The Relation Of Spirostomum Ambiguum To The Hydrogen ION Concentration (Alkaline Range)

The experiments to be described were undertaken at the suggestion of Mr J. T. Saunders. Their aim was to determine, if possible, the effect on the Spirostomum of the hydrogen ion concentration of any medium. Previous work (Saunders, 1924) had shown quite clearly that whereas various solutions of pH 7·3 were harmless to these animals, decreasing the hydrogen ion concentration to pH 8·0 or less proved fatal.

The question was, whether any more definite relation could be found between the pH of any solution and the length of life of the Spirostomum in that solution.

Two solutions, the hydrogen ion concentration of which could be controlled in different ways, have so far been employed: (i) a dilute solution of Palitzsch’s borax-boric acid buffer mixture, and (ii) Cambridge tap-water containing regulated quantities of carbon dioxide in solution.

Dale (1913) had shown that the time for which Paramecium survived in a buffer solution bore a definite relation to the pH and it was expected that somewhat similar results might be obtained with Spirostomum. According to Dale’s results, as shown graphically in her paper, Paramecium can survive indefinitely in solutions of pH varying between 6·o and 9·0. But the determination by Dale of the survival time is open to serious criticisms since she relied on direct observation only, taking the cessation of movement as indicating the death point. One of the greatest difficulties in work of this nature is the accurate determination of the death point. The appearance of the dying animals is not only different in different media but varies slightly even from one individual to another in the same medium. Cessation of movement is no criterion of death, for if the animals be transferred from the medium, in which they have ceased to move, to a medium favourable for survival they will sometimes recover and sometimes not. In earlier experiments direct observation was tried but was soon found to be quite unreliable. A method described by Packard (1925), for Paramecium, was tried. Packard found that Paramecium, if stained pink in “neutral red,” could be turned yellow again by immersion in a dilute solution of ammonia, the time required being inversely proportional to the permeability: hence the method could be applied to indicate approaching death. Unfortunately in the case of Spirostomum the stain apparently damaged the animals too severely, for maceration set in before the colour change in ammonia was completed.

A “recovery” method was finally adopted as being the most satisfactory. The survival time in a medium of given pH was determined as the longest exposure which the animals could withstand in that medium and afterwards recover their normal appearance when transferred back to a medium known to be favourable for prolonged existence. As the survival time of individuals in the same medium varied, the average time for a number of individuals was taken in determining the survival time for a given concentration of hydrogen ions.

Some preliminary experiments with Paramecium, Colpidium and Spirostomum led to the choice of the last-named for further work, as being easier to handle and showing greater susceptibility to variation of the hydrogen ion concentration than the other two.

The form used was Spirostomum ambiguum (Ehrenberg) major as identified by Miss Bishop in the case of the pure-line cultures supplied by her.

These cultures, together with a very prolific stock of the same form received from Glasgow, were kept and sub-cultured in test-tubes. The culture medium was made up with either decaying leaves or wheat grains in water by the method described by Miss Bishop (1923, p. 402). Some of the tubes were filled up with Cambridge tap-water (-0042 N Carbonates) and the others with Manchester tapwater (0·0002 N Carbonates) but I was never able to detect any marked effect upon the cultures attributable to this difference. If the cultures were left for long undisturbed marked unhealthiness appeared, but could usually be “cured” by bubbling carbon dioxide through the medium in the culture tubes. One reason for this, though there may have been others depending upon the bacterial processes concerned, was that such tubes often acquired small growths of green algae or flagellates, quite sufficient to lower the hydrogen ion concentration of a hard water to a harmful degree, by their photosynthesis.

All the experiments in the buffer solution were performed on the Glasgow cultures as these were very abundant at the time and seemed to give quite as consistent results as the pure-line cultures.

The experiments in Cambridge tap-water were done on the Glasgow culture at the end of July and repeated in September and October on a fresh culture received from Miss Bishop at that time. Though the same type of result was obtained in each case the absolute time for which the latter culture survived in any of the test solutions was very much less than that for the former.

As mentioned above two solutions have, so far, been used for providing the Spirostomum with a test medium of known hydrogen ion concentration, namely:

• Palitzsch′s borax-boric acid buffer mixture. This medium was made up from standard solutions of borax and boric acid, the proportions being varied to give the required pH, as originally described by Palitzsch (1915). It was then diluted ten times with glass distilled water and the resultant solution aerated for six hours, by means of an aspirator, with fresh air from outside the building, in order that the tension of oxygen and carbon dioxide might be similar in all cases.

