ABSTRACT
Locomotion. This does not occur to any appreciable extent in distilled water, acids, bases, KCN, H2O2, the leucobase of methylene blue, sugar, alcohol, papaverine hydro-chloride and quinine (the alkaloid, not its salt), when Amoebae are immersed in solutions of them in the concentrations mentioned. It does occur for varying lengths of time in certain concentrations of all the neutral salts used, and in an acid or base when a neutral salt is added. Salts which because of hydrolysis give an acid or basis reaction impede locomotion, depending on the degree of hydrolysis and the concentration.
The surface of Amoeba in various solutions. It is very adhesive in acids of a concentration of N/I to N/40,000, in salts which because of hydrolysis give an acid reaction, and in the leucobase of methylene blue. It is sufficiently adhesive in neutral salts in suitable concentrations to permit attachment to the substratum. Without such attachment, locomotion is impossible, therefore one of the conditions for locomotion is an adhesive surface, but the latter condition alone is no indication that locomotion will take place. The surface of Amoeba is not adhesive in sodium or potassium hydroxide, or in salts which because of hydrolysis give a basic reaction, and it is only indifferently adhesive in H2O2 and in the non-electrolytes and alkaloids used. It contracts in acids and considerably more in bases, although the surface is dentate in acids and smooth in bases, but undergoes no significant change in certain concentrations of neutral salts. The ectoplasmic surface ruptures readily in strong solutions of bases and salts. The time required varies with the concentration.
The ammonium salts used induce intermittent ejections of the endoplasmic contents, hence there is not a single rupture following which all the endoplasm is extruded, such as happens after a short or long period of immersion depending on the concentration in sodium or potassium hydroxide, or the salts used. The ectoplasmic surface is seen ·as a distinct morphological unit after it has ruptured in the reagents mentioned. In sodium or potassium hydroxide, it is preserved for varying lengths of time depending on the concentration, dissolving rapidly in strong solutions and more slowly in weak solutions. In salts, especially the neutral salts, it is preserved for several days, and is seen as a sort of vesicle containing the nucleus, some food-vacuoles, and a few crystalline particles attached to its inner surface.
The endoplasm of Amoeba in the solutions used. In acids and in the leucobase of methylene blue, it undergoes violent and unco-ordinated streaming. The streaming is conditioned in its violence and inco-ordination by the concentration of the acids, and by the degree of oxidation of the leucobase. It is more violent the stronger the acid and the less oxidation that has occurred in the leucobase at the time of immersion. Distilled water saturated with CO2 and HCN in fairly dilute solutions, as well as strong solutions, constitute media in which this does not occur. Streaming· is almost immediately stopped in CO2 and markedly impeded in HCN. In sodium or potassium hydroxide, KCN, H2O2, non-electrolytes and alkaloids, streaming is very slow even in very dilute solutions and stopped altogether in strong solutions. The inclusions of the endoplasm dissolve when Amoeba is immersed for some time in KCN and HCN, in alcohol and certain alkaloids in the concentrations mentioned, and in the leucobase of methylene blue. The food-vacuoles are distended in the solutions used of HCN, alcohol, ammonium salts, NH4OH, the leucobase of methylene blue and somewhat in LiCI, but are ejected only in NH4OH and ammonium salts, and with considerable endoplasm in many instances. The crystalline inclusions, which are ordinarily enclosed within a pellicle, i.e. each inclusion or crystal within a separate pellicle, come together in varying numbers within a common vesicle in certain solutions. There are usually two, three, or more such vesicles in a given specimen, each of which contains a dozen or more of such crystals. This sort of crystal aggregation occurs in KOH, N/4000; NH4OH, N/2000; NaHCO3, N/400; LiSO4, N/300, and a mixture of potassium nitrate, sulphate, and acetate, N/300. They may occur in other concentrations, but the above concentrations were the ones used when such aggregation was observed.
Time of inactivation or death of Amoeba in the various solutions. The time varies depending on whether or not the Amoebae were washed in distilled water (being shorter in the latter instance) as well as on the concentration. The following is for Amoebae that were washed in distilled water before immersion in the various solutions: (a) Acids, N/100 and N/1000, 7 minutes to 1 hour. (b) Sodium and potassium hydroxide, N/100 and N/200, 20 seconds to 1 minute; N/500 and N/1000, 2 to 3 hours; N/2000, 20 or more hours; N/4000, 48 or more hours. (c) KCN, molecular, made neutral to phenolphthalein by the addition of HCN, 12 minutes; KCN without the addition of HCN, 2 minutes; KCN, N/100 (made neutral as above), 2 or more hours; KCN, N/100, without HCN, 1 hour to hours. (d) Salts: NH4CI, N/300, 32 hours; NaCl, N/300, 266 hours; NaCl, N/100, 124 hours; a mixture of equal parts of KCl, N/500 with KOH, N/4000, 64 hours; a mixture of equal parts of KCl, N/500 with HCl, N/6000, Sr hours; CaCl2, N/300, 211 hours; NaCl and CaCl2 mixed in equal parts, N/300 each, 266 hours; NaCl and CaCl2 N/100,mixed in equal parts, 124 hours; LiCl, N/300, 116 hours; NaCl, KCl, and NH4CI, N/500, mixed in equal parts of each, 125 hours; BaCl2, SrCl2, and CaCl2 N/500, mixed in equal parts of each, 56 hours ; NH4N 08, N/ 300, 33 hours ; NaN 08, N/300, 92 hours; KNO8, N/300, 88 hours; RbNO8, N/300, 62 hours; Ca(NO8)2, N/300, 1 16 hours; NaNO8 and Ca(NO8)2, N / 300, mixed in equal parts of each, 119 hours ; NaNO8, KNO8, and Ca(NO8)2 N/300, mixed in equal parts of each, 132 hours; (NH4) 2SO4, N/300, 32 hours; Na2SO4, N/400, 60 hours ; Li2SO4, N/300, 30 hours ; LiCl and Li2SO.4 N /300, mixed in equal parts of each, 30 hours ; Na2SO4, K2SO4, and (NH4)2SO4, N/400, mixed in equal parts of each, 124 hours; NH4C2H8O2, N/300, 4 hours; NH4HCO8, N/300, 2 to 3 hours; NaC2H9O2, N/400, 35 hours; NaHCO8, N/400, 93 hours; NaC2H8O2 and KC2H8O2, N/300, mixed in equal parts of each, 52 hours; NH4Cl, NH4NOa, and (NH4)2SO.4, N/300, mixed in equal parts of each, 14 hours; NH4NO3, (NH4) 2 SO4, and NH4C2H8O2, N/ 500, mixed in equal parts of each, 3 hours; NaCl, NaNO3, and Na2SO4, N/300, mixed in equal parts of each, 92 hours ; Na CI, Na2 SO4, and NaC2H802, N/300, mixed in equal parts of each, 55 hours.
