contributions to the physiology of paramecium caudatum

CONTRIBUTIONS TO THE PHYSIOLOGY O F PARAAIECIUIlf CAUDATUM

C. ill. CHILD AND EZDA DEVINEY Hull Zoological Laboratory, The University of Chicago

SIXTY-SEVEN FIGURES

Some ten years ago data were published on the axial susceptibility to KCN of various ciliate protozoa, including Paramecium (Child, '14). At that time some evidence for the existence of a susceptibility gradient in Paramecium was found, though the KCN gradient was less distinct in Para- mecium than in most other forms examined. Since then numerous observations on the gradient of Paramecium have been made in this laboratory with various agents and by different persons. Some of the experiments have been repeated from year to year by students in class work. In 1923- 1924 Miss Deviney undertook a study of susceptibility in Paramecium and Opalina with reference primarily to pho- tolysis by ultraviolet radiation and to visible light after sensitization, but including also comparative work with a number of chemical agents, particularly bases and acids, and a study of the penetration of certain agents. The present paper includes both her data and other data accumulated in this laboratory on differential susceptibility along the axis, on the axial differential in staining and reduction with methylene blue and neutral red, the differential in penetration of certain agents, the indophenol reaction, and some data on rates of pulsation of the vacuoles. The data presented on susceptibility to ultraviolet and visible light, most of those on susceptibility to bases and acids, on vital dyes and on penetration represent independent work of both authors, and many

257 THE JOURNAL O F EXPERIMENTAL ZOOLOGY, VOL. 43, NO. 2

258 C. M. C H I L D A N D EZDh DEVINEY

of tlie experiments have also beer1 performed by students. Fo r the observations on the action of bases in increasing resistance to themselves, other data on apparent acclimation, those on susceptibility to acetic acid and neutral cyanide, those on the indophenol reaction, and those oil lack of oxygen, Child is responsible. Counts of rates of vacuolar pulsation have been made by both authors and by various students.

Since the paper is primarily concerned with the question of the existence or non-existence of an axial physiological gradient in Paramecium caudatum, many data of general physiological interest, e.g., concerning motor reactions, survival time, rate of cytolysis, rate of penetration, etc., with different concentrations or intensities, are in large par t omitted in the interests of brevity.

METHODS

Since it seemed desirable to obtain evidence first from miscellaneous populations under various conditions, cultures from many different sources and in various media have been used in these experiments, but pure-line cultures under closely controlled conditions have not yet been studied. Aside from miscellaneous infusions composed of material from many localities, the medium most used was made by adding grains of boiled wheat to well-water and inoculating with Paramecium after four or five days. Such cultures are at first acid, but later approach neutrality, and gradually become more or less alkaline. I n the early stages of these cultures the animals are mostly aggregated close to the surface 011

tlie mall of the container, later farther from tlie surface arid distributed through the fluid, and still later mostly at the bottom. These differences in distribution are apparently associated with the decreasing acidity. When the animals are a t the bottom in this culture medium they are decreasing in numbers and on the way to disappearance.

As regards susceptibility, the experimental procedure in- i-ol\res first the determination of a range of concentration, or in the case of radiation, of illumination and time, which

PHYSIOLOGY OF PARAMECIUM 259

brings ahout death with cytolysis o r structural change of some sort in the ectoplasm, but which does not kill instantaneously or so rapidly as to obscure possible differences in susceptibility and which, on the other hand, does not permit acclimation. The effective range for this purpose of any agert must of course be determined experimentally, and the differences in susceptibility in different individuals and under different conditions make it evident that for exact determination of this range pure-line cultures must be used and cultural conditions maintained as constant as possible. Such determina- tions, being unnecessary for the present investigation, have not been made.

Agents used in the work on susceptibility are as follows: ultraviolet light and visible light after sensitization by eosin and in some experiments by neutral red and methylene blue; weak bases, NH,OH, NH,CI; a strong base, NaOH; weak acids , CH,COOH, H,CO,, (NaHCO,, NaHCO, plus CO,) ; KCN, alkaline and neutral ; strong acids, HC1, H,SO, ; neutral red and methylene blue as lethal agents. I n the work on ultraviolet light a Cooper-Hewitt 110-volt uviarc lamp was used and in exposure distance from light and time were varied. For sensitization to visible light eosin in concentrations ranging from 1/100000 to l/2000 and the various staining concentrations of neutral red and methylene blue were used.l

For the experiments with chemical agents solutions were made up in well-water, except when otherwise stated, this being also the water of the culture medium. I n the experiments the solution was diluted in definite proportions with the culture medium containing the animals. Conse- quently, the concentratioiis of the various agents as given are merely the concentrations indicated by amount of agent and volume of water and are only approximate. This pro-

We are indebted to Dr. M. A. Hinrichs for calling our attention to some of the earlier literature concerning the different toxicity of dyes in light and darkness. See, for example, L. Loeb, '07; Cooke and L. Loeb, '09; Boliii and Drzewina, '23.

260 C . M. CHILD AND EZDA DEVINEY

cedure is followed in these experiments because an environment approaching normal, except for the particular agent added, appears to be of far greater importance than quantitative exactness as regards concentration. Moreover, f o r a considerable range of concentration the differences as regards susceptibility are chiefly differences in time and in any miscellaneous population of Paramecium individual differences in susceptibility are considerable ; consequently, with any concentration the effect is not identical on all individuals. The data on susceptibility to weak and strong bases and acids are supplemented by data on the penetration of these agents with neutral red as indicator.2

In the study of susceptibility to lack of oxygen by Child sodium pyrogallate was used as the agent for removal of oxygen from the culture fluid. The following procedure has been found satisfactory. A very shallow paraffin cell, merely a thin band of paraffin, is made on a piece of microscope slide short enough to fit into a Syracuse dish. In this cell is placed enough of the Paramecium culture to bring the surface of the fluid well above the surface of the paraffin bounding the cell. Several grams of a mixture of sodium hydroxide and pyrogallic acid made by grinding together approximately equal quantities of these substances are placed in the Syra- cuse dish, the center of the dish being kept free from the powder f o r the passage of light. A short glass cylinder cut from a 20-mm. glass tube may be used to keep the powder away from the center. The slide containing the culture is placed on the cylinder or on the powder itself, in such posi- tion that the drop of culture lies directly above that part of the bottom of the dish which is free from the powder. The upper surface of the drop should be high enough to come into contact with the glass cover when that is sealed on. The sodium hydroxide-pyrogallic mixture is slightly moistened to hasten the reaction and a thin glass disk of proper size and already ringed with a mixture of paraffin and vaseline

2 See Harvey, '11, '13, '14; Crozier, '16 a, b ; Chambers, ' 2 2 a, b ; Jacobs, '20 b, '22.


in equal parts is at once sealed on, the surface of the culture drop coming into contact with this disk as it is pressed down. The whole preparation can be placed on the stage of the compound microscope and examined under as high a power as the thickness of the glass cover will permit.

Survival time varies of course according to the volume of air in the dish and the amount of pyrogallate. When the dish is well filled with sodium hydroxide-pyrogallic mixture deaths may begin to occur within three to four hours: with smaller amounts of pyrogallate and more air survival time is of course longer.

If there is an excess of NaOH in the mixture CO, may be removed as completely as 0,. In order to determine whether removal of CO, or the increase in alkalinity consequent upon its removal from the culture fluid has any toxic effect, control preparations were run with NaOH alone as absorbing agent. In these the animals remained normal and active until the culture fluid was reduced to one-tenth or less of its original volume (twenty hours or more) and then all individuals died within a short time-an hour or two. This survival time was three to six ,times as long as in the pyrogallate preparations and was limited only by increasing concentration of the culture fluid. In the pyrogallate preparations the animals died before any marked decrease in volume of the fluid occurred. The controls show then beyond question that removal of CO, is not a factor in the death of the animals in the pyrogallate preparations.

Unless the animals are removed from the culture medium and placed in a fluid of known constitution-a procedure which is not free from objection-the possibility cannot be completely excluded that removal of the oxygen from the culture fluid may bring about the formation or accumulation of toxic substances which would otherwise be oxidized. In view of the fact that the free oxygen content of many culture media is low and that Paramecia may aggregate in regions of a culture in which oxygen content must be below the general average of the fluid, it seems highly improbable that a

262 C. M. CHTTJI) AND EZDA DIWINEY

lethal coiicentratioii of toxic substances can result from the gradual removal of oxygen within so short a time as the oh- served survival time in the pyrogallate preparations.

With methplene blue and neutral red an axial differeiitial in rate arid depth of staining may occur; if staining is carried far enough, an axial differential susceptibility, indicated by cytolysis becomes evident ; with neutral red a differential in decoloration appears as cytolysis progresses. With low concentrations of methylene blue and neutral red the differential in depth of stain is the reverse of that observed in high concentrations, the anterior region being least stained or an- stained and the depth of stain increasing posteriorly. This differential appears while the animals are active and normal in behavior and is apparently due to the more rapid reduction aiid decoloratioii of the dyes in the anterior region. Similarly, after periods of staining not long enough to show toxic effect, return to culture fluid is followed in the course of fifteen to thirty minutes by more or less ectoplasmic decoloration, which occurs most rapidly in the anterior region.

As regards the indophenol reaction, the point of chief importance is the use of extremely dilute solutions of both agents, particularly the a naphthol, wliicli is extremely toxic. If the concentrations are too high, the animals are killed before the reaction proceeds f a r enough. With sufficiently low concentrations the animals become deep blue while still swimming actively and normal in form, but sooner or later die.

Counts of vacuolar rate have been made repeatedly from year to year in this laboiaatory by many different persons, besides the authors, aiid although the subject is unquestion- ably one which will repay extended investigation, the oppor- tunity for such a study has not yet appeared and the counts remain limited to small groups of data, usually on ten or twelve animals from one culture. Various counts of this sort have been made by students from year to year, and one of us lias also made repeated counts on lots from many different cultures. These counts have all been made on miscellaneous

PHYSIOLOGY O F PARAMECITJM 263

populations, and samples from many different cultures in various culture media have been used. Various methods for holding the animals quiet have served more or less satisfac- torily. Trapping in small spaces and very slight mechanical pressure have heeii found fairly satisfactory, but under these conditions changes in rate appear after a time. Gelatin, agar, and egg albumen have also been used. Certain agents which do not penetrate readily retard or stop locomotion heforc vacuolar rate is greatly altered. HC1, for example, in relatively low concentrations can be used in this manner, but under such conditions counts can of course be made only for a short time before progressive changes in rate begin. The counts have been made both by recording the period of each vacuole iii seconds and by determining the number of beats of each vacuole in a given length of time, usually the time required for the more rapid vacuole to gain one beat.