• Cambridge tap-water. The Cambridge tap-water is a “hard” water, having a carbonate concentration of -0042 N, which remains constant throughout the year. The pH is about 7·45 as it leaves the tap. On exposure to the air the alkalinity rises to pH 8·6, owing to the diffusion of carbon dioxide into the air, until that remaining in the water is in equilibrium with the atmospheric carbon dioxide. The solution remains practically constant at this pH although it is slightly below the theoretical value.

The pH of this water was adjusted for the experiments in the following way:

Water from the tap was put into test-tubes with some well-washed sprigs of the Canadian water weed, Elodea canadensis, corked and placed in sunlight. After a few hours the absorption of carbon dioxide by the plant, in the process of photosynthesis, increased the alkalinity of the solution often to as much as pH 9·6. At the same time a certain amount of calcium carbonate may be thrown out of solution ; but this process is slow, the solution tending rather to remain supersaturated. Therefore, if the water is not left longer than necessary in these tubes the reduction in quantity of salts in solution will not be serious in its effect upon the hydrogen ion concentration. In practice no water was used for experiment which had been for more than 30 hours in the Elodea tubes. The water was always filtered before being used.

Solutions of lesser alkalinity than pH 9·6 were obtained by taking a small quantity of the alkaline solution produced by photosynthesis and breathing alveolar air into it, in a test-tube, until the increase of carbon dioxide caused a fall in alkalinity to about pH 7·6. This latter solution was then slowly added, with shaking, to more water of pH 9·6 until the desired hydrogen ion concentration was reached. Although it was comparatively easy to prepare these solutions, it was found that only a temporary stability of hydrogen ion concentration was attained, lasting at most for 24 hours.

In all solutions of carbon dioxide in Cambridge tap-water, however they were prepared and despite the utmost efforts to establish an equilibrium at the required hydrogen ion concentration, the value of the latter was found to increase slowly.

In practice this difficulty has so far prevented any very reliable measurement of the survival time between pH 7·5 and 8·5 as in this region the time is comparatively long and the increase in hydrogen ion concentration is large in consequence. Water can be kept constant at pH 8·6 on exposure to the atmosphere; this was made use of by placing Spirostomum in water, the carbon dioxide content of which was in equilibrium with that in the air, held in shallow open dishes in the water bath. The animals were subsequently pipetted from these dishes into cells containing freshly drawn tap-water of pH 7·45 and left to recover in the usual way (see below). In solutions of greater alkalinity than pH 8·5 more reliable results were obtainable, since the survival time was shorter and the increase in hydrogen ion concentration of the solution, during the experiment, consequently was not so great (i.e. at most 0·15-0·2 pH).

The hydrogen ion concentration of these solutions can be conveniently measured by the colorimetric method of Clark (1922). The indicators used throughout this work were thymol blue, cresol red and phenol red, none of which showed signs of having any harmful effect upon the Spirostomum in the dilutions used. Correction has been made for the “salt error” of the indicators, throughout.

It may be noticed in passing, that twice in the course of these experiments there have been periods when the Cambridge tap-water caused maceration of the Spirostomum within a few hours at any hydrogen ion concentration, although no apparent cause could be found. No account has been taken, in this paper, of experiments carried out during these periods.

The experimental solution of known hydrogen ion concentration was put into glass cells, mounted, by means of marine glue, upon ordinary glass slips and of such a size as to be conveniently closed by $78$ inch glass circles (see Fig. 1).

These could be observed under the microscope with a $23$ inch objective and No. 2 (× 6) eyepiece, speed and completeness of observation being facilitated by the use of a mechanical stage. There is one disadvantage of these cells. The marine glue, being opaque and black, darkens the walls of the cell so that they cast a heavy shadow round the edge. In this shadow the animals tend to collect, especially in the more alkaline solutions, for, as has been observed (Saunders 1924), their phototropic response (negative) is much more marked in solutions of low hydrogen ion concentration. This leads, not only to the animals escaping from observation, but also from such illumination as may be intended for them by the experimenter. Canada balsam, though having the advantage of greater transparency, was otherwise unsuitable, apparently having a slightly harmful effect upon the animals as it dissolved in the water and also cracked off the slide very easily.

As it was found that both light and temperature exercised a certain influence upon the survival time of the animals it was necessary to maintain these factors as constant as possible throughout the series of experiments.