The effect of acids. They seem to make the surface of Amoeba gelatinous and adhesive, and this condition obtains in very dilute solutions, e.g. N/20,000. The effect is reversible if the Amoebae are transferred to culture fluid or a weak neutral salt solution before the action becomes too prolonged. HCN is different in its action on Amoeba from the other acids used. It is similar in effect to alcohol and KCN when the latter is made neutral by the addition of HCN. Distilled water saturated with CO2 apparently causes a rapid entrance of fluid, so that the granular endoplasm is surrounded by a broad hyaline area.
The effect of bases. Sodium and potassium hydroxide seem to liquefy the surface of Amoeba so that it is inadhesive and very susceptible to rupture. Ammonium hydroxide apparently neither liquefies nor gelates the surface, but evidently passes rapidly into the cell causing marked changes in the endoplasm, which is manifested chiefly in the eruptive character of streaming and the ejection from time to time of the food-vacuoles and other endoplasmic contents. The effect is apparently dependent, as Jacobs4 indicates, on the absolute concentration of the free base present.
The effect of salts. All of the neutral salts used are for varying lengths of time depending on the concentration and the salt favourable to normal activity. The cations, in so far as the effect of salts is ionic, produce marked effects, while the anions are relatively inactive, although it appears that the chlorine ion is less toxic than the nitrate ion, and the nitrate ion than the sulphate ion. The cations differ in their action, and likewise the anions and univalent and bivalent ions also differ among themselves as well perhaps as a group. The sodium ion seems less toxic than lithium, and lithium than potassium, potassium than rubidium, and rubidium than ammomum. Calcium is less toxic than barium, strontium, or magnesium. The carbonate and acetate ions are more toxic than the chlorine, nitrate, or sulphate ions, and the acid carbonate less toxic than the basic carbonate.
The effect of oxidising and reducing agents. Hydrogen peroxide appears to induce an irreversible permeability. The leucobase of methylene blue makes the surface of Amoeba gelatinous and adhesive, and stops almost immediately all activity. The endoplasm becomes hyaline, and at death the Amoeba is seen as a spherically lobose vesicle.
The effect of non-electrolytes. Cane sugar and glucose impede locomotion depending on the concentration. No significant abnormalities were noted. Alcohol seems to liquefy the surface and make the cell permeable. The endoplasm becomes after a time gradually hyaline. Streaming is impeded although at times it is eruptive and unco-ordinated.
The effect of alkaloids. The alkaloidal salt, quinine hydrochloride, causes no apparent abnormalities, but the alkaloid, quinine, and papaverine hydrochloride prevent locomotion and reduce streaming. The endoplasm in the latter solutions becomes partially hyaline. The surface of Amoeba is apparently made more permeable than normally, and also partially liquefied.
The effect of the chemicals used on enucleated Amoebae. Enucleated Amoebae disintegrate more readily in all of the solutions used than nucleated ones. The PH of the endoplasm seems also to be lowered in enucleated Amoebae.
The ectoplasmic surface of Amoeba is elastic. Mechanically induced pseudopods that do not contract are prevented from contracting by adhesion to the substratum. Protoplasm from a ruptured Amoeba was not observed to form a new surface membrane on coming in contact with the surrounding medium. Torn ectoplasm heals by a drawing together of the torn surface at the ruptured place. The endoplasm extruded during a temporary rupture takes no part in closing the rupture.
1. Introduction†
After subjecting Amoebae to localised stimlation, the results of which are presented in section I of this investigation,1 it was thought profitable to ascertain the general effect produced by immersing Amoebae in the various solutions which had been previously used in obtaining reactions to localised stimulation. The following presentation of the results of such experiments indicate the nature of the reactions observed.
The writer gratefully acknowledges the aid received from Professor S. O. Mast in carrying on this investigation, and that received from him and Professor H. S. Jennings for helpful criticism of the manuscript.