The figures are drawn from individual cases, but are semi- diagrammatic, being reduced to the lowest terms consistent with showing the essential features of the general character and course of cytolysis and other effects. An attempt to re- produce the microscopic details of the ectoplasmic changes has not heen made and the entoplasm is not shown. Vacuolar changes also have not been shown. Many of these features are of course of interest, but are somewhat aside from the chief purpose of the paper, and description and discussion of them woiild increase considerably the volume of the text.

SUSCEPTIBILITY

General

With most agents used the first effect observed in concentrations or degrees of action which are toxic, but not im- mediately lethal, is a change of shape, beginning with shortening and rounding of the anterior end and progressing posteriorly, with more or less complete disappearance of the oral groove. This change seems to be associated with a change in physical condition of the ectoplasm, heginning an-

264 C . M. CHILD AND EZDA DEVINEY

teriorly, but in some cases it may be a physiological contraction. When the action of the agent producing such change is relatively slow, the loss of normal shape may progress until the body becomes almost or quite spherical, the last part to lose its morphological form being the tail. This change in shape may be interrupted or complicated at any stage by cytolytic disintegration of the anterior ectoplasm, by the appearance of clear droplets or vesicles on the external surface, or of one large vesicle in the anal region, into which most or all of the entoplasm may be extruded as the ectoplasm shrinks. I n high concentrations of various agents, e.g., bases, this form change may be almost instantaneous, and bursting may occur within a few seconds. With decrease in concentration the change occurs more slowly, bursting occurs later or less frequently, and with further decrease may not occur at all or only after complete cytolysis inside the pellicle. With some other agents, the lower concentrations bring about marked shortening and rounding, beginning anteriorly, while concentrations above a certain range kill with little o r no change of shape and act essentially ' as histological fixing agents. In the strong acids, HCl and H,SO,, cytolysis occurs with little change of shape and becomes more rapid and complete with increase in concentration up to a certain point, above which histological fixation occurs. The change of shape, as it appears after action of ultraviolet radiation, of strong bases and various other agents is shown in its earlier stages in figures 1 to 3, and following figures show other stages and modifications to be described below. The initia- tion of this change of shape at the anterior end and its progress posteriorly suggest an anteroposterior differential of some sort in the physiological condition of the ectoplasm.

When the agent does not act so rapidly as to stop all cilia at once, cessation of movement occurs first at the anterior end and progresses posteriorly, another indication of a physiological gradient of some sort. In some individuals the cilia of the extreme posterior end also show a high susceptibility. If the agent brings about discharge of the trichocysts in any

PHYSIOLOGY O F PARAMECIUM 265

region, the discharge begins or is more complete in the anterior region and, except in very high concentrations, is commonly limited to the anterior one-half or third.

The appearance on the surface of vesicles or droplets of clear fluid surrounded by a surface film (figs. 15 to 18) is characteristic of a certain range of concentration or intensity of most agents used. With some agents they appear in the higher, with others in the lower concentrations or intensities. The extrusions from the anal region which result from increase in internal pressure, either because of ectoplasmic shrinkage or because of imbibition of water, are also surrounded by surface films (figs. 19, 20), which appear to be continuous with the pellicle of the animal. If, however, they represent only the original pellicle, it must be capable of enormous extension, for example, in such a case as figure 20. Moreover, under conditions giving rise to such extrusions, the pellicle over the rest of the body may undergo extensive shrinkage. And, finally, the vesicles sometimes become completely separated from the body without bursting, but the pellicle of the region where the vesicle appeared may show no visible injury. This fact seems to make it clear that the boundaries of such vesicles may be wholly or in part new surface films resulting from exposure of the colloid sol com- posing the vesicle to the external medium. On the other hand, it is clearly evident that under some conditions elevation of the pellicle occurs by the accumulation of fluid beneath it, and it is probable that there is no sharp distinction between elevation and stretching of the original pellicle and formation of a new film. That is, when the pellicle is stretched beyond a certain point or when it ruptures locally, some addition of substance may occur or a new film continuous with the old pellicle may be formed at the point of rupture.

When the increase in internal pressure is sufficient, most or all of the entoplasm may be extruded into the anal vesicle, while the ectoplasm undergoes extreme shrinkage. The anal vesicle may finally burst, and in some agents, e.g., NaOH, ectoplasmic cytolysis, beginning with the surface film of the

266 C. M. C H I L D AND EZDA DEVINEY

vesicle may follow bursting. The localization of extrusion in the anal region appears to be largely the result of mechanical weakness of this portion of the ectoplasm rather than of higher susceptibility in the physiological sense. Undw conditions which do not induce ectoplasmic shrinkage or otherwise increase internal pressure, the anal vesicles do not appear. Paralyzed vacuoles sometimes become very large and burst through the ectoplasm or give rise to vesicles o r entoplasmic extrusions in the vacuolar regions. Vacuolar vesicles or ruptures, however, do not seem to be general results of increase in internal pressure, but appear only with certain agents and under certain conditions, e.g., in relatively low concentrations of KCN, m/50 and lower.

After shortening of the anterior end the ectoplasm of this region appears distinctly thicker than the lateral ectoplasm, and at the posterior end the ectoplasm is normally thicker than in lateral regions. Under conditions which increase internal pressure, rupture of the thinner lateral ectoplasmic regions may occur before cytolysis proper begins. In high concentrations cytolysis often begins in the ruptured region, but in lower concentrations or in less susceptible individuals cytolysis may proceed from the anterior end, even though rupture has already occurred laterally.

I n many agents the clear vesicles arise more frequently in the lateral o r posterolateral regions tliaii anteriorly, even though cytolysis begins at the anterior end. Apparently the thinner ectoplasm of posterolateral regions permits the formation of vesicles more readily than the anterior ectoplasm, particularly after anterior shortening. The appearance of the vesicles may precede actual cytolysis of the ectoplasm by a longer or shorter time. Vesicles may appear while the aiiimals are swimming actively and locomotion may continue f o r at least two hours after their appearance in the individuals observed.

It is evident that the appearance of a vesicle in the anal region or elsewhere does not necessarily mean that that particular area of the ectoplasm is physiologically more sus-

PHYSIOLOGY OF PARAMECITJM 267

ceptible. In many cases thc vesicles evidently represent fluid portions of the cytoplasm forced through the ectoplasm in mechanically weak regions. For example, under conditions which give rise to vesicles, individuals in which the anal region gives way early usually show no vesicles on other regions of the body, while others without anal vesicles give rise to others on various parts of the surface. When bursting and outflow of a large part of the eiitoplasm occur early, the ectoplasm has not been seen to give rise to vesicles in any case. Moreover, the fact that anal o r other vesicles may appear in consequence of the shrinkage or contraction of the cctoplasm, which begins in the most susceptible ectoplasmic region, the anterior end, while the vesicles arise elsewhere, indicates that the susceptibility gradient is not necessarily concerned in localizing the vesicles. And, finally, with a certain range of concentration of various agents which show the ectoplasmic gradient clearly no vesicles appear, while in other concentrations of the same agents they are characteristic phenomena. It may be that some surface change must occur before the vesicles appear and that local differences in condition depending on nutritive or other metabolic factors and differing in different individuals may be concerned in localizing the region of formation of a vesicle, either by mak- ing the region concerned mechanically weaker or changing it otherwise. But however they are localized, there can be no doubt that mechanical pressure is an important factor in their formation.

The range of susceptibility in the individuals of a culture is wide, but whether pure-line culture would shorn greater uniformity has not been determined. With a given concentration or degree of action, survival time may range in some agents from a few minutes to seve+al hours, and a few individuals may survive for even longer periods in concentrations which kill most of the animals within a few minutes. Moreover, the character of the visible changes preceding or accompanying death or cytolysis may differ widely with the susccptihility of the individual and the concentration o r de-

268 C. M. CHILD AND EZDA DEVINEY

grce of action of the agent. For example, in relatively high concentrations of certain agents, e.g., bases, HC1, a complete disintegration, a ‘melting’ of all ectoplasmic structure, including the pellicle, may occur. In lower concentrations of the same agents the pellicle may remain intact over the whole body length and cytolytic changes take place inside it. Certain agents in certain concentrations apparently alter susceptibility to themselves in such a way that after a certain period of exposure reversal of the cytolytic gradient may occur. This seems to be particularly the case with the bases used (pp. 274-280). The eifect of a particular concentration or intensity may differ in different regions of the individual body : for example, complete disintegration, including the pellicle, may occur in anterior regions (figs. 4 to 9), while posterior to a certain level the pellicle may remain more o r less completely intact and the cytolytic changes occur inside i t ; in other cases elevation of the pellicle or vesicle formation may precede cytolysis in one region, but not in another.

Conditions in the culture medium also influence the degree of susceptibility to a particular agent. The susceptibility of the same culture may change in course of time, and that of different cultures in different media and even in the same medium, as far as origin is concerned, may also differ. Con- cerning differences in susceptibility of different races, no positive statement can be made at present, though incidental observations leave little doubt that such differences exist and may be considerable. As regards the influence of cultural conditions and of differences in hereditary constitution on susceptibility, our study of Paramecium has raised rather than answered questions. Such questions, as not directly concerned with the chief purpose of the study, have been left in large part for the future. *

Because of the variation in degree and character of cytolytic changes, the individual, cultural, and probably racial differences in susceptibility and the incidental complications, such as bursting in consequence of general internal pressure or by enlargement of a vacuole, anal extrusion of ento-


plasm, vesicle formation on one part or another of the body, and in general the very different effects in many cases of different concentrations of the same agent, the use of a wide range of concentration or intensity is always desirable. It has seemed unnecessary, however, to load the present paper with quantitative data concerning concentrations, survival times, rate of progress of cytolysis, etc., which can have little or no general significance and which do not concern the chief point at issue.

In spite of the complications and variations in character of the changes associated with cytolysis and death, there can be no doubt of the existence in general of an anteroposterior gradient in physiological condition in which the differences in susceptibility are non-specific for different agents used and are paralleled by other apparently non-specific physiological differences.

Ultraviolet radiation

More than a hundred lots of animals from various cultures were exposed at distances ranging from 10 to 90 em. and during periods ranging from one minute to five hours, but 30 em. was finally taken as a convenient standard distance and exposure times at this distance ranged from one to twenty minutes. With a given exposure time and distance results differ, of course, with the intensity of ultraviolet radiation from the arc and to some extent with different cultures .

With adequate exposure (e.g., 12 minutes at 30 em.) the effects observed on removal from the light and during one to two hours following are indicated in figures 1 to 14. The most extreme effects consist of complete disintegration and coagulation, extending a greater or less distance posteriorly from the anterior end (figs. 4 to 7) . In cases in which the disintegration has not progressed far or has not begun at

Doctor Hinrichs demonstrated the existence of the susceptibility gradient in Paramecium to ultraviolet radiation before our work began, but left more extended investigation to US.

