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 MECHANISM OF GRANULAR GROWTH OF RABBIT SEPTICEMIA BACILLUS TYPE G

The acid agglutination optimum of Microbes D and G is not independent of the nature of the buffer mixture. Glycocoll-HCl buffer mixtures cause complete flocculation at high C_H+ (2.7 to 2.4), at which points little or no flocculation occurs with the Na lactate-lactic acid buffer series. Beef infusion has the property of broadening the acid agglutination optimum of both Microbes D and G, bacilli of rabbit septicemia. This extension is in the direction of a lower C_H+. There is no evidence that the beef infusion has the power, per se, of agglutinating these organisms. It would seem merely to increase their sensitiveness to sedimentation in the presence of H ions. The data presented explain the mechanism of the granular growth character of Microbe G in liquid media as compared to the diffuse growth of Microbe D. Peptone (Fairchild), contrary to beef infusion, shifts the acid agglutination optimum of Microbes D and G in the direction of a higher C_H+. Strong concentrations of peptone exhibit an inhibitory effect on the agglutination of Microbe D in the optimum zone.     

THE AGGLUTINATION OF RED BLOOD CELLS

1. Unsensitized sheep cells suspended in sugar solutions are agglutinated by electrolytes whenever the potential is depressed to 6 millivolts or less, except in the case of MgCl₂ or CaCl₂. 2. With these salts no agglutination occurs although there is practically no potential. The presence of these salts prevents acid agglutination. This is presumably due to a decrease in the "cohesion" between the cells. 3. Cells which have been sensitized with specific antibody, ricin, colloidal stannic hydroxide, or paraffin oil, are agglutinated whenever the potential is decreased below about 12 millivolts. 4. The agglutination by electrolytes is therefore primarily due to a decrease in the potential whereas agglutination by immune serum, ricin, etc., is due primarily to an increase in the critical potential.     

THE RATE OF REDUCTION OF METHYLENE BLUE BY BACILLUS COLI

This paper deals with the relation between substrate concentration and velocity in the case of the reduction of methylene blue and of the other oxidation-reduction indicators of Clark by B. coli in the presence of succinic acid and glucose. This system is compared with starch and barley amylase. Reasons are given for considering the mechanism as an adsorption phenomenon.     

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.     

INCREASE IN BACTERIOPHAGE AND GELATINASE CONCENTRATION IN CULTURES OF BACILLUS MEGATHERIUM

1. The increase in bacteria, phage concentration, and gelatinase concentration in cultures of B. megatherium has been determined. 2. With lysogenic cultures the phage concentration, gelatinase concentration, and bacteria concentration increase logarithmically at first. The phage and gelatinase concentration then decrease while the bacteria concentration increases to a maximum. 3. The results are the same with sensitive cultures if the ratio of phage to bacteria is small. If the ratio of phage to bacteria is large phage, gelatinase, and bacteria concentration all increase at first and then decrease. The maximum rate of increase coincides approximately with the maximum rate of oxygen consumption of the culture. 4. 60–90 per cent of the phage is free from the cells. 5. The amount of phage produced is determined by the combined phage and not by the total phage. 6. Phage is produced during growth of the cells and not during lysis. 7. In a very narrow range of pH near 5.55 no increase in bacteria occurs but large increases in phage may be obtained.     

Distinct lectin activities from six species of cellular slime molds.

Extracts of cohesive cells of four species of cellular slime mold, D. mucoroides, D. purpureum, D. rosarium and P. violaceum agglutinate erythrocytes in a manner that is similar to that previously observed with extracts of D. discoideum and P. pallidum. We determined inhibitory activity of a series of sugars on the agglutination activity of each of these extracts, using both semiquantitative and quantitative agglutination assays. The inhibitory potency of this series of sugars was distinct for each extract, although only slight differences were found between several species, especially D. discoideum and P. violaceum. A possible role of these agglutinins in species-specific cell cohesion is considered.     

