the results obtained with cells producing virus, non-virus-producing transformed hamster and mouse cells that synthesize virus-specific RNA are either not agglutinated or are agglutinated to a lesser degree. These results suggest that the cell surface alterations responsible for agglutination are not necessarily associated with the transformed state of the cell, but rather with the possession of sarcoma virus-specific information.     

Membrane Potential in Phaeoceros laevis: Effects of Anoxia, External Ions, Light, and Inhibitors

Gametophyte cells of Phaeoceros laevis (L.) Prosk. have vacuole electric potentials (PDs) of about −175 millivolts; the steady PD is not affected by light but small transient PDs result after changing from light to darkness or darkness to light. The PD is more negative than the Nernst potentials for any of the permeating ions. Changes in the concentration of any one of the external ions between 0.1 and 10 mm have only a very small effect on the PD. Increases in external pH cause the PD to depolarize by a few millivolts. Azide, 2,4-dinitrophenol, and NH₄Cl each cause rapid and reversible depressions of the PD; the effects of these agents are similar in magnitude in the light and in the dark. Anoxia depolarizes the PD by about 30 millivolts in the light and by about 60 millivolts in the dark. Ouabain and 3-(3,4-dichlorophenyl)-1,1-dimethylurea have no effects on the PD. It is concluded that the membrane potential is controlled by an electrogenic efflux pump, possibly for H⁺. It is also concluded that the source of energy for the pump is respiration and not photosynthesis.     

Anti-human erythrocyte antibodies in horse-derived antivenoms used in the treatment of snakebite envenomations

This work examined the presence of antibodies reacting with human erythrocytes in horse-derived antivenoms used in the treatment of snakebite envenomations, and assessed the efficacy of various fractionation protocols in the elimination of agglutinating antibodies. A number of antivenoms produced by various fractionation protocols were tested for direct agglutination of human erythrocytes. Reactions were observed visually and microscopically, and an indirect anti-equine globulin test was also used. In addition, rabbits and mice were injected intravenously with antivenoms to observe possible intravascular hemolysis and erythrocyte sequestration. All tested antivenoms agglutinated human erythrocytes, albeit to different extent, and also gave a positive anti-globulin test. Agglutination was due to IgG(T) subclass of antibodies. Pepsin digestion of horse IgG, to obtain F(ab′)₂ fragments, reduced the direct agglutination, but not the indirect anti-globulin test. Ion-exchange chromatography of IgG in a strongly basic quaternary ammonium cellulose membrane abrogated direct agglutination and reduced the indirect anti-globulin test. Binding of antivenom antibodies to erythrocytes in vivo was demonstrated in rabbits, although there was no evidence of intravascular hemolysis or erythrocyte sequestration in rabbits and mice. It is concluded that anti-human erythrocyte antibodies are present in horse-derived antivenoms, and that fractionation of horse plasma by pepsin digestion, and especially by anion-exchange chromatography, reduces the titer of these antibodies. Our in vivo experimental results do not support a role for these antibodies in early adverse reactions occurring after antivenom administration.     

Divalent cation enhancement of the agglutinability by soyabean lectin of liposomes prepared from total lipid of erythrocytes and of erythrocyte membranes

CaCl₂ or MgCl₂ but not NaCl enhances the soyabean lectin-induced agglutination of liposomes prepared from total lipids of erythrocyte membranes. The addition of purified phosphatidylserine to the total lipids of erythrocyte membranes before the formation of liposomes inhibits lectin-induced agglutinability of the preparation in the absence of CaCl₂, but not in its presence. When preformed phosphatidylserine liposomes are added to liposomes of total lipids of erythrocyte ghosts, they do not inhibit agglutination, indicating that phosphatidylserine does not inhibit the lectin directly. CaCl₂ or MgCl₂ but not NaCl also stimulates the soyabean lectin-induced agglutination of human erythrocyte membranes.Electron micrographs indicate that the liposome preparations are multilamellar and separate even in the presence of CaCl₂. When such liposomes are treated with lectin with or without CaCl₂, the electron micrographs show significant agglutination without apparent fusion. The reversal of the agglutination of liposomes by specific sugars followed by turbidimetric and electron microscopic techniques supports the conclusion that CaCl₂ stimulated lectin-induced agglutination is unaccompanied by fusion.The stimulation by divalent cations of lectin-induced agglutination of erythrocyte ghosts or of our liposomes may be due to a decrease in apparent surface charge of these membrane systems.     