270 C. M. C H I L D AND EZD.4 DET’INEY

the time of removal, its progress in the posterior direction may be observed. F o r example, progress from the stage of figure 5 to that of figure 6 or 7 has been repeatedly observed iii fifteen to twenty-five minutes. The complete disintegration extends over more or less of the body, but usually stops sliort of the posterior end, which retains its form, but undergoes coagulation and loss of structure. A varying percentage of individuals, up to 30 or 40 per cent, shows a second region of disintegration at the posterior end (fig. 8), or disintegration of the vesicle extruded from the anus (fig. 9 ) as well as the anteroposterior disintegration. Many other individuals in the same lot, apparently somewhat less susceptible, show conditions indicated in figures 10 to 14. In these cases disintegration and coagulation of the ectoplasm progress posteriorly o r sometimes obliquely (fig. 12) inside the pellicle. This series of changes may stop at the stages of figures 10 to 12, but usually progresses to stages like figures 13 and 14, or eveii t o completely spherical forms. Repeated counts have shown 75 to 95 per cent of the individuals undergoing cytolysis as indicated in figures 4 to 14, when exposure time is adjusted to material. The remainder in sucli cases show variations as described below.

With low susceptibility or with exposure time so short that cytolysis begins only some time after removal from the light, if at all, the appearance on the body of clear vesicles is usually the first change (figs. 15 to 18). Apparently the vesicles may arise on any part of the surface, but they seem to appear somewhat more frequently on the lateral surfaces than on the ends. The vesicles may appear while the animals are swimming actively, and there may be no further cytolysis, at least for hours. Even though the vesicles appear first in lateral regions, ectoplasmic cytolysis may be anteroposterior, though this is not always the case. Differences in illumination of different parts of the body undoubtedly play a part in localizing the vesicles arid even the further cytolytic changes. As movement is retarded in the ultraviolet light, the animals all come to lie with long axes at right angles to

PHYSIOLOGY OF PAILAMECIUM 271

the direction of the light. Revolution on the long axis con- tinues for a time, but finally ceases, arid unless the fluid is agitated the animals lie with one side toward the light, and even though the ultraviolet penetrates the whole body, the

2 3 5

7 8 9 10 6

rr' 12 13 Figures 1 to 14


side toward the light must be somewhat more intensely ra- diated. Comparison of lots continuously agitated with those left undisturbed shows clearly enough that unequal radiation may be a factor in localizing the effects. Sometimes the formation of an anal vesicle is followed by gradual outflow of the entoplasm and contraction of the ectoplasm (figs. 19, 20) which may or may not undergo anteroposterior cytolysis.

With low intensities the effects of regional difference in intensity become more evident. For example, with exposures of twenty to thirty minutes at 60 em. and fifty to sixty minutes at 90 em. the animals undergo slowly the usual change in shape and continue to revolve on the longitudinal axis for a considerable time. Under these conditions, the midlateral regions are most exposed, the bulging increases there and cytolysis may begin in, or be limited to this part of the body, as high as 50 per cent of forms like figures 21 to 23 being observed in some lots exposed in this way.

With long time exposure to low intensity some degree of what appears to be differential acclimation occurs and some degree of differential recovery may follow temporary exposure not too long continued. In these cases the change of form begins posteriorly instead of anteriorly and occurs very slowly, and when cytolysis finally takes place, it progresses anteriorly from the posterior region (figs. 24, 25). Appar- ently in such cases the anterior region is able to acclimate or acquire tolerance during the exposure, or to recover after removal to a greater degree than the posterior region. These data, however, are only incidental, and extended investigation of this aspect of the gradient problem in Paramecium as well as in other forms is necessary for definite conclusions.

Visible 1 iglai with semitixation Cytolysis by light with eosiri as sensitizer has been repeat-

edly used as a class experiment. Various coiicentrations of eosin, ranging from 1/100000 to 1/2000, have been used for sensitization. None of the eosiii concentrations used is toxic


in darkness within periods mucli longer than the experimental periods. The cytolytic changes are not essentially different from those observed with ultraviolet radiation, but the differences in susceptibility along the axis are usually somewhat

26 27 28 Figures 13 to 28

more sharply defiiied than in the ultraviolet. Cytolysis commonly begins with elevation of the pellicle or shrinkage and disintegration of the ectoplasm in the anterior region (figs. 26, 27) and the changes progress posteriorly as in ultraviolet (figs. 5 to 14). In experiments during the winter of 1924-


1925 thirty minutes’ exposure to noon sunlight through win- dow glass with four drops eosin 1/100 to 10 ce. culture fluid gave some degree of cytolysis in 95 per cent of the individuals, the course of cytolysis in 94 per cent being anteroposterior or somewhat oblique in consequence of unequal illumination and 1 per cent bursting in the anal or oral region. As a particular body level cytolyzes, it begins to stain with eosin, so that an ant eropos t erior staining gradient follows cyt olysis, particu- larlj7 in the higher eosin concentrations. With very high concentrations of eosin, e.g., 1/2000, 50 to 60 per cent of the animals may show cytolysis beginning in the mouth region (fig. 28). This is believed to be the result of ingestion of eosin with bacteria or other food particles or perhaps in solution.

The greater toxicity for Paramecium of neutral red and methylene blue in light has been repeatedly obse&ed, but since the differences in cytolysis in light and darkness are differences in rate rather than in character, the description in later sections of the action of these dyes is sufficient (pp. 288-292).

Weak bases: NH,OH, NH,Cl

Afore than a hundred tests of susceptibility to NH,OH have been made at various times with many concentrations in both well-water and distilled water, ranging from the un- diluted ammonia water (Baker & Adamson, C.P.) to 1/150000, the various concentrations being in some case slightly further diluted by culture fluid (one to three drops culture fluid to 10 cc. NH,OH). In most cultures examined the characteristic effects with concentrations ranging from l/2000 to 1/10000 are as follows. The first change is anterior shortening and rounding (figs. 1 to 3) , usually accompanied for a longer or shorter time by backward locomotion. Following this, swelling and loss of form begin at the anterior end and progress posteriorly (fig. 29), the earlier stages of swelling often occurring while the animals are still swimming, the ciliary activity of each level ceasing as it undergoes swelling. The


swelling may progress posteriorly over the whole body length and the shape may approach o r become spherical, the tail being the last region to lose its form. I n such cases cytolysis occurs entirely within the pellicle, but rupture of the pellicle may take place sooner o r later. I n other cases in the same concentrations cytolysis of the pellicle may ac- company the swelling and proceed a greater o r less distance from the anterior end. I n many individuals the swelling stops at some level and death of the posterior regions occurs without much change of form. I n such cases the pellicle covering the swollen region may undergo complete cytolysis,

Figures 29 to 33

while that of more posterior regions remains more or less intact (figs. 30, 31). In a varying percentage of individuals the posterior end appears as a second region of high susceptibility with early stoppage of cilia often followed by early swelling and cytolysis (fig. 32). With concentrations adjusted to susceptibility of culture anteroposterior cytolysis appears in 70 to 90 per cent of the individuals, the remainder bursting o r giving rise to anal vesicles because of high susceptibility, or undergoing gradual cytolysis inside the pellicle without marked swelling.

With concentrations of NH,OH below 1/10000 cytolysis is, of course, less rapid, and with decrease in concentration an increasing proportion of individuals remains unaffected for


at least several hours. With very high concentrations (1/200 to l / 2 ) cytolysis consists in a ware of complete disintegration running rapidly over the body, i.e., in a few seconds to one to two minutes. The disintegration progresses from the anterior end posteriorly, except when the extreme contraction or shrinkage of the ectoplasm results in early bursting, either of an anal vesicle or some other region, in which case disintegration often progresses from the region of bursting.

As might be expected, NH,C1 is in general very similar to NH,OH in its cytolytic action, except in very high concentrations. All the effects described above for N K O H have been observed with NH,C1 in concentrations from m/5 to m/30 (Child). It makes little difference whether the solution is slightly acid or slightly alkaline, since in both cases the cell protoplasm becomes alkaline, as is shown by staining with neutral red (pp. 292-294). In high concentrations the animals show little change of form at first, but begin to cytolyze anteriorly, and in some cases at the tail within one to two minutes, and cytolysis progresses posteriorly.

There is a wide range of variation in the effects of NH,OH and NH,Cl on different individuals and different cultures, and it has not been possible thus far to correlate the differences with differences in acidity or alkalinity of the culture medium. It seems probable that the history of the culture as regards various factors, e.g., bacteria present, character of infusion, perhaps exposure to light (Packard, '25), etc., may play a part in determining the condition of the animals at any given time. At any rate, the data indicate that considerable differences in physiological condition of the protoplasm may exist in different individuals or populations of Paramecium, apparently belonging to the same species.

One example, a series of boiled wheat cultures several weeks old, will serve to illustrate the point. These cultures were started at the same time and had been kept under the same laboratory conditions, except that one had stood in sunlight several hours each day f o r several days, while the others remained in diffuse daylight. Some of the cultures


were slightly acid (pH 6.6 +-), others alkaline (pH 7.3 k).

The susceptibility of these cultures to NH,OH and NH,Cl was found by Child to differ considerably from that recorded for the same cultures in earlier stages and f o r most other cultures. In NH,OH, 1/2000 to 1/10000 anterior shortening was slight at first arid the cytolytic change consisted in a gradual loss of structure, thickening, and increase in transparency of the ectoplasm progressing posteriorly .from the

34 33

~ 36

~ _--.. :.,

37

Figures 33 to 38

anterior end (figs. 33, 34) without disintegration or rupture of the pellicle. In NH,C1 in various concentrations from m/10 to m/2 the animals first underwent elongation (fig. 3 5 ) which was followed by gradual shortening and cytolysis, beginning anteriorly (figs. 36 to 38) and progressing over the whole length without rupture of the pellicle, the whole series of changes occurring mere or less rapidly (five to thirty minutes), according to concentration. The result was essentially the same in both acid and alkaline culture media and in acid and alkaline NH,Cl.


In one old alkaline culture with animals at the bottom and decreasing in numbers, complete reversal in direction of cytolysis was observed in very high concentrations of NH,OH (1/2 to 1/10 ammonia). I n these animals anterior contraction was more extreme than in any other case observed, but disintegration began a t the posterior end and progressed anteriorly. I n low concentrations this culture showed the usual anteroposterior cytolysis. A possible inter- pretation of these aberrant results suggests itself: these animals having been in a slightly alkaline culture medium f o r some time may have been more permeable to the base, but the extreme ectoplasmic contraction or shrinkage induced by its rapid entrance decreased this permeability, and this decrease was greatest anteriorly where the shrinkage was greatest. Further experiment is necessary f o r definite conclusions concerning such cases.