PROTOPLASMIC POTENTIALS IN HALICYSTIS : III. THE EFFECTS OF AMMONIA

The nature and origin of the large "protoplasmic" potential in Halicystis must be studied by altering conditions, not only in external solutions, but in the sap and the protoplasm itself. Such interior alteration caused by the penetration of ammonia is described. Concentrations of NH₄Cl in the sea water were varied from 0.00001 M to above 0.01 M. At pH 8.1 there is little effect below 0.0005 M NH₄Cl. At about 0.001 M a sudden reversal of the potential difference across the protoplasm occurs, from about 68 mv. outside positive to 30 to 40 mv. outside negative. At this threshold value the time curve is characteristically S-shaped, with a slow beginning, a rapid reversal, and then an irregularly wavering negative value. There are characteristic cusps at the first application of the NH₄Cl, also immediately after the reversal. The application of higher NH₄Cl concentrations causes a more rapid reversal, and also a somewhat higher negative value. Conversely the reduction of NH₄Cl concentrations causes recovery of the normal positive potential, but the threshold for recovery is at a lower concentration than for the original reversal. A temporary overshooting or increase of the positive potential usually occurs on recovery. The reversals may be repeated many times on the same cell without injury. The plot of P.D. against the log of ammonium ion concentration is not the straight line characteristic of ionic concentration effects, but has a break of 100 mv. or more at the threshold value. Further evidence that the potential is not greatly influenced by ammonium ions is obtained by altering the pH of the sea water. At pH 5, no reversal occurs with 0.1 M NH₄Cl, while at pH 10.3, the NH₄Cl threshold is 0.0001 M or less. This indicates that the reversal is due to undissociated ammonia. The penetration of NH₃ into the cells increases both the internal ammonia and the pH. The actual concentration of ammonium salt in the sap is again shown to have little effect on the _P.D. The pH is therefore the governing factor. But assuming that NH₃ enters the cells until it is in equilibrium between sap and sea water, no sudden break of pH should occur, pH being instead directly proportional to log NH₃ for any constant (NH₄) concentration. Experimentally, a linear relation is found between the pH of the sap and the log NH₃ in sea water. The sudden change of P.D. must therefore be ascribed to some system in the cell upon which the pH change operates. The pH value of the sap at the NH₃ threshold is between 6.0 and 6.5 which corresponds well with the pH value found to cause reversal of P.D. by direct perfusion of solutions in the vacuole.     

COMPARATIVE STUDIES ON RESPIRATION : VIII. THE RESPIRATION OF BACILLUS SUBTILIS IN RELATION TO ANTAGONISM.

1. In relatively low concentrations of NaCl, KCl, and CaCl₂ the rate of respiration of Bacillus subtilis remains fairly constant for a period of several hours, while in the higher concentrations, there is a gradual decrease in the rate. 2. NaCl and KCl increase the rate of respiration of Bacillus subtilis somewhat at concentrations of 0.15 M and 0.2 M respectively; in sufficiently high concentrations they decrease the rate. CaCl₂ increases the rate of respiration of Bacillus subtilis at a concentration of 0.05 M and decreases the rate at somewhat higher concentrations. 3. The effects of salts upon respiration show a well marked antagonism between NaCl and CaCl₂, and between KCl and CaCl₂. The antagonism between NaCl and KCl is slight and the antagonism curve shows two maxima.     