Agglutination of Erwinia stewartii Strains with a Corn Agglutinin: Correlation with Extracellular Polysaccharide Production and Pathogenicity

A bacterial agglutinin was extracted from ground corn (WI hybrid 64A × W117) seed with phosphate-buffered saline (pH 6.0) and precipitated with (NH₄)₂SO₄ at 70% saturation. The activities of this agglutinin against 22 strains of Erwinia stewartii (agent of bacterial wilt of corn) that varied in virulence were determined. Specific agglutination (agglutination titer per milligram of protein per milliliter) values were correlated negatively with virulence ratings. Strains with high specific agglutination values (15 or higher) were avirulent or weakly virulent; strains with low specific agglutination values (10 or lower) were highly virulent, with two exceptions. Avirulent strains produced butyrous colonies and released only small amounts of extracellular polysaccharide (EPS) into the medium, and the cells lacked capsules; virulent strains produced fluidal colonies and released large amounts of EPS, and the cells were capsulated. There was a strong correlation between the amount of EPS produced by each strain (as determined by increase in viscosity of the medium) and the specific agglutination value; in contrast, lipopolysaccharide compositions were similar in all strains. When cells of six fluidal strains were washed by repeatedly centrifuging and resuspending them in buffer, they were agglutinated more strongly by corn agglutinin than were unwashed cells. When avirulent cells were washed, their specific agglutination values did not increase significantly. Eight EPS-deficient mutants of E. stewartii, selected for resistance to the capsule-dependent bacteriophage K9, had lower virulence but higher specific agglutination than did their corresponding wild-type parents. Production of EPS appears to be essential for virulence; EPS may prevent agglutination of bacteria in the host, thus allowing their multiplication.     

STABILITY OF SUSPENSIONS OF SOLID PARTICLES OF PROTEINS AND PROTECTIVE ACTION OF COLLOIDS

1. It is shown that the concentrations of different salts required to precipitate suspensions of gelatin-coated collodion particles in water are practically identical with the concentrations of the same salts required for the "salting out" of gelatin from aqueous solutions. Neither effect shows any relation to the electrical double layers surrounding the particles. 2. It is shown that at the isoelectric point of gelatin, suspensions of gelatin-coated collodion particles are not stable and it had been shown previously that gelatin is least soluble at the isoelectric point. The addition of salt increases both the solubility of gelatin in water as well as the stability of suspensions of gelatin-coated collodion particles in water, and both effects increase with the valency of one of the ions of the salt. 3. This latter effect is not due to any charges conferred on the gelatin particles by the salts, since the cataphoretic experiments show that salts like NaCl, Na₂SO₄, or CaCl₂, which at the isoelectric point of gelatin increase the solubility of gelatin as well as the stability of suspensions of gelatin-coated collodion particles, leave the particles practically uncharged in the concentrations in which the salts are efficient. 4. It follows from all these facts that the stability of suspensions of gelatin-coated particles in water depends on the solubility of gelatin in water; e.g., on the chemical affinity of certain groups of the gelatin molecule for water. 5. Though crystalline egg albumin is highly soluble in water, the stability of collodion particles coated with crystalline egg albumin does not depend upon the affinity of the albumin molecule for water, but depends practically alone on the electrical double layer surrounding each particle. As soon as the P.D. of this double layer falls below 13 millivolts, the suspension is no longer stable. 6. The critical potential for the stability of suspensions of collodion particles coated with genuine egg albumin is the same as that for particles of boiled (denatured) white of egg. Since through the process of heating, egg albumin loses its solubility in water, it is inferred that egg albumin undergoes the same change when it forms a film around a solid particle like collodion. 7. The influence of electrolytes on the stability of suspensions of collodion particles coated with casein or edestin was similar to that of collodion particles coated with egg albumin. The experiments are, however, complicated by the fact that near the isoelectric point CaCl₂ and even NaCl cause a suspension again at concentrations of about M/2 or 1 M, while still higher concentrations may cause a precipitation again. These latter effects have no connection with double layers, but belong probably in the category of solubility phenomena. 8. These experiments permit us to define more definitely the conditions for a general protective action of colloids. Protective colloids must be capable of forming a durable film on the surface of the suspended particles and the molecules constituting the film must have a higher attraction for the molecules of the solvent than for each other; in other words, they must possess true solubility. Only in this case can they prevent the precipitating action of low concentrations of electrolytes on particles which are kept in suspension solely by the high potentials of an electrical double layer. Thus gelatin films, in which the attraction of the molecules for water is preserved, have a general protective action, while crystalline egg albumin, casein, and edestin, which seem to lose their attraction for water when forming a film, have a protective action only under limited conditions stated in the paper.     