A stromq base: NaOH Earlier experiments with NaOH seemed to show a rather

puzzling lack of uniformity in course and character of the effects, but more extended study with a wide range of concentrations has made it evident that, so far as the gradient is concerned, this apparent lack of uniformity is, at least in part, due to incidental or secondary factors. And, first of all, Child has found that exposure of Paramecium for a short time tp concentrations of NaOH which are not lethal decreases its susceptibility to higher concentrations which are rapidly lethal to animals not previously subjected to lower concentrations. Even in concentrations which kill a considerable proportion of the individuals, e.g., 30 to 50 per cent, within the first few moments, some animals which remain unaffected during this time may remain alive for hours and show increased resistance to higher concentrations which are rapidly lethal for other lots from the same culture. This increase in resistance has been confirmed by iiumerous experiments, one of which is cited. Animals brought into NaOH m/300+ showed 60 to 70 per cent active a i d normal after

PEIYSIOLOGY O F PARAMECIUM 279

two hours. During the next three hours the concentration was gradually raised to approximately m/75, and after two hours in this concentration 40 to 50 per cent were still moving actively, though most of them showed some effect, either en- larged vacuoles or extrusion of entoplasm from the anus, or both. Animals from the same culture brought at once into m/75 underwent cytolysis in one to three minutes. Ap- parently NaOH in sufficiently high concentration brings about a surface change which decreases susceptibility to its action. In animals stained with neutral red this increase in resistance occurs without change in color of the dye, even in the ectoplasm; that is, the surface change apparently takes place without penetration of the base (p. 293). In the higher concentrations, e.g., m/10 to m/50, in which rapid shortening of the anterior region and thickening of the ectoplasm occurs, the increase in resistance is apparently greatest in the short- ened anterior region. In such concentrations cytolysis begins with bursting at some point, followed by complete disintegration, progressing from that point over the whole body in a few seconds. The earlier cases of cytolysis are almost in- variably anteroposterior in direction, except as regards the tail, which is a region of relatively high susceptibility in some individuals. In the later cases, however, cytolysis begins somewhere in the posterior half of the body and progresses in both directions, or begins at the posterior end and progresses anteriorly, the anterior end usually cytolyzing last of all. In these cases the increased resistance of the anterior region is probably associated with the extreme contraction, as suggested in the case of reversal in NH,OH (p. 278).

With decrease in concentration from m/50 to m/100 bursting becomes less frequent and is often preceded by anal cx- trusion of entoplasm or formation of clear vesicles on the body surf ace. With further decrease in concentration the vesicles and the reversal in direction of cytolysis become less frequent, and an increasing percentage shows anteroposterior cytolysis inside the pellicle, with the tail as a second region of high susceptibility in some individuals. In m/100 cytolysis

280 C . M. CHILD AND EZDA DEPINEY

in most individuals occurs within five minutes, but in m/250 to m/300 it usually begins only after two hours or more and requires several hours f o r completion. Below m/300 little or no cytolysis has been observed in most of the cultures used, but some cultures are much more susceptible than others. We have a record of cytolysis in NaOH m/500, but later work suggests that this record may be incorrect as regards concentration.

In concentrations about m/ 100 cytolysis without bursting begins at the anterior end with shrinkage of ectoplasm and appearance of a clear area, apparently fluid, beneath the pellicle, often accompanied by one or more small vesicles (figs. 39, 40). A short time later, an ectoplasmic change con- sisting in increase in transparency and loss of the yellowish color begins to progress posteriorly and a similar change in color of the entoplasm follows slightly later. Figures 40 and 41 indicate diagrammatically two stages in the posterior progress of this change, the broken line representing the approximate boundary between more transparent and unaltered portions. Actually the boundary is less sharp than the figures indicate, for the change is gradual. In a considerable number of individuals the tail also undergoes similar change early (fig. 40). Following this cytolysis, the shape may gradually approach o r become spherical and the pellicle may finally burst. In lower concentrations the changes are somewhat similar, but of course slower. In m/250 to m/300 a clear area beneath the pellicle at the anterior end is usually the first indication of cytolysis after two to three hours. The pellicle apparently becomes somewhat soft and sticky, being often drawn out into a point or otherwise distorted (figs. 42 to 44). In some individuals the pellicle of the tail is simi- larlg altered (fig. 43). These animals are still motile, but many are apparently stuck to the bottom or held by the tail, or are dragging debris which has stuck to them. When further cytolysis occurs, it progresses posteriorly as in figures 40 and 41, but very slowly, requiring several hours for completion. The vacuoles are paralyzed and usually become very


large, the anterior very commonly larger than the posterior, though sometimes only one is visible. Occasionally a vacuole enlarges to such an extent that it bursts the body. The per- centages of individuals which show any signs of cytolysis within five to six hours in m/250 to m/300 varies greatly with different cultures and does not depend solely on alkalinity or acidity of the cultures. Even in m/250 some individuals from some cultures are alive and normal after twenty-four hours.

Figures 39 to 44

Weak acids: CH,COOH, CO, I n a few experiments with acetic acid (Child) it was found

that concentrations of m/150 or higher killed and fixed at once. In m/200 anteroposterior cytolysis occurred in a few individuals with anterior bursting, vesicle .formation, or wholly within the pellicle. In m/300 more or less cytolysis occurred in 25 to 30 per cent of the animals, the remainder being apparently more or less fixed. I n m/450 60 to 70 per cent showed some stage of cytolysis after two hours. Cytol- ysis consists in swelling and increase in transparency, sometimes accompanied by vesicle formation (figs. 45 to 47). In the lower concentrations the pellicle remains intact at first, but may finally become granular. The whole body sooner or later becomes stiff through coagulation. Cytolysis begins anteriorly and progresses posteriorly, usually as a rather

282 C. M. C H I L D A N D EZDA DEVINEY

definite wave of swelling and loss of structure, which may reach the posterior end ten to twenty minutes after beginning, the pellicle often undergoing less alteration in posterior than in anterior regions. The beginning of cytolysis at the anterior end is preceded by the usual anteroposterior gradient in stoppage of cilia, then often by discharge of trichocysts, beginning anteriorly, but usually occurring only over the anterior one-third to one-half the body length (fig. 47). The individuals which cytolyze during the first two hours in m/430 show in general the usual gradient (figs. 45, 46), but among those which begin to cytolyze after two hours more or less reversal appears, cytolysis beginning at both ends or at the posterior end first. This reversal suggests that the anterior region has become to some extent acclimated or has increased its resistance to the acid to a greater extent than the posterior region. In m/600 all individuals remained normal in form and motility during twenty-four hours.

NaHCO, solutions and NaHCO, saturated with CO, were used as sources of CO, or H,CO, (Jacobs, '12, '20 a, b). The solutions to which CO, has been added are of course the more toxic, and in high concentrations the bicarbonate produces, at least temporarily, more or less complete fixation followed by loss of color and decrease in refractive index progressing posteriorly from the anterior end. Tn m/20 NaHCO, + CO, anteroposterior slowing and cessation of cilia and anterior shortening occurred and cytolysis began at the anterior end in 90 per cent within forty minutes. Progress of cytolysis from anterior to posterior end required fifteen to forty minutes. In a few individuals vesicles appear, but cytolysis usually consists in swelling and loss of structure within the pellicle, though the latter finally loses its sharp contour and becomes granular. The visible changes are not widely different from those in acetic acid (figs. 45 to 47). With this agent there is no marked irritation and no reversal in direction of locomotion.

In NaHCO, m/20 the first cases of cytolysis occur only after one hour or more; after two and one-half hours 50 per


cent show some stage of cytolysis, the remainder being intact and a few still swimming. The cytolytic changes resemble those in acetic acid. The progress is slow-an hour or more from beginning to end. In NaHCO, m/10 there is considerable irritation with backward locomotion. In samples from some cultures iiumerous vesicles appear and the pellicle very commonly separates from the ectoplasm, beginning at or near the anterior end, with a second region of separation often appearing at the tail. Separation of the pellicle is followed by disintegration of the ectoplasm. I n other cultures shorten-

c/ 45

v 46

I' \u 47

Figures 45 to 47

ing, swelling, and cytolysis begin anteriorly without vesicles, but in the posterior half or third cytolysis is often preceded by vesicles or by separation of the pellicle. I n m/10 cytolysis begins two to ten minutes after exposure and with few exceptions is complete four to thirty minutes later. NaHCO, m/5 appears to be slower in action'than lower concentrations, apparently because of a temporary fixing action.

Pot assiwnz c yauid e

Although HCN is chemically a weak acid, its physiological action is not simply that of a weak acid; therefore, it is considered separately. Since the earlier data were published

284 C. M. C H I L D AND E Z D S DEVINEY

(Child, '14) further work with KCN has shown that the effect on the vacuolar regions described in the earlier paper is incidental and characteristic of relatively slow lethal action in alkaline solutions. Higher concentrations of KCN neutralized with HCI give very uniform results similar to those obtained with other agents. In neutralized KCN, m/10 indicated concentration, there is no excitation at any time. Locomotion gradually becomes slower and ceases, stoppage of cilia progressing from the anterior end posteriorly, with the tail sometimes also showing early cessation of cilia. After thirty to forty-five minutes, 50 per cent show some stage of cytolysis. This progresses posteriorly and may involve marked swelling and disintegration, including the pellicle in anterior regions (figs. 48, 49), and in some individuals it is preceded by the appearance of clear vesicles. Other individuals show much less swelling, and ectoplasmic cytolysis takes place inside the pellicle (fig. 50). With concentrations which are not too high, anteroposterior cytolysis occiirs in 90 per cent or more of the individuals, the remainder bursting or showing entoplasmic extrusion in anal, oral, o r vacuolar region. In KCN m/5 most individuals appear to be fixed, at least temporarily, but a few undergo cytolysis. The lower limit of concentration of neutralized KCN which produces cytolysis has not been determined.

Strong acids : HCI, H,SO, In concentrations of HC1 ranging between m/50 and m/200

approximately cytolysis begins after a few seconds to two or three minutes and consists of swelling and loss of structure of pellicle and ectoplasm (figs. 51 to 53). Cytolysis begins at the anterior end without preceding change of shape. Usually there is a slight pause after cytolysis of the extreme anterior tip (fig. 51), then a wave of cytolysis progresses posteriorly with a sharp boundary between cytolyzed and uncytolyzed regions. In the higher concentrations this wave may pass over the whole length in a few seconds; in the lower concentrations, in a minute or two. Coagulation ac-

PHYSIOLOGY O F PARAMECIUM 2,85

companies or follows cytolysis, the general body form being retained and 110 outflow of entoplasm taking place. Over a considerable range of concentration the only differences observed in many samples from different cultures were differences in time. In concentrations below m,/200 the pellicle usually retains its sharp contour, at least for some hours, and cytolysis occurs inside it. In concentrations above m/50 to m/100 fixation usually occurs, the limit between cytolysis arid fixation varying considerably in different individuals and cult II r e s .