2. Material and Methods
The Amoebae used in the following experiments correspond with Schaeffer’s 8 description of Amoeba proteus, except that the nucleus is usually biconcave. They were obtained originally from a stream in the vicinity of Baltimore and transferred to cultures prepared as previously described in section I.1
For all the following experiments, the Amoebae in a few drops of culture fluid were taken from the culture dish and washed in three changes of distilled water. The total time employed in washing did not exceed thirty minutes. Specimens were then distributed to each of several Syracuse watch-glasses containing the solution whose effect it was desired to observe. Usually for a given series of experiments, a hundred or more Amoebae were taken from the same culture dish and washed in distilled water. Twenty or more were then immersed in different solutions at the same time in order to observe the comparative effects produced. Amoebae were similarly treated when testing the effect of different concentrations of a given chemical. Observations were made under a Leitz binocular microscope and a Bausch and Lomb monocular microscope with water immersion lens.
3. Observations and Results
1. Acids
The following acids were used — HCl, HNO8, H2SO4, HCN, (COOH)2, and H2CO8.The effect of all of these and of salts such as ferric, zinc, or nickel chloride, which because of hydrolysis give an acid reaction, is essentially the same except the effect of HCN and H2CO8. The Amoebae become sticky at their surface and a slight general contraction takes place, the degree of stickiness and contraction depending on the concentration. Locomotion is markedly impeded and streaming becomes violent and unco-ordinated. There is no apparent indication that the intake and outgo of fluids is appreciably disturbed. The antero-posterior differentiation is preserved and made more conspicuous by the fact that usually immediately before death, after one or more reversals in streaming, the posterior end becomes strikingly contracted so that practically no endoplasm is visible in it. It then appears as a collapsed tube with villous processes. Eruptive advances are made consecutively in various directions around the anterior end. The eruptive character increases as the contraction of the posterior third increases. When this latter third has become markedly contracted it begins to coagulate and turn yellow. This dead posterior third is frequently eliminated from the living portion or dragged behind attached by a filament. The remainder, if transferred to culture fluid, resumes normal activity after a time. Occasionally, coagulation sets in at the first reversal in streaming, so that the specimen at death is, as it were, hind part before. In the more dissociable acids,· e.g. HCl, the effect seems to be owing to the H-ion.
In HCN, 2 per cent. Amoebae die within a short time, and their appearance at death is similar to that seen in other acids (fig. 3). In a more dilute solution N/150, N/250 HCN induces a hyaline appearance throughout the endoplasm, but death usually follows before all of the solid particles such as crystals and granules have disappeared (fig. 4). The few that remain are located at the posterior end near the lower surface. If the Amoeba is turned over, they slowly settle again. The crystals evidently dissolve within the spherule which surrounds them, for the spherules are still easily distinguishable after the crystals have disappeared. Locomotion is very slight and streaming is restricted to a slow churning movement. Amoebae placed in culture fluid or dilute sea-water recover slowly. If Amoebae are immersed in distilled water saturated with CO2, the condition shown in fig. 2, A and B, results and death follows unless transferred to culture fluid or salt solution, i.e. NaCl. Recovery when transferred is fairly rapid (fig. 2, C, D and E). These results indicate that CO2 enters and leaves cells readily and substantiates the conclusion reached by Jacobs.8
The average length of life of Amoebae in the different acids used, varies considerably for the same concentration; also, if Amoebae are transferred to dilute sea-water, when they are nearing the lethal point, those in the various acids become normal again at varying intervals. Those treated with HNO8 and H2SO4 remain inactive and irregularly lobose for twenty or more hours after which they slowly resume normal activity.
Within thirty-six hours they are not distinguishable from Amoebae in the culture dish. Those immersed in hydrochloric and oxalic acid become normal sooner by six to ten hours than those immersed in nitric or sulphuric acid. During the period of inactivity following transference to dilute sea-water or culture fluid, Amoebae immersed in HCl and (COOH)2 remain for a few hours in a lobose form, then almost imperceptibly they creep together until all, if not too widely separated originally, flatten out and form a closely interlaced protoplasmic mat (fig. 1). After an hour or more, separation occurs and pseudopod formation and locomotion are slowly resumed.
The phenomenon just described is also seen when Amoebae are immersed in a mixture of equal parts of N/300 potassium chloride, nitrate, and acetate. In this solution the specimens also creep closer and closer together, just as those immersed in acid do after transference to sea-water or culture fluid. They remain in this state of aggregation for an hour or more, and then slowly separate by assuming a more spherical and apparently a more turgid appearance. They do not become active and are very soft and non-resistant to manipulation with a blunt glass needle, and rupture very easily either with or without such manipulation.
Acidification of the endoplasm is indicated as follows : If to Amoebae in culture fluid, neutral red (not the salt of the dye) is added, the food-vacuoles alone become bright red in colour. Only the food-vacuoles become coloured if neutral red is added to Amoebae in neutral salt solutions, e.g. NaCl or CaCI2; but in dilute acids Amoebae with coloured food-vacuoles are also seen with a pink endoplasm. The part of the endoplasm which first becomes coloured is that contained within the spherules surrounding the crystals. Such crystals constitute a considerable portion of the endoplasmic inclusions and are very similar to those described by Leidy.4 The crystals themselves usually do not, in such instances, become appreciably coloured. It is the normally uncoloured fluid substance contained within the spherule surrounding the crystal that becomes coloured. In advanced stages of acidification or its equivalent, other still more minute and normally colourless spherules become coloured pink, so that what appears at first as a diffuse pink distributed throughout the substance of the endoplasm, is seen upon closer inspection to be localised in definite units of the endoplasmic constituency. When Amoebae thus coloured are replaced in a neutral solution or in culture fluid, the pinkness disappears save for the food-vacuoles which remain of about the same intensity of colour.