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 PARASPORAL BODY OF BACILLUS LATEROSPORUS LAUBACH

On sporulation the slender vegetative rods swell and form larger spindle-shaped cells in which the spores are formed. When the spores mature they lie in a lateral position cradled in canoe-shaped parasporal bodies which are highly basophilic and can be differentiated from the surrounding vegetative cell cytoplasm with dilute basic dyes. On completion of sporulation the vegetative cell protoplasm and the cell wall lyse, leaving the spore cradled in its parasporal body. This attachment continues indefinitely on the usual culture medium and even persists after the spores have germinated. In thin sections of sporing cells the bodies are differentiated from the cell protoplasm by differences in structure. Whereas the protoplasm has a granular appearance, in both longitudinal and cross-sections the parasporal body comprises electron-dense lamellae running parallel with the membranes of the spore coat and less electron-dense material in the interstices of the lamellae. The inner surface of the body is contiguous with that of the spore coat as if it were part of the spore, rather than a separate body attached to the spore. The staining reactions of the parasporal body are not consistent with those of any substance described in bacteria. With Giemsa the bodies stain like chromatin, but the Feulgen reaction indicates that they do not contain the requisite nucleic acid. With an aqueous solution of toluidine blue they stain metachromatically, but with an acidified solution the results are variable. Neisser's stain for polyphosphate is negative. The basophilic substance is removed from the body with some organic solvents. This basophilic substance has not been specifically identified with any material seen in ultrathin sections, but it is suggested that it might be the less electron-dense material in the interstices of the lamellar structure. In contrast to the spore coat of B. laterosporus, those of its two relatives B. brevis and B. circulans take up basic stain like the parasporal body. Thin spore sections of these species have shown that the walls are thicker than those surrounding the spores of B. laterosporus, and it is suggested that the outer stainable layer of brevis and circulans spores is an accessory coat which in laterosporus may have been deformed to give a parasporal body.     

COMPARATIVE STUDIES ON RESPIRATION : XXIV. THE EFFECTS OF CHLOROFORM ON THE RESPIRATION OF DEAD AND OF LIVING TISSUE.

1. Chloroform in low concentration (0.25 per cent) causes an increase in the rate of production of CO₂ in Ulva; this is followed by a decrease. In higher concentration (0.5 per cent) only a decrease is observed. 2. Assuming that the normal oxidation depends on the action of peroxide and peroxidase, experiments were made by placing Ulva in 1.0 per cent H₂O₂ and in Fe₂(SO₄)₃ (which acts like a peroxidase). The former diminishes the rate, the latter increases and subsequently decreases it. 3. When Ulva is killed in such a manner as to destroy the oxidizing enzymes, no CO₂ is produced unless H₂O₂ and Fe₂(SO₄)₃ are present. If to this mixture chloroform is added, the effect depends on the concentration of the iron. If the concentration is low there is an increase in the production of CO₂ followed by a decrease. If the concentration is high the rate appears to decrease from the start.     

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).     

THE FLOCCULATION OF BACTERIA BY PROTEINS

1. The effect of adding pure proteins to bacterial suspensions at different H ion concentrations has been studied. 2. The zone of flocculation of protein-treated bacteria bears a significant relationship to the isoelectric point of the protein used. With the higher concentration of protein, agglutination occurs at or near the isoelectric point of that protein; at reactions acid to this, the bacteria carry a positive charge and are not agglutinated. With diminishing concentration of protein, the zone of flocculation shifts toward and goes beyond that characteristic of the untreated bacteria. This occurs both in the presence and absence of salts. 3. A diversity of other suspensions, such as sols of gold, mastic, cellulose nitrate, cellulose acetate, Fe(OH)₃, oil emulsions, and erythrocytes, have been found by ourselves and others to exhibit a similar altered stability when treated with proteins in the same way.     

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.     