49 51 52 53 Figures 48 to 53

A few experiments with H,SO, showed concentrations above m/1000 2 lethal, but without marked cytolytic changes. In the lower concentrations within this range a wave of change in aggregation passes rapidly over the body soon after cessation of ciliary movement, but the disintegration involved is less complete than in HC1. I n the earlier cases of cytolysis this wave progresses posteriorly, but among the individuals which cytolyze later cases of reversal in direction appear, suggesting differential action of the acid on different body lerels in altering susceptibility to itself.

286 C. M. C H I L D AND EZDA DEVINEP

Lack of oxygeit

As noted in the description of the method (p. 260), the survival time varies widely in different preparations according to amount of pyrogallate and volume of air. With relatively large amounts of pyrogallate anterior shortening begins in some individuals in one to two hours and all are dead in four to five hours. With decreasing amounts of pyrogallate the survival time increases indefinitely.

Shortening and rounding of the anterior region are the first visible effects and occur while the animals are swimming actively. Before cytolysis begins the shape is usually approximately that indicated in figure 54. Cessation of ciliary movement begins at the anterior end and shows the usual anteroposterior gradient. After cessation of ciliary activity jerking movements of the body may occur in consequence of sudden discharge of trichocysts. Such movements have been observed more frequently in P. aurelia than in P. caudatum. In P. aurelia the body is often suddenly pushed backward by discharge of the anterior trichocysts, then irregular movements occur as trichocysts are discharged from lateral regions, and a few seconds later the body is pushed forward by discharge of the posterior trichocysts. In such cases the discliarge of the trichocysts is followed almost at once by cytolysis, In both species, however, cytolysis may occur without discharge of trichocysts.

In P. caudatum cytolysis begins in the anterior region inside the pellicle. The first visible indication is a slight shrinkage of ectoplasm away from the pellicle or a slight elevation of the pellicle with appearance of fluid between the two. This change begins at the anterior tip or laterally about the extreme tip (figs. 54,59), and after an interval of one to several minutes in cases observed progresses a greater or less distance posteriorly, sometimes, though not uniformly, more rapidly on one side of the body (figs. 56, 57). In the majority of individuals observed this general separation of pellicle and ectoplasm ceases at a greater o r less distance from the


anterior end and at more posterior levels clear vesicles appear. Figure 56 shows an early stage; figure 57, the same individual two minutes later. Figures 58 to 60 show three successive stages occurring within two minutes in the same individual. In many animals early separation of pellicle and ectoplasm also occurs in the tail region (figs. 55, 59). As cytolysis occurs at a particular level, the ectoplasm loses its morphological structure and a complete breakdown o f the cytoplasm into a granular mass gradually occurs. During

59 60 Figures 54 to 60

this process the pellicle may burst somewhere in the postoral region or may gradually approach o r attain spherical outline.

The point should be emphasized that conclusions concerning the course of cytolysis cannot safely be drawn from examination of the animals only after the ectoplasmic changes have occurred, for even though cytolysis occurs first a t the anterior end and progresses posteriorly, the gross changes, such as vesicles, anal extrusion, bursting, and sometimes even separation of pellicle, may be more extensive in posterior


than in anterior regions. Actual obserration of the course of cytolysis is necessary in all cases.

A few observations on P. aurelia showed in some cultures a course of cytolysis very similar to that described for P. caudatum : in other cultures the breakdown of ectoplasmic structure progressed very rapidly from anterior to posterior end and was usually followed within a few seconds by separation of pellicle and ectoplasm over most or all of the body length. Such separation may begin about the anterior end and progress posteriorly or may appear first in the postoral region.

In both species the visible cytolytic changes differ in different cultures and probably with different races. Extensive separation of pellicle and ectoplasm is characteristic of some, while in others such separation is limited to the extreme anterior region. Some of the experiments indicate, or at least suggest, that animals from certain cultures may be much more capable of anaerobic respiration than animals which have lived under other cultural conditions, but further work is necessary for definite conclusions on this point.

T H E ACTlON O F NEUTRAL RED

Three samples of neutral red used were all highly toxic and much more toxic in light than in darkness. Several different aspects of the action of this dye require consideration. First, as regards staining and lethal effect of the higher concentrations, it was found that concentrations from l/SOOO to 1/1000 stain rapidly with toxic o r lethal effect after a few minutes, even in darkness, 1/1000 retarding locomotion almost instantaneously. Neutral red evidently penetrates the ectoplasm with extreme rapidity, for entoplasmic staining begins almost at once, the ectoplasm apparently not being able to hold the dye against the greater avidity, adsorptive capacity, or whatever it may be, of the entoplasmic constituents. When distinct staining of ectoplasm does begin, in all except very low concentrations the color appears first in a more or less sliarply defined region at the extreme anterior


end, aiid in 20 to 40 per cent the tail begins to stain soon after the anterior end. These early staining ectoplasmic regions show first a distinctly less acid color than the entoplasm and often appear to be neutral o r slightly alkaline, but this difference disappears as staining progresses. In the high concentrations an anteroposterior gradient in depth of color sooii becomes visible, but later the anterior tip rather sudden17 becomes almost or quite opaque black in consequence partlj- of increase in acidity and partly of deeper staining. Dis- charge of the trichocysts in this region may occur at the same time (fig. 61, optical section). The tail undergoes similar deep staining in those individuals in which it stains early. A little later this deep staining progresses posteriorly from the anterior tip until all levels of the ectoplasm are deeply stained, though the anterior tip usually appears more deeply colored than the rest. In these high concentrations this deep staining is associated with the death changes in the protoplasm and these in most individuals lead to fixation rather than cytolysis, particularly in the preoral region. After death the dye is given off much more slowly from these fixed individuals than from those which die in lower concentrations.

In concentrations about 1/100000 staining of entoplasmic constituents occurs as in high concentrations, but the extreme anterior ectoplasmic region and sometimes the tail stain only very slightly or remain completely mistained and the depth of color in the ectoplasm increases from this region posteriorly (fig. 62, optical section). This condition persists as long as the animals remain normal and active. Apparently decoloration of the dye by reduction takes place more rapidly in the anterior ectoplasm aiid in the tails of some individuals than elsewhere.

Toxic effect is indicated by alteration, retardation, aiid finally cessation of locomotion and by shortening and rounding of the anterior region. As these changes occur the anterior ectoplasm rather suddeiily becomes more deeply colored and more acid and the tail may show similar change. At this stage the anterior end and often the tail are the most

THE JOURNAII O F EXPERIMENTAI> ZOOI.OG1,, \701). 43, N O . 2

290 C. M. CHILD A N D EZDA DEVINEP

deeply stained and apparently the most acid portions of the ectoplasm, and the depth of color decreases posteriorly (fig. 61, optical section). Death apparently passes slowly over the body with, or soon after the deeper staining, arid finally an apparent decrease in acidity and a leaching out of the dye take place beginning at the anterior end and perhaps the tail.

The grosser cytolytic changes, elevation of the pellicle and vesicles, are usually more coiispjcuous in lateral and posterolateral regions. Large anal vesicles appear frequently, bnt the anterior region usually dies and coagulates with little visible cytolytic change. Moreover, the entoplasm often shrinks into a coagulum with separation of fluid between it and the ectoplasm before the latter dies. As the internal pressure increases, this fluid is apparently forced through the less hardened portions of the ectoplasm or the anal region and forms vesicles or elevates the pellicle. This series of changes may occur within a few moments or extend over several hours, according to concentration of the neutral red and illumination. F o r example, in 1,/200000 neutral red 80 to 90 per cent of the animals die between two and four hours in darkness, in thirty to forty-five minutes in dim sunlight tlirough glass and heat filter, arid in twenty to thirty miiiutes in strong sunlight through glass and heat filter.

THE ACTION O F METHYLENE BLrE

As regards cliff ereritial actioii, methylene blue resembles neutral red. The ectoplasm stains slowly, but the dye evidently passes through it to the entoplasm, constituents of which stain rapidly. Some differences in toxicity of different samples of methylene blue have been observed. The data recorded below were obtained with a Coleman and Bell preparation ‘for bacilli. ’ In very high concentrations, e.g., 1/2000, in darkness locomotion is at oiice altered, the anterior tip (fig. 61) and often the tail become opaque black in a few miiiutes arid an ectoplasmic gradient of decreasing depth of color extends posteriorly. The deep color or opacity which indicates occwrrence or approach of death also progresses

PHYSIOLOGY O F P A R A M E C I U M 291

posteriorly, but in the high concentrations there is little visible cytolysis. With decreasing concentration staining is less rapid and reduction of the dye and cytolysis occur to a greater extent.

In coiicentrations of 1,/100000 o r lower the ectoplasmic staining gradient is at first reversed because anterior regions reduce the dye more rapidly than other parts and remain un- stained or only slightly stained. The anterior tip may remain entirely colorless and a gradient of increasing depth of color

61 62 63 Figures 61 to 63

64

extends posteriorly, the stain being deepest postorally (fig. 62). As with neutral red, this condition may persist until toxic effects appear, or the anterior region may gradually stain until it becomes more deeply colored than other parts and an ariteroposterior gradient of decreasing depth of color exists. Apparently the anterior region gradually loses its ability to reduce the dye more rapidly than other parts. In these low concentrations the anterior region may become deeply stained and the extreme tip opaque while the animals are still active, but while the depth of stain and the extent of the opaque black are much less than in high concentrations,

292 C. M. CHILD BND EZDA DEVINEY

cytolysis begiiis. Evidently the reduced leuco-compound is toxic as well as the urireduced dye. The first distinct evi- dences of cytolysis are usually clear vesicles which with rare exceptions appear first at or near the anterior end (fig. 63) , or an elevation of the pellicle beginning on each side of the anterior end and progressing over the whole body length in two to three minutes (figs. 64, 65). As in other cases in which shrinkage of ectoplasm or imbibition occurs, cytolysis may be complicated by vesicles from the anal region o r elsewhere or by bursting.

The toxicity of methylene blue is much greater in light than in darkness. In 1/100000 in darkness, for example, the first indicatioiis of toxic effect were observed after three hours, and after six hours 20 to 25 per cent were still active. In dim sunlight through glass all animals underwent extreme shortening with extrusion of most of the eiitoplasm through the anal region in thirty minutes. I n strong sunlight death occurred in five to fifteen minutes with extreme shortening, anal extrusion, and bursting.