2. Bases
Sodium and potassium hydroxide in sufficiently strong concentration (N/1 to N/500) induce a cessation of locomotion which is quickly followed by one or two rapid reversals in streaming, and then the ectoplasmic membrane ruptures in one or more places simultaneously. The place of rupture whether anterior, posterior, or lateral, is not predictable. After the surface membrane has ruptured the endoplasmic contents pass through the opening and go into solution in the surrounding medium. The nucleus on coming in contact with the basic medium swells momentarily and bursts with considerable violence. Observations were not possible concerning whether the nuclear membrane is preserved for an appreciable length of time after its rupture. Apparently it dissolves completely along with the other inclusions and substance of the endoplasm. The ectoplasmic membrane is seen as a distinct morphological unit along with the food-vacuoles, whose vacuolar membrane is of ectoplasmic origin for varying lengths of time depending on the concentration.
In weak concentration, N/500 to N/4000, the specimens are elongated, monopodal, and travel very slowly, if at all. Apparently their inability to travel is conditioned by an inadhesive surface preventing attachment to the substratum. The form assumed is spatulately club-shaped. So long as the surface membrane does not rupture, an indefinite period of immersion is not followed by injurious effects, as judged by their rapid recovery when restored to culture fluid.
Ammonium hydroxide has an effect quite different from that of Na or K hydroxide, and that of the neutral ammonium salts which will be described later. Attachment to the substratum and locomotion are slight, food-vacuoles are ejected, streaming is rapid but unco-ordinated, and there are many changes in the direction of locomotion through the extension of blunt protuberances in various directions. Some enlarged vesicles develop. In some instances the food-vacuoles are segregated at the posterior end, and the portion on the Amoeba containing them is constricted off. This reagent is apparently as active in causing cytolysis as KOH, N/500 or N/1000, although the way in which cytolysis is accomplished is not similar in any significant way to that observable in the effect of KOH or NaOH. It is probable that the reactions occurring under the influence of this reagent are attributable to an increase in intracellular alkalinity, such as Jacobs 8 finds in plant as well as animal cells. He finds by using NH4OH that the intracellular PH depends rather on the absolute concentration of the free base than on the PH of the surrounding medium. Therefore the effect of NH4OH is probably not ionic but molecular. The surface membrane does not appear to differ from that observed normally.
3. Salts
(1) The Neutral Salts of Ammonia
The reactions of Amoeba to ammonium chloride, nitrate, and sulphate, N/100 to N/500, are in general similar. Locomotion is rapid and other activities are normal for varying lengths of time depending on the concentration, being shorter for N/100 than N/300 or N/500, and so on. In NH4Cl, N/200 for example, Amoebae react normally for about thirty minutes after they have been immersed. At the end of this period, locomotion and streaming become irregular and slower. Periods of complete inactivity (fifteen to twenty minutes) alternate with periods in which Amoebae are active. Within an hour or more the food-vacuoles begin to distend, and the form of the Amoebae changes from a monopodal and elongated one to a heteropodal one, in which several pseudopods are extended or withdrawn consecutively. Within five hours a dozen or more large vesicles develop. These probably resulted from the fusion of several smaller ones which are seen distributed throughout the endoplasm before the larger ones arise. Locomotion is impeded almost proportionately as these vesicles increase in size and number and finally stops altogether, although there persists rather violent streaming as the endoplasm rushes into various blunt protuberances which are extended from time to time in various directions. Within four hours the larger food particles are ejected. Periods of reduced locomotion and an apparent increase in turgidity accompanied by violent streaming in many directions through the formation of blunt protuberances, are followed fairly regularly by the ejection of the greatly distended food-vacuoles. After such an ejection the Amoeba may be quiet for a few minutes or not and then resume normal locomotion with the resumption of a monopodal form. The pressure within the cell seems to increase more rapidly than the ejection of food-vacuoles and some endoplasm serves to relieve it, so that presently nearly all the observable food-vacuoles are ejected and occasionally some of the enlarged vesicles. By this time the Amoeba is rather club-shaped or lobose and studded with these vesicles (fig. 13). The Amoeba becomes inactive save for occasional local advances of the endoplasm into protuberances formed by a local expansion of the ectoplasm. The endoplasm in some instances and usually shortly before death is squeezed through the surface membrane as through a sieve in varying amounts and at different intervals. At death Amoebae are honeycombed with large vesicles, between which are seen a few crystals and other inclusions of the endoplasm which remain (fig. 13). The nucleus is not ejected. It becomes distended and remains in the ectoplasmic “shell.”
The contractile vacuole of four Amoebae in the culture dish was observed to contract on an average in minutes. One of these specimens, which is in general typical of what was observed in the other three, when transferred to NH4CI, showed the following variations in time between consecutive contractions when observed continuously for five hours, at the end of which time the vacuoles stopped contracting entirely: 12′, 10′45″, 7′5″, 6′55″, 7′10″, 55″4′45″, 30′50″, 49′40″, 16′15″, 5′15″, 6′45″, 8′5″, 6′40″, 7′45″, 6′10″, 11′25,″ 7′10″, 10′10″, 12′10″, 7′30″, 10′30″, 11′40″, 13′-20″, 7′, 9′. Contractions were frequently only partial, i.e. the vacuole contracted so that it appeared approximately 1/5, 1/4, 1/2, 2/3 as large as when the contraction began, and then again became refilled.