INHIBITORY ACTION OF PARATYPHOID BACILLI ON THE FERMENTATION OF LACTOSE BY BACILLUS COLI. I

Bacteria of the paratyphoid group may be divided into two classes according to the behavior of 4 day cultures in lactose bouillon after a second inoculation with certain types of Bacillus coli. One class includes all true hog-cholera bacilli, the other nearly all true paratyphoid and enteriditis types. Under the imposed conditions Bacillus coli produces the usual amount of gas in the presence of the first group. In the presence of the second no gas or only a bubble appears. The production of acid is not interfered with. The significance of the inhibition was investigated in a variety of ways suggested by the particular hypothesis entertained at the time. Two main possibilities presented themselves; first, the direct association of the inhibition with living paratyphoid bacilli, and, second, the existence of a ferment or other product of growth as the inhibiting agent. The theory that the living bacilli or those killed at the lowest possible temperature are responsible was favored by a number of experiments. Thus the complete removal of bacteria by filtration, or by centrifugation combined with the use of kaolin to produce a clear fluid restored gas production. The presence of a fine cloud of bacteria was sufficient to inhibit. On the other hand, the addition of large numbers of living bacteria from agar slants or from lactose bouillon after the requisite incubation period to fresh lactose bouillon failed to inhibit gas production when Bacillus coli was added simultaneously. When the inhibiting culture was heated at 62°C. for 35 minutes to sterilize it, gas production was still largely inhibited. But it was restored when higher temperatures were used, completely at 100°C. and above. It was also gradually restored by exposing the heated culture to 37°C. for 3 or more days. The presence of variable amounts of lactose, or even the complete absence of lactose did not interfere with the development of the inhibitory factor. The activity of the inhibition factor presents itself in the form of a curve, beginning at 0 when both paratyphoid and colon bacilli are inoculated simultaneously and rising as Bacillus coli is inoculated at longer intervals from the paratyphoid bacilli. The maximum of inhibition is reached at about the 4th day; thereafter it remains at the same level for a few days and then gradually falls until it is lost within 3 or 4 weeks. The curve of the hog-cholera group is delayed in that the maximum inhibition is reached at the end of 3 weeks. These curves have not been accurately determined. Taking into consideration all the accumulated data the writers tentatively present the hypothesis that the inhibitory factor is some metabolic product of the paratyphoid bacillus, possibly an enzyme, which is destroyed at a temperature somewhat above the thermal death point of the bacilli and which more gradually disappears from incubated cultures. The substance fails to pass Berkefeld filters. It is carried down mechanically with substances clearing the culture fluid. The experiments support current theories which hold that the acid-producing and gas-producing entities in cultures are distinct.     

PHOTOMETRIC EVIDENCE FOR THE OSMOTIC BEHAVIOR OF RAT LIVER MICROSOMES

Electron microscope observations are consistent with the interpretation that the elements of the endoplasmic reticulum are osmotically active in situ as well as after isolation. More recently, it has been reported that microsomal suspensions equilibrate almost completely with added C¹⁴-sucrose and that no osmotic behavior is evident from photometric data. These findings were considered at variance with the electron microscope data. However, equilibration with added label simply attests to a relatively high permeability, and, in addition, the photometric data need not be critical. Osmotic volume changes, measured photometrically, may be masked by concomitant events (e.g., changes in the refractive index of the test solutions at varying osmotic pressures, breakdown of the particles, and agglutination). For these reasons the photometric experiments were repeated. In this work, the reciprocal of optical density of microsomal suspensions was found to vary linearly with the reciprocal of concentration of the medium at constant refractive index. These changes probably correspond to osmotic volume changes, since the effect was found to be (a) independent of substance used and (b) osmotically reversible. The transmission of the suspension was found to vary with the refractive index of the medium, the concentration of particles, and the wavelength of incident light, according to relationships that are similar to or identical with those obtained for mitochondrial suspensions.     