PENETRATION O F BASER AND ACIDS

The penetration of bases and acids was studied by the indicator method with neutral red as the i n d i ~ a t o r . ~ It was hoped that this .study might throw some light on the question whether, or to what extent, axial differences in susceptibility may be associated with differences in permeability of the living cell surface. After staining with neutral red, usually in darkness, sometimes in dim diffuse daylight, the penetra- tions of bases and acids, as indicated by change in color of the neutral red, primarily in the ectoplasm, was observed. Since the color of the stained animals is somewhat on the acid side and appears to become more acid as staining progresses, the change produced by bases is more distinct than that produced by acids. I n solutions of weak bases, e.g., NH,OH and NH,Cl (compare Jacobs, '22; Chambers, '22 a) ,

*See, for example, Harvey, '11, '13, '14; Crozier, '16 a, b; Jacobs, '20 h, '22; Chambers, '22 a, b.


the animals may become entirely yellow while normal in form and swimming actively. Any coiicentration of NH,OH low enough not to penetrate all regions at once, e.g., l/2000 and lower, shows the change in color appearing first in the ectoplasm of the anterior end and progressing posteriorly, the progress being rapid or slow according to the concentration. In a varying percentage of individuals the tail appears as a second region of early change. Change of color in the entoplasm follows more slowly that in the ectoplasm, evidently because the entrance of the base into the entoplasm depends largely on its penetration of the ectoplasm. I n very low concentrations of NH,OH (1/30000 to 1/50000) the color change occurs over more or less of the preoral region, but is usually followed after a few minutes by a return to a distinctly acid color which persists. Apparently the animals are able after a few minutes either to neutralize the base more rapidly than it enters o r else to retard or prevent its further entrance and to neutralize what has entered. After this return to acid color has occurred, considerably higher concentrations are necessary to produce again the change to alkaline color than in animals freshly exposed after staining. This fact indicates that a sort of acclimation occurs very rapidly in consequence of a decrease in permeability to the base, or else that the intracellular acidity increases with the approach of death or with other changes brought about by the agent.

With concentrations of NH,Cl below m/40, animals stained with neutral red show the same gradient in penetration of the basic ion, as indicated by the color change in the ectoplasm. In the lower concentrations of NH,C1, as in NH,OH, the return to acid color takes place after a few minutes.

In NaOH no change in color of the neutral red occurs until the animals show marked toxic effects. In NaOH m/250, for example, change to yellow in the ectoplasm at the anterior end has been observed only when anterior shortening occurs and locomotion becomes abnormal. I n this case the color change is followed in a few minutes by cytolysis. In the less


susceptible individuals surf ace changes apparently occur gradually which still more completely exclude the base, and in these individuals color change may not occur until cytolysis is advanced. Even when large anal vesicles are formed, as in figure 20, the color of the dye indicates that the base does not penetrate the vesicle until cytolysis approaches or begins. These experiments on the penetration of bases have been performed by students in laboratory work as well as by both of us.

When animals are stained with neutral red in darkness and then brought into NaOH m/250 to m/300, 30 to 50 per cent remain normal and active after two hours. I f these are then placed in sunlight, they usually show reversal in direction of change in color of the neutral red and of cytolysis ; in other words, the differential susceptibility to NaOH has un- dergone reversal (compare p. 279).

Since Paramecium, and particularly the ectoplasm, becomes more acid in color as staining with neutral red progresses, the color change accompanying penetration of acids is less distinct than with bases. Il’evertheless, it has been possible to observe repeatedly the appearance of the extreme acid color, purple, at the anterior end and its progress posteriorly in both acetic acid and CO,. Moreover, as death occurs, this color change is followed by a decolorization gradient in the same direction, the neutral red being no loiiger held by the protoplasm. HCI certainly penetrates less readily than the weak acids and, so far as could be determined, does not appreciably alter the color of the dye until toxic effect of the acid appears. Some cases of reversal have been observed in HCl when entrance of the acid occurs only after fifteen to thirty minutes or more in the solution.

THE INDOPEIENOL REACTION

Without entering into discussion concerning the value of the indopheiiol reaction as an indicator of intracellular or regional localizations or difTerences in oxidase activity, the results of experiments (Child) are presented. Since both the


reacting substances are toxic, 0: naphthol highly so, dimethyl- parapheiiylciiediamine less so, though rapidly lethal in the higher concentrations, they must be used in very low concentrations to avoid killing the animals before the intracellular reaction takes place. I n the low concentrations the animals attain a brilliant blue color while still swimming actively, but are finally killed. In the experiments described below freshly made solutions of dimethylparaphenylenediamine hydrochlor- ide (Eimer and Amend) 1j1000 to lj4000 served as stock solutions, one to four drops of such solution in 5 to 10 cc. of culture medium being used. Since u naphthol is not readily soluble in water, solutions were made by shaking the crystals in water in the proportion of 1 mgm. to 1 cc. and usiiig one to four drops of this solution in 5 to 10 cc. of culture medium. 'VCTithin these limits the results are essentially similar with considerable differences in concentration except as regards time and in the extremely low concentrations with numerous animals, depth of the blue color.

When both reagents are added to the culture medium at the same time, the blue color appears gradually, the ectoplasm staining at first almost uniformly, but showing after ten to forty minutes somewhat deeper blue in the anterior region. The animals from old cultures commonly show no more marked gradient than this in any concentrations, but in cultures with abundant food and animals increasing rapidly the slight aiiteroposterior ectoplasmic gradient appears first as in the other animals; but as the reaction proceeds, deeply colored granules begin to appear in the inner portions of the ectoplasm and along the ectoplasm-entoplasm boundary. These are often so numerous at the anterior end that this becomes opaque black, and from this region their number decreases posteriorly (figs. 66, 67) . A varying proportion of individuals shows a second region of such granules at the tail (fig. 66). Thus far this granule gradient has been oh- served universally in animals from thriving cultures, but as conditioiis in the culture change and the animals begin to decrease in number, it becomes less and less marked, until


only tlie more or less diffuse ectoplasmic color gradient appears.

The granule gradient seems to have no relation to a gradient in penetration. It follows almost uniform coloration and tlie granules do not become more numerous in the more posterior regions as the reaction proceeds. Moreover, it appears while the animals are still actively swimming and normal in form. The granules appear to be indophenol crystals or masses; that they are preformed seems improbable, since the vital dyes show no indication of them. If the intracellular differential in the indophenol reaction has any significance in relation to oxidase activity, this granule gradient indicates clearly the existence of a gradient in such activity. Moreover, the gradient corresponds to what we might expect as regards the localization and the axial differential in oxidative activity in Paramecium.

Both of the reacting substances penetrate the ectoplasm of Paramecium rapidly, the a naphthol apparently almost instantaneously. If animals are placed in dimethylparaphenylenediamine for fifteen to thirty minutes and a naphthol is then added, the indophenol color begins to appear at all levels of the body within a few seconds. If the animals are first placed in a naphthol and the diamethylparapheiiyleiiediamine added later, the color appears more slowly and at about tlie same rate as when both reagents are added together.

The intracellular indophenol reaction is inhibited by KCN. According to concentration arid length of time in KCN, the inhibiting action may be merely a retardation with main- teiiance of the original gradient, or with greater effect the granules may be fewer or entirely absent in the anterior region while some appear in posterior regions ; that is, some degree of reversal of the indophenol gradient may occixr. With still higher concentrations of KCN, the reaction may not occur at all in the ectoplasm, but some blue granules may appear in the entoplasm. With highly toxic concentrations of RC", it may be completely inhibited in all parts.


VACUOLAR RATE

A few data on vacuolar rate are included here as illustrat- ing what we have observed from year to year. Counts of small numbers of individuals, usually lots of ten each, have been made from many different cultures by students and others and Child has made repeated counts. The data thus far are consistent in showing that in animals in good condition the rate of the anterior vacuole is higher than that of the posterior, except in occasional individuals. Sometimes the anterior vacuole has been seen to drop below the posterior for a few beats and then to recover its higher rate.

66 67 Figures 66 and 67

Conditions which are toxic o r injurious usually decrease the vacuolar rate and often bring about early paralysis. The anterior vacuole is the more susceptible to such conditions and, so fa r as observations have heen made, is retarded more tliaii the posterior, so that its rate becomes equal to or lower than that of the latter. The data given here represent merelr a few sample lots, the records from different cultures being given separately because the rates often differ widely.

In table 1 counts from four cultures, I to IV, are given- I to I11 made by Child; IV, by Mr. I. W. Sander. Counts of ten individuals each are given for I to I11 and of five individuals f o r IV.

298 C. M. CHILD AND EZDA DET’INEY

11.8 I 44 11.6 i 31.5 13.7 1 34.5 11.2

In the case of culture I the counts are given as the number of beats occurring while one vacuole gains one entire beat on the other. Here in all cases the anterior vacuole is the faster. The records f o r cultures I1 to I V are given as number of secoiids between successive contractions, the figures being the averages of from five to twenty beats. In culture I1 the anterior vacuole is faster in eight, while the two are equal or nearly so in two individuals. In culture 111 the anterior vacuole is faster in the first seven individuals; the posterior, in the last three. These three were individuals which had heen two to four hours under slight pressure. Cili-

53 45.4 54

__ I

Anterior 1 Posterior

TABLE 1

11 I .interior Posterior I Anterior

32 : < i . A 42.5 I 42.5 36 36.7 ~ i i .3 38 30 33 3 1 37 2 1 . i 22.7 21.7 24 16.7 16.5

1

6 .9 10.2 9.4

10.8 11 9 . 8

15 12.7 33. ti 42

111 I 1 I 1 Postprior 1 Anterior Posterior I

ary activity had almost ceased, the vacuolar rate was decreasing arid the animals were evidently approaching death. The differential retardation of the vacuoles under pressure or when trapped in small spaces was confirmed in other lots from the same culture and has been repeatedly confirmed with other cultures. Culture 111 shows in gencral a much higher vacuolar rate than I1 or IV. This culture was supposed to consist of P. aurelia, but unfortunately the diagnosis was not confirmed by micronuclear examination. Later observations indicate that the high vacuolar rate does not necessarily possess specific significance, though the form regarded as P. aurelia has in general a considerably higher rate than P.

PHYSIOLOGY OF PA4RAMECIUM 299

caudatum. The data under I V are given because they show unusually large difference in rate of the two vacuoles, those of the first two and the fifth individuals being the largest occurring in our data. One count by Mr. T. S. Eliot on an animal with four vacuoles, presumably P. multimicronucleata, gave as averages for seventeen beats of the anterior and thir- teen of the other vacuoles : 17.3, 21.4, 22.3, 22.4.

A few observations have been made on the differential retardation in toxic agents. Table 2 gives a few cases in KCN and HCl.