The inhibition of contraction of the vacuole is probably not owing to an appropriation of its function by the enlarged vesicles which begin to be conspicuous at the time the vacuole stops pulsating. If so, it would seem that there would be an increasing length of time between contractions as these supernumerary vesicles develop. What seems to happen is that the contractile vacuole is unable to adhere to the inner ectoplasmic surface when it has obtained a certain size, and thus the reaction between contractile vacuole and the ectoplasm is prevented from occurring which permits the discharge of the contents of the vacuole to the exterior.
(2) Basic Salts of Ammonia
Ammonium acetate and carbonate are similar to NH4OH in their effect on Amoeba, the chief difference being that the effects are induced more slowly.
(3) The Neutral Salts of Sodium, Lithium, and Potassium
The neutral sodium salts have no pronounced action on Amoebae, and they live as long or longer than they do in sterile culture fluid.
In lithium chloride and sulphate (the only lithium salts used) the reactions observed are similar to those observed in NaCl and Na2SO4. Lithium seems to differ from sodium in the apparent tension it causes in the ectoplasm. When Amoeba ruptures in LiCl, for example, the ruptured place gapes open and the endoplasmic contents in a sort of coagulum protrude through this opening.
The general effect on Amoeba of the neutral salts of potassium is to induce a monopodal form, a rather uniform locomotion, and the aggregation of some of the ordinarily separate crystalline particles within a common vesicle. At the time locomotion stops, the Amoebae assume a short, clubshaped form, appear very compact and dark with the contractile vacuole more or less central, distended, and non-contractile; or if contractile, it is so slow in contraction that periods of thirty to forty minutes intervene between contractions. Considerable time may elapse after locomotion and streaming have stopped before the ectoplasmic membrane ruptures and the endoplasm disappears. This is not true for Amoebae immersed in solutions of neutral sodium salts. In the latter streaming and pseudopod formation, although much reduced, are appreciable until within a few hours of death. Again, Amoebae in neutral potassium salts appear pudgy, soft at the surface, and club-shaped, whereas in the neutral salts of sodium Amoebae are heteropodal or show a marked tendency to become so ; the ectoplasm is relatively rigid and there is a gradual reduction in size. The appearance at cessation of locomotion is somewhat tortuous or gnarled, and there is an enlargement of the contractile vacuole or failure to pulsate.
There are distinct differences between the action of the salts of ammonium, sodium and lithium, potassium and rubidium. The action of ammonium is clearly different from that of sodium, lithium, potassium, or rubidium. Sodium and lithium while similar to each other, are different in effect from potassium and rubidium which, while they resemble each other, also differ from ammonium. This is clearly demonstrated in a paper by Edwards and Forgrave.2
(4) Basic Salts of Sodium and Potassium, i.e. the Acetate, Carbonate, and KCN
The effects manifest are closely comparable to a dilute solution of NaOH or KOH, i.e. in sodium or potassium acetate or carbonate N/300 or N/500 the effect is approximately that observed in N/2000, NaOH, or KOH. Streaming and locomotion are very much reduced or stopped, and the Amoebae appear very sluggish. Potassium acetate or carbonate causes a greater inactivity than sodium acetate or carbonate when used in the same concentration. The acid carbonate of Na and K are more favourable to normal activity in Amoeba than the basic carbonate.
In KCN, N/100 to N/500, however, there is a marked similarity to KOH. Locomotion and streaming stop and a general contraction of the ectoplasm occurs and an inability to attach to the substratum. It is unlike KOH in the following: (1) Amoebae in KOH, N/100, for example, cytolyse within two minutes, whereas in KCN, N/100, they do not cytolyse until after two or more hours ; (2) Amoebae in KCN, N/100, develop mushroom-like protuberances uniformly over the surface which later appear like cilia, whereas in KOH, N/100, they do not (fig. 5, B). Those in the cyanide do not survive as long or become normal as quickly after being transferred to culture fluid as those in the hydroxide. Fig. 5, A and B, represents an Amoeba in KCN, N/100, before neutralisation by the addition of HCN with phenolphthalein as indicator; C and D, the same Amoeba after it has been restored to culture fluid.
The effects not attributable to the OH-ion are apparently due to the CN-ion, since HCN in so far as its effect is not due to the H-ion is comparable to KCN in so far as this effect is not accounted for by the OH-ion. The addition of HCN to a solution of KOH causes the effect to be more nearly like that of KCN. This similarity increases with varying additions of HCN until almost identical effects are obtained. KCN, N/100, when it is made neutral to phenolphthalein by the addition of HCN, causes Amoeba to become monopodal, elongated, inadhesive, unattached to the substratum, slow in streaming and locomotion. The posterior end is greatly contracted, while the anterior end is greatly distended. In one and a quarter hours the specimens are constricted into one to three segments, which are connected by a protoplasmic filament (fig. 7). These segments are sometimes bulbous and sometimes shaped like a mushroom. The endoplasm becomes hyaline. Finally, blisters arise on one or more of the distended segments which rupture in one or more places. Specimens survive in a neutral solution of KCN, N/100, about half to one and three-quarter hours; in KCN, made neutral as indicated above, Amoebae stop all movement at once. Hyaline blisters arise at various parts of the surface, and one or more of these rupture causing complete cytolysis (fig. 7).
Experiments with certain alcohols and alkaloids indicate greater similarity of neutral KCN with them than with any other chemicals whose effects on Amoeba are presented in this paper.