THE BIOGENESIS OF PRIMARY SEX HORMONES : I. THE FATE OF ESTRINS INJECTED INTO THE RABBIT

1. A method is given for the extraction and fractionation of rabbit urines which frees these urines of inactive chromogens but permits a quantitative recovery of estrone and estriol for the colorimetric determination of these compounds. 2. Estrone and estriol content of rabbit urine extracts can be determined by the concentration of the colored compound they form upon diazotization with sulfanilic acid and by the modified phenolsulfonic acid test of Cohen and Marrian. Estriol can be determined by the specific reaction first described by David. The technique for these tests is presented. 3. Estriol (300 micrograms) injected into rabbits (a) in heat, (b) pregnant, (c) pseudopregnant, (d) hysterectomized in heat, (e) hysterectomized pseudopregnant, (f) ovariectomized, is excreted in the urine as estriol. Rabbit does in the luteal phase (b, c, and e) excrete 3 to 4 times the amount of estriol excreted by females without corpora lutea (a, d, and f). 4. When estrone (300 micrograms) is injected into the same types of rabbit does types a, b, and c excrete both estrone and estriol, type f excretes both estrone and estriol shortly after ovariectomy, but only estrone at 2 months after castration. Hysterectomized animals (types d and e) never excrete estriol after estrone injection. The total urinary estrin (estrone plus estriol) in estrone-injected animals is increased 2 to 3 times in animals in the luteal phase (b, c, and e). 5. It is concluded that the uterus is the site of conversion of estrone to estriol, and that the conversion cannot take place in a uterus completely free of ovarian control (e.g., in long time ovariectomized animals). 6. In neither estrone-injected nor estriol-injected females is all the injected hormone recovered in the urine. The maximum recovery is 66 per cent. When estrone-benzoate (600 micrograms) is injected 94–98 per cent of the hormone is recovered from animals in the luteal phase (types c and e) and about 79 per cent in an ovariectomized female (type f). These data are taken to indicate that luteal secretions give partial protection against destruction to the hormones. 7. The observation that in certain of the urine extracts the hormone titer by bioassay is somewhat higher than the colorimetric titer may indicate that there is a slight conversion of estrone to estradiol, particularly since no equilenin was found in any of the extracts by colorimetric test. 8. The simultaneous injection of 300 micrograms of estrone and 500 micrograms of progesterone 4 days after an initial injection of 300 micrograms of estrone results in: (1) an increased estrin excretion in females in heat, hysterectomized unmated, and ovariectomized, and a slight decrease in the pseudopregnant female; (2) the appearance of estriol in the urine of the long time ovariectomized animal with no urinary estriol in a control ovariectomized animal receiving no progesterone. These findings are taken to prove that the conversion of estrone to estriol occurs in the uterus under the influence of progesterone. Since animals in heat produce small amounts of estriol after estrone injection it is inferred that the ovaries of estrus rabbits produce small amounts of corpus luteum hormone in the absence of formed corpora lutea.     

THE KINETICS OF PENETRATION : V. THE KINETICS OF A MODEL AS RELATED TO THE STEADY STATE

An organic potassium salt, KG, passes from an aqueous phase, A, through a non-aqueous layer, B, into a watery solution, C. In C it reacts with CO₂ to form KHCO₃. The ionic activity product (K) (G) in C is thus kept at such a low level that KG continues to diffuse into C after the concentration of potassium becomes greater in C than in A. Hence potassium accumulates in C, the osmotic pressure rises, and water goes in. A steady state is eventually reached in which potassium and water enter C in a constant ratio. The rate of entrance of potassium (with no water penetrating into C) may fall off in a manner approximately exponential. But water enters and may produce an exponential decrease in concentration. This suggests that the kinetics may be treated like that of two consecutive monomolecular reactions. Calculations made on this basis agree very well with the observed values. The rate of penetration appears to be proportional to the concentration gradient of KG in the non-aqueous layer and in consequence depends upon the partition coefficients which determine this gradient. Exchange of ions (passing as such through the non-aqueous layer) does not seem to play an important rôle in the entrance of potassium. The kinetics of the model may be similar to that of living cells.     

THE ACCUMULATION OF ELECTROLYTES : II. SUGGESTIONS AS TO THE NATURE OF ACCUMULATION IN VALONIA

It is suggested that K enters chiefly as KOH, whose thermodynamic potential (proportional to the ionic activity product (a _K) (a _OH)) is greater outside than within. As this difference is maintained by the production of acid in the cell K continues to enter, and reaches a greater concentration inside than outside. KOH combines with a weak organic acid which is exchanged for HCl entering from the sea water (or its anion is exchanged for Cl^-), so that KCl accumulates in the sap. Na enters more slowly and its internal concentration remains below that of K. The facts indicate that penetration is chiefly in molecular form. As the system is not in equilibrium the suggestion is not susceptible of thermodynamic proof but it is useful in predicting the behavior of K, Na, and NH₄.