The two groups of table 2 came from different cultures. In both the anterior vacuole was faster under normal conditions. In addition to counts numerous records have been made

Anterior

82.5 80 23 22.5

Posterior Anterior

75 55 20 21

25.3 25 .3

Paralyzed 23 33 13

Posterior

20.8 20.4 22.4 13 27.5 13 .6

without counts of individuals in various toxic agents in which the anterior vacuole was slower than the posterior or paralyzed while the posterior was still beating, and no case has been observed thus fa r in which the rates of the two vacuoles do not become approximately equal or the posterior faster when the toxic action is not too rapid and survival time is more than a few minutes. In a lack of oxygen preparation with sodium pyrogallate after three hours the posterior vacuole was found to be faster in almost 50 per cent of the animals observed (fifty). These data on the diflerential susceptibility of the two vacuoles to toxic agents possess in their present form no great significance. The differential retardation should be followed in the individual with repeated counts


at regular intervals from the beginning of action of the toxic agent. Our data are merely incidental observations and are recorded here because they appear suggestive f o r further investigation.

Recently Unger ('23) has reported that in P. aurelia the posterior vacuole is the faster and that in P. calkinsi there are no significant differences. Mr. Unger ' s observations were made on pedigreed races under controlled cultural conditions, and under those conditions the species with which he worked evidently differ as regards relative rates of vacuoles from P. caudatum. It is an interesting question whether any of these apparently specific differences in vacuolar rate can be altered by cultural conditions which may be regarded as within the range of normal environment. At present it is possible only to call attention to the fact that A h . Unger does not find in his two species the situation as regards vacuolar rate that we have observed in P. caudatum and in individuals which we believe to be P. aurelia.

EXPERIMENTS ON OTHER SPECIES

Except as regards Opalina obtrigonoides, work on other species has been in large part incidental and occasional, as the species happened to appear in sufficient numbers in cultures. Thus far evidence of the existence of an anteroposterior physiological gradient has been obtained in all species examined and with all agents used. All the work on Opalina was done by Deviney, that on the other species by Child. As regards most species, repeated confirmation has been obtained hy various observers. The species examined and agents used are as follows : Opalina obtrigonoides, ultraviolet and visible radiation, NH,OH, NH,Cl, NaOH, NaHCO,, HCl, neutral red, methylene blue, penetration of weak and strong bases after neutral red ; Frontonia leucas, ultraviolet ; Spirosto- mum ambiguum, KCN, H,S04, methylene blue, KMnO, ; Spirostomum sp. (marine), KCN, Janus green; Colpidium sp., KCN; Dileptus gigas, KCN; Stentor coeruleus, S. poly- morphus, KCN, KMnO, ; Vorticella (three species), KCN ;


Carchesium polypinum, KCN ; Stylonychia (two species, KCN ; Euplotes ( t~7o species) , KCN ; Onychodromus grandis, NaOH, H,SO,, methylene blue, neutral red ; Oxytricha sp., ultraviolet, KCN, methylene blue. In Noctiluca an oral-aboral gradient has been demonstrated by means of KCN, KMnO,, and neutral red. In most of these forms the gradient is anteroposterior without a secondary posterior region of high susceptibility. In Opalina the posterior region about the opening of the excretory apparatus shows high susceptibility which is probably connected with a sphincter function. In Spirostomum some individuals show a secondary short gradient from the posterior end.

CONCLUSION

In the attempt to avoid giving the impression of a higher degree of uniformity than exists, particularly as regards the disintegration gradient, we have called attention to, and fig- ured the departures from, uniformity which have been observed and have given data concerning their frequency and the conditions under which they occur. As a matter of fact, there is less uniformity in the course of cytolysis in Para- mecium than in any other ciliate thus far examined. In most of the forms in the list above the cytolytic gradient is uniform in 100 per cent of the individuals. Elevation of the pellicle, the formation of vesicles, and anal extrusion, all of which are of frequent occurrence in Paramecium, are much less frequent in the other forms. Even between Paramecium and Opalina there is a marked contrast in these respects. In Opalina cytolysis, under almost all conditions observed, consists of a wave of disintegration, including the pellicle and passing posteriorly over the body with a second region of cytolysis about the excretory pore.

I t appears evident from the study of the other species that the gradient is less highly developed in Paramecium than in most other ciliates. If the gradient represents physiological polarity, this accords with expectation, f o r Paramecium does not exhibit a high degree of polar differentiation. The de-

302 C. M. C H I L D AND EZD.4 DEVINEY

partures from uniformity in the course of cytolysis are in large part, if not wholly, depeiident on surface conditions and appear to be associated with peculiarities of the pellicle which are not present or much less marked in most other ciliates examined. hlariy of these irregularities are probably due to local temporary differences in physiological condition of different regions of the body surface. If it is true that the gradient is less highly developed in Paramecium than in most other ciliates, we should expect that it would be more readily altered temporarily by local conditions in Paramecium, and that seems to be the case. The physical properties of the pellicle may also be concerned in some of the irregularities: the bulging of the anal region and extrusion of entoplasm through it seem in many cases to be results of mechanical weakness of this region.

But in spite of the various irregularities observed and described, it cannot be doubted that a physiological differential of some sort is characteristic of the longitudinal axis of Para- mecium. This differential is non-specific as regards susceptibility to the agents used, that is, for certain ranges of cow centration o r degree of action, the direction of the gradation is similar with all. The susceptibility gradient is paralleled by a gradient in staining and in rate of reduction of vital dyes, a gradient in permeability to weak bases and probably to weak acids, an indophenol gradient, and a difference in vacuolar rate.

Siiice differences in permeability to certain agents exist along the axis, the question which first arises is whether a gradient in permeability can be the physiological basis of the observed gradients. A brief consideration will show that other factors than permeability must be concerned. First, with the higher eoncentrations of the weak bases penetration is practically instantaneous, but a distinct gradient in susceptibility to these concentrations occurs. Second, the strong base, NaOH, which, according to neutral red as indicator, does not penetrate until a toxic effect is produced, gives essentially the same susceptibility gradient as the weak bases and


other agents. Third, neutral red, methylene blue, and a naphthol apparently penetrate the ectoplasm almost instantaneously, yet an ectoplasmic gradient in staining and toxic effect results. Fourth, the fact that in low coiicentratioiis of these dyes the staining gradient is opposite in direction to the permeability gradient shows that some other factor than permeability is concerned, and this factor is apparently a gradation in the rate of reduction of the dyes, decreasing in the posterior direction. Fifth, it has been shown above that the indophenol gradient cannot be simply a matter of permeability. Sixth, to agents such as CO, and HCN, which penetrate very readily and act chiefly from inside the cell (Jacobs, '12, '20 a, b; Bodine, '24) the differences in susceptibility along the axis are great. Seventh, there is a distinct gradient in susceptibility to lack of oxygen which does not involve penetration of any agent. Eighth, the reversals of the gradient in the lower concentrations o r degrees of action of various agents, whether they are the result of superficial o r internal changes, indicate the existence of other physiological differences than merely differences in permeability.

Undoubtedly, the permeability gradient to certain agents is an expression of a gradient in certain factors of protoplasmic constitution, and it is probable that this protoplasmic gradient determines, at least f o r certain agents, the cytolytic gradient. In other words, the permeability gradient is one, the cytolytic gradient another, expression of the protoplasmic differences along the axis. Child is able to confirm Packard's recent conclusion that light increases permeability to NH,OH in Paramecium (Packard, '25). It is clear, however, that this increase in permeability does not account for the greater toxicity of neutral red and methylene blue in light, for these dyes penetrate almost at once even in darkness, and the greater toxicity in light is as evident when the actual penetration and staining take place in darkness as when the animals are exposed to light from the beginning of staining.

In short, it seems evident that differences in permeability to certain agents are features of the physiological gradient,

304 C. M. CHILD AND EZDA UEVINEY

rather iliaii that the gradient is merely a permeability gradient, as has sometimes been assumed (B. L. Lund, '18; E. J. Lund, '18). The reduction gradient with low concentrations of methylene blue and the indophenol reaction suggest ectoplasmic differences in oxidative activity along the axis. The fact that the susceptibility gradient is essentially the same for strong and fo r weak bases and acids suggests that graded physical differences, doubtless at least in part colloidal, are also features of the gradient. As in the case of other phenomena characteristic of living protoplasm, the physiological gradient appears in various aspects or expressions which are mutually related and interdependent. The question whether this or that expression is the primary or fundamental factor of the gradient is like the question whether structure or function is the primary factor in life, or whether the flowing water or the limiting channel is the fundamental feature of the flowing stream.

The physiological gradient has often been called a metabolic gradient because the evidence indicates clearly that quantitative differences in metabolism are characteristic features of it. In the case of Paramecium, the existence of a metabolic gradient corresponding to the susceptibility gradient is indicated by various lines of evidence, and in various other cases the existence of the metabolic gradient has been directly demonstrated (p. 306).

The question of the significance of differences in susceptibility as indicators of quantitative differences in physiological condition has been considered elsewhere (Child, '24, pp. 78, 79; Bellamy and Child, '24), but certain points must be emphasized here. In the first place, it seems evident that the non-specific differences in susceptibility which are similar for different agents cannot depend on the particular method of action of any one agent. Some agents act through surface changes, others penetrate readily, either as ions or molecules, and act within the cell. Some act in one way o r another on the respiratory or other metabolic processes, while others apparently do not affect these processes, except as they bring


about cytolysis and death through other changes. Some undoubtedly act primarily o r chiefly on the fatty constituents of protoplasm, others presumably on the proteins. Many agents alter water and salt content and many probably act through adsorption or other surface tension effects. In organisms with highly specialized parts qualitative or specific differences in different organs or tissues may determine dif- f erences in susceptihility which are specific for particular agents. On the other hand, the differences in susceptibility which we find predominantly in the simpler organisms and the earlier developmental stages are not specific for different agents, but are similar f o r certain ranges of concentration o r degree of action of all agents thus far used. The only conclusion possible in the light of all the facts seems to be that such differences in susceptibility depend neither upon specific differences in constitution of different regions of the organism, nor upon the particular method of action of the agent, but rather upon the relation between the organism as a system in process of equilibration and disturbances. Whatever the na- ture of a particular disturbance, the system, or part of the system in which the energy changes are occurring more rapidly will be more susceptible to disturbances adequate to produce irreversible changes leading to disruption or persistent alteration. On the other hand, to certain lower ranges of concentration or degree of action the more active system or region will undergo equilibration, that is, will acclimate or acquire tolerance, and after temporary exposure will recover more rapidly or more completely than the less active. Rela- tions of this sort are characteristic of non-living systems- e.g., a flowing stream-an automobile-as well as of living systems.

In this non-specific susceptibilitjf, then, the external factor appears primarily as a disturbance of a certain degree, and the differential effect depends on quantitative differences in the physiological condition of the protoplasmic system; that is, on differences which are associated with differences in rate rather than in kind of living.