Calcium Chloride and Nitrate
These two salts are very similar in their action on Amoeba to that of the neutral sodium salts. They are probably slightly more toxic, since the time at which inactivity is reached is shorter than in the sodium salts mentioned.
At the time of inactivation their form is either short and club-shaped, or that of a rosette. They are more sluggish than Amoebae in neutral sodium salts and show less tendency to extend even a blunt protuberance. The recovery of Amoebae when replaced in culture fluid is slower for calcium than for sodium.
In magnesium chloride and sulphate no pronounced effects were observed, and recovery was rapid when replaced in culture fluid.
Concentration of the Cation
(a) The NH4-ion when concentrated by mixing equal parts of the same concentration of NH4Cl, NH4NO8 and (NH4)2SO4 induces the same effects as any one of these salts when used alone, except that they are induced in about half the time. (b) The sodium, lithium, and potassium-ion when similarly concentrated is without appreciable effect.
Concentration of the Anion
The following anions were concentrated by appropriate mixtures: Cl, NO8, SO4, C2H8O2, COB8 A mixture of equal parts in the same concentration of barium, strontium, and calcium chloride is favourable for Amoeba. The concentration used was N/500. But it is not so favourable as a mixture containing equal parts in the same concentration (N/300 and N/500) of sodium, potassium, and ammonium chloride. No significant effects were noted. The Cl-ion as judged by this method of varying the cation and concentrating the anion is not harmful to Amoeba, and is in general more favourable than the nitrate, sulphate, carbonate, or acetate ion.
A mixture of Na-K and Ca(NO3)2 N/ 300 and N/ 500 seems to induce a more rapid and uniform locomotion than any salt or salt mixture used. No abnormalities are seen except in some Amoebae in such a solution, some supernumerary vesicles develop, but their size is not as large as those vesicles that develop in the neutral ammonium salts.
A mixture of Na-K- and (NH4)2SO4 is much more favourable to Amoeba than using a solution of any one of them separately. No significant effects were observed. A mixture of Na- and K-acetate is also better for Amoeba than when either is used separately. The effect of an increased alkalinity in the medium is apparent in both instances, judging from the similarity of reaction of Amoeba in Na- or K-hydroxide to what is observed in the acetate solution.
These results indicate that of the anions used they are relatively non-toxic, and that while the cations are the more reactive, they may be toxic or not depending on the cation. Univalent cations differ in their effect on the locomotion (and other activities) of Amoeba as was shown by Edwards and Forgrave.2 Bivalent cations also differ similarly, hence it does not seem possible to draw a conclusion concerning the differences that may be in general attributable to the effects of either univalent or bivalent cations. For calcium is less toxic to Amoeba than potassium when calcium and potassium chloride are used in the same concentration.
Evidence of antagonism are variously shown. A mixture of sodium and potassium chloride is less toxic than KCl alone, so also is a mixture of sodium and ammonium chloride less toxic than NH4Cl alone. Calcium and sodium chloride in a mixture is better for Amoeba than calcium chloride alone, but not as good as NaCl alone.
4. Oxidising and Reducing Agents
(a) Hydrogen Peroxide
Amoebae in dilute solutions of H2O2, M/100, rapidly become elongated, monopodal, and unattached to the substratum. Within an hour, ten to twelve tapering pseudopods are extended from various places on the surface of Amoeba. The tips of these are free of endoplasm, i.e. hyaline, for about one-third their length (fig. 8). It appears that there is a rapid intake of fluid so that a turgescence is manifest. Within two hours the Amoebae are almost spherical, the endoplasm is centrally massed, i.e. its coarser and more differentiated parts, and a broad hyaline area is seen between it and the outer ectoplasmic surface. The Amoeba is yellow at death but the surface membrane does not rupture. Amoebae restored to culture fluid after having been immersed for a few minutes in H2O2 were not observed to recover. This reagent seems to induce an irreversible permeability and probably through the solution of the surface membrane of the Amoeba.
(b) The Leucobase of Methylene Blue
The dye methylene blue bas apparently no injurious effect on Amoeba. The leucobase, however, has marked effects which are similar to HCI and which vary in intensity, depending on the degree of oxidation that is allowed to take place before Amoebae are immersed in a solution of it. The effects are more marked and toxic the less the leucobase has become oxidised when Amoebae are immersed in it. The surface very soon becomes adhesive, and particles in contact with it are slowly ingested (fig. 9, A, B, C, and D), although no vacuole forms around such ingested particle. Locomotion is stopped and streaming is restricted to eruptive advances in diverse directions. All the granular inclusions of the endoplasm soon disappear. At death Amoebae are seen as large, blue, hyaline vesicles studded with deeper blue food-vacuoles. The food-vacuoles are distended and the blue colour largely centred in the food mass which is enclosed by the vacuole. The leucobase seems to gelate the surface of Amoeba.
5. Non-Electrolytes
(a) Sucrose (cane sugar)
M/500, has practically the same effect as distilled water, i.e. Amoebae become radiate and inactive except for slow changes in form through the extension of rather long and slender pseudopods. There is no locomotion or attachment. In great concentration, M/250, the pseudopods extended are shorter and some slight locomotion occurs. Amoebae have more “body” and less pseudopods. In glucose, M/200, Amoebae are somewhat more active than when they are in the cane sugar.