INFLUENCE OF A SLIGHT MODIFICATION OF THE COLLODION MEMBRANE ON THE SIGN OF THE ELECTRIFICATION OF WATER

1. It is shown that collodion membranes which have received one treatment with a 1 per cent gelatin solution show for a long time (if not permanently) afterwards a different osmotic behavior from collodion membranes not treated with gelatin. This difference shows itself only towards solutions of those electrolytes which have a tendency to induce a negative electrification of the water particles diffusing through the membrane, namely solutions of acids, acid salts, and of salts with trivalent and tetravalent cations; while the osmotic behavior of the two types of membranes towards solutions of salts and alkalies, which induce a positive electrification of the water particles diffusing through the membrane, is the same. 2. When we separate solutions of salts with trivalent cation, e.g. LaCl₃ or AlCl₃, from pure water by a collodion membrane treated with gelatin, water diffuses rapidly into the solution; while no water diffuses into the solution when the collodion membrane has received no gelatin treatment. 3. When we separate solutions of acid from pure water by a membrane previously treated with gelatin, negative osmosis occurs; i.e., practically no water can diffuse into the solution, while the molecules of solution and some water diffuse out. When we separate solutions of acid from pure water by collodion membranes not treated with gelatin, positive osmosis will occur; i.e., water will diffuse rapidly into the solution and the more rapidly the higher the valency of the anion. 4. These differences occur only in that range of concentrations of electrolytes inside of which the forces determining the rate of diffusion of water through the membrane are predominantly electrical; i.e., in concentrations from 0 to about M/16. For higher concentrations of the same electrolytes, where the forces determining the rate of diffusion are molecular, the osmotic behavior of the two types of membranes is essentially the same. 5. The differences in the osmotic behavior of the two types of membranes are not due to differences in the permeability of the membranes for solutes since it is shown that acids diffuse with the same rate through both kinds of membranes. 6. It is shown that the differences in the osmotic behavior of the two types of collodion membranes towards solutions of acids and of salts with trivalent cation are due to the fact that in the presence of these electrolytes water diffuses in the form of negatively charged particles through the membranes previously treated with gelatin, and in the form of positively charged particles through collodion membranes not treated with gelatin. 7. A treatment of the collodion membranes with casein, egg albumin, blood albumin, or edestin affects the behavior of the membrane towards salts with trivalent or tetravalent cations and towards acids in the same way as does a treatment with gelatin; while a treatment of the membranes with peptone prepared from egg albumin, with alanine, or with starch has no such effect.