THE JOURNAL OF EXPERIMENTAL ZOOJ~OGY, VOL. 43, NO. 2


In most protoplasms, under the usual conditions of life, quantitative differences in the oxidative reactions appear to be factors of fundamental significance in the 'rate of living,' and differential susceptibility may serve as a general comparative indicator of such oxidative differences, even though the agents used do not directly affect the oxidations. As already pointed out, the susceptibility gradient of Parame- cium parallels other gradients which suggest a quantitative differential in oxidative reactions, e.g., the lack of oxygen gradient, the reduction gradient, the indophenol gradient and, according to our observations, the rate of vacuolar pulsation. Moreover, a gradient in respiratory rate paralleling the susceptibility gradient has been directly demonstrated f o r various other forms as a gradient in oxygen consumption o r CO, production, o r b0th.j

Indications of differential acclimation to various agents, e.g., to bases and acids, have been noted in Paramecium, but all data concerning this aspect of susceptibility are merely incidental. Extended investigation of both acclimation and recovery remains for the future. The apparent axial differential in acclimation is in complete accord with other facts concerning the gradient.

The axial differences in susceptibility in Paramecium are predominantly non-specific, but certain more or less specific or apparently specific differences appear. Some of these, such as the extrusion of entoplasm through the anal region, or bursting in this region, are apparently incidental mechanical effects of various agents. The more complete cytolysis of the pellicle in anterior regions with some agents and the more frequent appearance of clear vesicles in lateral and posterior regions with other agents evidently do not indicate specific differences at different levels, because the one effect may give place to the other at any level of the body. Burst- ing in the oral region or cytolysis beginning there is some-

; Grantia, Hyman, '24; Corymorpha palma, Child, '23, Hyman, '23 a ; Tubularia. crocea, Hyman, '24 ; Planaria. dorotocephala, Hyman, '23 b, Robbins and Child, '20 ; Lumbrieulus inconstans, Nereis virens, N. vexillosa, Hpman a i d Gali- glier, '21.


times seen on exposure to visible light with a sensitizer. This apparently specific regional effect seems to be merely the result of ingestion through the mouth and accumulation in the oral region of the sensitizer, either on the surface of bacteria or other particles or perhaps in solution. With certain agents more or less specific effects may appear with the llower concentrations and non-specific effects with higher concentrations. For example, with the lower concentrations of KCN, the regions of vacuole discharge give rise to vesicles, the anterior usually earlier than the posterior, before other parts are affected. With high concentrations of KCN, alkaline neutral 01" slightly acid, this effect has not been observed.

The extreme anterior ectoplasmic region seems to be more or less definitely marked off from the rest of the body as a region of specially high susceptibility, high rate of reduction of methylene blue and neutral red and greatest accumulation of indophenol. Moreover, this region appears to possess considerable importance as a factor in coordinated motor reactions. As soon as it begins to be affected by agents, the character of these reactions changes and as the anterior region approaches death, definite motor reactions disappear, though movement of cilia at more posterior levels and locomotion may still continue. The facts suggest that this region represents a certain degree of sensory specialization. This is of course the region where, if anywhere, we might expect to find a receptor apparatus and in its very high susceptibility this region resembles the specialized sensory regions of ineta- zoa. In the neuromotor apparatus of Paramecium, as described by Rees ( '21), numerous fibrillae extend between this anterior region and the center, and it is not impossible that some of them are afferent.

In the descriptions of experiments it has been noted repeatedly that the tail region possesses high susceptibility, high permeability, high reducing power, and high indophenol content in some individuals, but, according to our observations, not in all. This difference in individuals may be a matter of different varieties or possibly in some cases species

308 C. M. CHILD A N D EZDA DEVINEY

or it may represent differences in physiological condition. In P. aurelia the posterior end, SO far as we have observed, does not show these characteristics, and the possibility is not entirely excluded that some of the individuals in which physiological specialization of the posterior end did not appear may have been P. aurelia instead of P. caudatum, though we have endeavored to avoid confusion on this point. Pure-line cultures will be necessary to decide the question. If the differences at the posterior end are merely individual physiological differences, as they appear to be, they may depend on the degree of development of the tail region since the preceding division. The anterior member of a fission pair develops a new tail, the posterior member possesses a fully developed tail. But whether the earlier stages of tail development appear as the more active because they are younger, or whether the tail represents some degree of specialization, e.g., a secondary sensory region, which becomes more active as its special function develops, must be left f o r the future to determine.

Our data indicate that regional specific differences in physiological condition are not very great in the ectoplasm of Paramecium, but the use of other agents may bring to light specific differences not yet observed. It seems to be evident at any rate that the quantitative physiological gradient is a characteristic feature of the longitudinal axis. As regards the presence of other gradients, we have found it very diffi- cult to attain certainty. To some agents the region of the oral groove appears to be slightly more susceptible than the aboral side, but very commonly the oral groove disappears completely in coiisequence of the change of form preceding cytolysis, and when the vacuoles become paralyzed and en- large, the mouth remains the chief landmark, and even that is not always clearly visible when cytolysis begins. Bills ( '24), working with the lower alcohols, finds susceptibility higher on the aboral side, but we have not observed such a differential as a general feature of susceptibility.


In view of the differences between Unger's results (Unger, '25) and our own concerning vacuolar rate, the question of the relation of vacuolar rate to the gradient cannot be answered in any definite way at present. Further investigation is necessary to determine how far such differences are really specific and how far they are due to cultural conditions or to the conditions under which the counts were made. I n our counts we have found that in animals confined in small spaces reversal of the original difference in rate usually occurs after a time, the posterior vacuole becoming the faster. Differences in rate of the two vacuoles may conceivably result, not merely from their positions at different levels of the gradient, but from difference in size of the regions which they drain, from differences in nitrogen metabolism in these regions, from differences in entoplasmic content, and perhaps from differences in digestive activity. The gradient exists only i n the ectoplasm and various other physiological conditions may overbalance the ectoplasmic differences in influencing vacuolar rate.

The authors are acutely aware that the present paper is incomplete in many respects. It raises many questions without answering them; it leaves many lines of investigation with nothing more than a few incidental observations; it omits many details of the processes of cytolysis; it does not attempt quantitative presentation of data because an adequate basis f o r such presentation has not yet been attained. These characteristics of the paper are in part a consequence of the attempt to limit it to consideration of evidence bearing upon the problem of the gradient. But even with respect to this problem the paper represents little more than a preliminary collection of data which may serve as a starting-point f o r more exact and extended investigation. Concerning the existence of the gradient there can be no doubt, but it is evident that we must know more concerning the physiological effects of different cultural conditions and that we must control such cultural conditions more completely than we have done thus f a r before we can proceed very fa r in study of the physiology

310 C. M. C H I L D AND EZDA DEVIXEY

of Paramecium. We have merely attempted to show that even in miscellaneous populations under different cultural conditions the gradient can be demonstrated to be a characteristic feature of the longitudinal axis.

SUMMARY

1. Paramecium caudatum shows a differential susceptibility, decreasing from the anterior end posteriorly, to the following agents : ultraviolet radiation ; visible light after sensitization by eosin, methplene blue or neutral red; the weak bases, NH,OH, NH,Cl; the strong base, NaOH; the weak acids, acetic, CO, ; KCN ; the strong acids, HCl, H,SO,, the dyes neutral red, and methylene blue. Susceptibility to lack of oxygen shows a similar differential.

2. As indicated by the color of neutral red within the cell, weak bases and apparently weak acids penetrate the ectoplasm of living normal animals. With low concentrations the rapidity of penetration decreases from the anterior end posteriorly. A strong base and a strong acid apparently do not penetrate until the surface is injured, but when they do entcr the penetration gradient is similar to that f o r weak bases and acids.

3. Neutral red and methylene blue apparently penetrate the ectoplasm almost at once, but stain it only slowly. With thc higher conceiitratioiis an anteroposterior gradient in staining, a similar susceptibility gradient, and with neutral red, a decolorization gradient accompanying cytolysis are observed. 4. I n low concentrations of both methyleiie blue and neutral

red the anterior region stains least or not at all and the depth of staining increases posteriorly. Similarly, after temporary exposure to these dyes sufficient to stain the ectoplasm, but not to be appreciably toxic, the rate of decoloration decreases from the anterior end posteriorly. These facts indicate an axial differential in rate of reduction of methylene blue and apparently also of neutral red in the ectoplasm.

5. I n the indophenol reaction deep blue granules appear in the ectoplasm and on the ecto-entoplasm boundary, being most

PHYSIOLOGY 02 PARAMECIUM 311

numerous in well-fed animals. The depth of blue color and the number of granules are greatest in the anterior region and decrease posteriorly.

6. The various methods indicate that the tail region differs somewhat in physiological condition in different individual 8. It is suggested that this difference may result from the relation of tail development and fission.

7 . In general, the anterior vacuole beats more rapidly than the posterior, but in the presence of toxic agents the rate of both vacuoles may be decreased, that of the anterior more than that of the posterior, so that the two are equal in rate o r the anterior slower than the posterior.

8. The various data indicate the existence of a physiological gradient in the longitudinal axis. Differences in permeability along the axis may account for some aspects of the gradient, but it is evident that the gradient is not merely a permeability gradient. The evidence points to the conclusion that the gradient is primarily a quantitative differential including both metabolic and physical factors. There is no evidence for the existence of any other basis of physiological polarity than this gradient.

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-~ 1913 A criticism of the indicator method of determining cell

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H Y X ~ N , L. H. Oxygen consumption with respect to level, size and re- Proc. Amer. Soc.

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~~ 1924 Respiratory differences along the axis in Grantia and Tubu- laria.

IIY~XAN, L. II., AND GALIGHER, A. E. 1921 Direct demonstration of the existence of a metabolic gradient in annelids. Jour. Exp. Zool., vol. 34, p. 1.

JACOBS, M. H. 1912 Studies on the physiological characters of species. I. Jour. Exp. Zool., vol. 12, p. 519.

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--___ 1920 b The production of intracellular acidity by neutral and alkaline solutions containing carbon dioxide. Amer. Jour. Physiol.,

1922 The influence of ammonium salts on cell reaction. Jour. Gen. Physiol., vol. 5, p. 181.

1907 Uber den Einfluss des Liclites auf die Farbung und Entwicklung von Eiern von Asterias in Losungen verschiedener Farbstoff e. Arch. f . Entwickelungsmerh., Bd. 23, S. 3.59.

LUND, BARBARA L. The toxic action of KCN and its relation to the state of nutrition and age of the cell as shown by Paramecium and Di- dinium.

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ROBBINS, HARRIET S., AND CHILD, C. M. 1920 Carbon dioxide production in relation to regeneration in Planaria. dorotocepliala. Biol. Bull., vol. 38, p. 103.

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