(b) Methyl and Propyl Alcohol
Methyl alcohol seems to induce a rigidity in the surface of Amoeba when in a 4 to 20 per cent. solution. There is slight movement within a small radius, owing to a slow change of form accompanied by slow streaming. This rigidity, probably caused either by a condition of the ectoplasm or a turgescence, is what appears to prevent attachment to the substratum. The pseudopods formed are hyaline from the tips of the pseudopods toward their base for a considerable distance, and the protoplasm becomes slightly buff-coloured in a 4 per cent, solution. This condition remains for three hours after which it disappears, and the specimens change from a very slender, elongated, heteropodal form to one less elongated, broad, and monopodal. There is a slight apparent increase in the rate of streaming and locomotion the longer the specimens are in the solution, until within fiftee Hours the specimens appeared to be normal. If they are then transferred to 20 per cent. methyl alcohol the endoplasm within thirty minutes becomes centrally massed and extends from anterior to posterior, surrounded by a hyaline area about twice the size of the more solid central mass. The specimens at death assume the appearance of being plasmolysed.
Propyl alcohol in a concentration of 2.5 per cent. by weight causes a cessation of locomotion, an inability to attach to the substratum, and a gradual dissolution of the crystals and granules of the endoplasm (fig. 10). Streaming does not occur at the posterior end but is confined to the anterior half. There are no visible particles at the anterior tip and the substance there is apparently homogeneous. This clear zone merges almost imperceptibly into a slightly more differentiated one which consists of extremely minute vesicles or spherules. This latter zone in turn merges into another which contains some minute granules and so on, until a normal appearing endoplasmic constituency is seen. The nucleus, food-vacuole, and contractile vacuole become distended (fig. 10).
The dissolution of crystals and other solid particles in the endoplasm is accompanied by the appearance of conspicuous globules. Just before death the endoplasm is practically colourless, the only markedly differing constituents being the nucleus and the few food-vacuoles which have not been ejected. The latter are distended by the intake of fluid, so that the food contained within the vacuole is surrounded by a clear homogeneous liquid. The membrane probably ruptures at death, although this was not ascertained with accuracy. The contractile vacuole is usually centrally located within the Amoeba, just on the edge of the hyaline area. It becomes somewhat larger than it does at the time of contraction in a normal specimen and remains thus. If when some of the specimens in a given experiment have died and others seemed about to do so, they are transferred to culture fluid; there is within seventeen hours more uniform distribution of the endoplasm, the contractile vacuole begins to contract irregularly, streaming occurs from anterior to posterior, the few remaining food vacuoles become reduced in size, the nucleus changed from a spherical to a biconcave form, and within twenty-four to fortyeight hours the specimens appear normal. Alcohol seems to increase unduly the permeability of the surface membrane. The effect on the ectoplasm through observable changes in viscosity indicative of gelation or solation is not clear, although it is probably solating.
6. Alkaloids
Quinine hydrochloride and quinine, M/500, and papaverine hydrochloride, N/1000, were used chiefly. In quinine hydrochloride Amoebae change within two hours from an initially contracted and dentate form (probably due to the change from distilled water to the quinine solution) to one more elongated. They live more or less normally in this solution for several days. At death they have the appearance of those that die in HCl.
In quinine, Amoebae become globular with irregular indentations occurring uniformly over their surface. There is no locomotion or streaming. The granular portion of the endoplasm is surrounded by a sharply defined and rather broad hyaline area (fig. II). The contractile vacuole enlarges and stops contracting, bits of protoplasm are constricted off, and in some specimens several ordinarily separate food masses are seen within one common vacuole. There are frequently several of these, all of which, as well as those with only one food particle, are distended. The crystal inclusions of the endoplasm dissolve and globules are seen. It is probable that these clear homogeneous globules arise as another form of the dissolved crystals. At death, the outer surface of the ectoplasm seems to liquefy leaving the endoplasm as a semigelatinous mass.
In papaverine hydrochloride, N/1000, Amoebae are unattached to the substratum and locomotion is inappreciable. Streaming is intermittent and more or less unco-ordinated. The food-vacuoles become distended and there is some indication that one or more unite, so that the originally separate food masses are contained within a common vesicle. All of the food-vacuoles in a given Amoeba do not in any instance coalesce, only some of them, and the process was not actually observed, but deduced from the fact that no such composite vacuoles existed in the Amoeba when first put into the solution. The crystals or granules also run together, so that in a given Amoeba one or more vesicles are seen containing eight or ten or more crystals in close contact with each other and surrounded by a hyaline area. A dozen or more large colourless vesicles filled with an apparently homogeneous fluid are seen distributed here and there throughout the centrally massed endoplasm (fig. 12). Those crystals that remain particulate appear to dissolve, and simultaneously globules appear. From this and other observations it is believed that the crystals become liquid within the spherule which normally surrounds them, and in this way the globules arise. The large clear vesicles probably owe their origin to the failure of the contractile vacuole to pulsate, or are caused by the same condition which prevents the contractile vacuole from pulsating.
One-third to one-half of a given number of Amoebae cytolyse within five hours. Those that do not, survive for ten to twelve hours or longer. Some of these when transferred to culture fluid either cytolyse immediately or else eject all of their food, and after forty-eight hours become normal ; others assume a more or less spherical form. The surface seems gelatinous and within an hour death occurs without appreciable change in form. Whenever the membrane ruptures permitting extrusion of the endoplasm rather than a gelatinous surface and no apparent rupture, the ectoplasmic membrane remains with a large number of globules adhering to its inner surface. The action of the above-mentioned reagents is to induce a liquefaction of the ectoplasmic surface.
4. Miscellaneous Observations
1. The Effect of Chemicals on Enucleated Halves of Amoebae
Preliminary
ln order to divide Amoebae into approximately equal halves the following method was used. A 5 per cent. agar agar solution was prepared and then poured while warm into a Syracuse watch-glass. After it has hardened, Amoebae in culture fluid were transferred to the watch-glass. When they are elongated and travelling, which they do readily on such a substratum, a fine glass needle is brought down at an angle just over a given Amoeba and poised by its point being inserted in the substratum. At the precise moment desired, the needle is quickly lowered and the Amoeba cut without any appreciable disturbance being manifest, Frequently both halves continue travelling much the same as before, but for only a short time in the enucleated half which extends a pseudopod immediately adjacent to the cut surface.
This method enables one to divide Amoebae into small or large parts, with or without the nucleus or contractile vacuole or both in a given part. One may obtain a small amount of cytoplasm containing the nucleus and contractile vacuole; or a large amount of cytoplasm with or without the contractile vacuole or nucleus. A contractile vacuole is formed whether the half is with or without a nucleus. If the original contractile vacuole remains in the enucleated half, it functions as in the undivided Amoeba. If it is contained within the nucleated half, another forms in the enucleated half and vice versa.
When neutral red is added to a solution containing Amoebae that have been divided, the halves without a nucleus do not become coloured with respect to their food-vacuoles as readily or as intensely as the nucleated halves. In a basic or NH4CI solution the food-vacuoles in both nucleated and enucleated halves of Amoebae in the majority of instances remain uncoloured. Also when divided Amoebae whose food-vacuoles have been previously coloured while in culture fluid are immersed in NH4CI, the colour gradually disappears. The colour is seen longest in nucleated halves. This is taken to indicate an increased alkalinity within the food-vacuoles, and with probably a still greater increase within the endoplasm itself.
In distilled water, HCl, KOH, NH4CI, KCl, methyl and propyl alcohol, the length of life is invariably shorter for enucleated than for nucleated halves.
2. The Elasticity of the Ectoplasmic Surface of Amoeba
If a capillary pipette with a lumen a few micra in diameter and a bulb attached is brought near the surface of an Amoeba, a protuberance is extended toward the capillary. On coming in contact with it, the surface membrane is gradually drawn up the capillary, causing the endoplasm to become compressed into a spherical sack which is held at the opening of the capillary by the drawn-in membrane. If the Amoeba is now released by pressing the bulb, it resembles in appearance a dipper with a long handle. Within a minute or more the “handle” is with-drawn and the Amoeba resumes a normal appearance and activity. If the Amoeba is not released, the part covering the endoplasm soon ruptures and the endoplasmic contents are extruded. The membranous portion remains in the capillary until ejected into the solution, where it remains for some time colourless and collapsed. Also if a glass needle is inserted into an Amoeba and pulled outward against the surface, a protuberance is formed which may not exhibit appreciable contractility. This is owing to the adhesion of the protuberance to the substratum. Under certain conditions the surface of Amoeba adheres so firmly that it can be torn or pulled in any direction without its detaching or contracting. It is this condition that seems to have led Seifriz,6 for example, to conclude that the membrane of a plasmodium is inelastic.
Rupture of the ectoplasmic surface permitting partial or complete extrusion of the endoplasm, induced by the action of either sodium or potassium hydroxide, or certain salt solutions, or mechanically, is instructive in connection with the claim of several investigators that a new membrane forms over torn protoplasmic surfaces. In these instances it does not appear (in the case of Amoeba) that a new membrane forms serving to close the rupture, but rather that a decrease in internal pressure results from the loss of endoplasm which suffices to permit the normally contractile ectoplasmic surface to reunite. When an Amoeba is torn by using a glass needle, the same phenomenon is frequently seen. The rupture is closed or not, depending on whether the internal pressure is sufficient to cause a temporary extrusion of the endoplasmic contents followed by a closing of the rupture ; or an extrusion which continues until all of the endoplasm disappears, thus preventing the effective action of solution pressure and ectoplasmic contractility.
Likewise the contention that a new surface forms around extruded endoplasm, and that bits of Amoeba when cut off assume a spherical shape through the formation of a surface membrane, is not supported by the observations and experiments of the writer. What happens when an Amoeba is cut into small pieces is that in the cutting, the upper and lower surfaces are pressed together causing them to adhere. If the cutting is too rapid, some extrusion of endoplasm may occur, owing either to a too rapid bringing together of the ectoplasmic surfaces resulting in imperfect adhesion, or to such a disturbance in the surface-volume relation within the separated parts that the cut surface is temporarily or permanently unable to withstand it. No surface film or membrane has been observed to form around the endoplasm extruded from an Amoeba, although superficially such may seem to occur. Such surfaces existed before the extrusion. This can be tested by inducing a small rupture in the surface of an Amoeba either mechanically or chemically, and observing with a water immersion lens, if need be, the endoplasm as it passes through the rupture and subsequently.
References
Description of Plate
Figs. 1, 4, 5A, 5B, and 5C, 9, 10, 11, 12, and 13 are camera drawings. The others are free-hand drawings. Abbreviations used: C, vesicle containing crystals; C.V., contractile vacuole; F.V., food-vacuoles; g, globules; N, nucleus; V, vesicles.
The writer was enabled in part to carry on this investigation by means of a grant from the American Association for the Advancement of Science.