For temperature control a shallow tin bath was used, having a false bottom allowing of a water circulation. The whole was painted dead black inside and covered with two sheets of glass, placed side by side. A maximum and minimum thermometer showed that the temperature variations within this arrangement were considerably less than the corresponding changes in the room temperature.

The most convenient form of illumination was obtained from a 200 v. 60 w. blue-tinted globe placed directly over the water bath at a distance of 12 inches above the cells in a dark room. This amount of light was certainly not harmful to the animals and probably had only a very small effect upon them, though they tended to avoid it as far as possible by remaining in the shadow of the marine glue in the cells.

An attempt was made to repeat the experiments in complete darkness, only using a red lamp for the necessary microscopical observations. The results only differed from those obtained in the light by what was clearly a much greater experimental error arising from the conditions of work. It is however hoped to extend some such method as this for examining the effect of more widely varying intensities of illumination upon the animals.

A number of Spirostomum in about I c.c. of fluid were pipetted off from the culture tube into 10 c.c. of glass-distilled water, in a test-tube, and left until the animals were swimming freely in their new environment. This usually required an hour or so, but after that they appeared normal and would live for several days if left untouched, after which they died, apparently of starvation. Once “acclimatised” no harmful effect was apparent such as might have been expected from a consideration of Blättner’s (1926) work, but the adulteration of the distilled water with an appreciable quantity of culture fluid may easily have determined this difference, without, I think, being sufficient to render the washing of the animals ineffective.

Ten numbered cells (see above) were meanwhile filled with the solution, of known pH, to be tested. Four washed Spirostomum were pipetted, with the smallest possible quantity of fluid, into each cell¹. It is important to have a sufficiently wide-mouthed pipette with no sharp edges, for this purpose, or the animals may be easily damaged, as pointed out by Blättner (1926).

The cells were closed with cover glasses, care being taken to avoid the inclusion of air-bubbles. They were then put into the water-bath and the time recorded.

Preliminary experiments having indicated roughly the survival time, for the given solution, further treatment was timed accordingly, subject to modification as a result of observation.

If, for instance, the survival time was expected to be between two and three hours the first cell would be removed after $112$| hours and another after every succeeding 20 minutes or half hour, the time being noted in each case.

Each cell, when it was removed from the water bath, was examined under the microscope, and the appearance and number of the Spirostomum noted. The coverslip was next removed and washed, and the greater part of the liquid pipetted out of the cell, every care being taken to avoid touching or removing the animals. The cell was quickly refilled with freshly drawn tap-water (pH 7·45) and the process repeated before the coverslip was replaced and the whole returned to the water bath for a further period of 24 hours. This length of time was found to be about the least which would ensure the animals recovering as far as they could: at the same time, animals, left even longer, altered very little more in appearance. At the end of this period the cells were re-examined and the condition of the contents noted.

The appearance of the animals, or their remains, at this stage, could be placed fairly easily in one of four groups, namely :

• I. Animals looking quite healthy and swimming freely, having recovered completely.

• II. Animals nearly normal in shape, possibly slightly contracted, and with the cilia beating, but resting, as a rule, on the floor of the cell. They often appeared slightly more opaque than the normal, but they were considered to be well on the way to recovery.

• III. Abnormal animals appearing:

  • Only swollen at the posterior end, which can readily be distinguished at all stages by the presence of the excretory vacuole. The anterior part in these cases has alone regained the normal form and they may be regarded as having been unsuccessful in recovering from the considerable swelling which they had undergone in the original solution.

  • Much contracted, sometimes distorted and often quite rounded off. The contractèd and distorted forms were usually found to correspond with animals which had been severely damaged but did not swell. (This was commonly the case in the buffer mixtures.) The rounded forms were those animals which had become even more swollen than III a in the original solution.

• IV. This group includes all those cases of extreme exposure to the solution of damaging hydrogen ion concentration such that the transfer to the favourable solution was unable to arrest the progress of maceration, of which, however, traces usually remained visible.

Stages I and II were considered to have recovered, whereas III and IV represented those animals which had been too severely damaged to do so (see Fig. 2).

The “survival time” of the animals was taken, for the purposes of this work, to be the longest time for which the animals could remain in the solution of harmful hydrogen ion concentration and afterwards “recover,” as defined above, when transferred back to a solution, the hydrogen ion concentration of which was known to be harmless.

An example of an actual result obtained with a set of ten cells containing the buffer solution is given on the following page (Table I) to show the determination of this end-point. In this case it is quite clear¹, but often the individual variations of the Spirostomum amounted to one-and-a-half or two hours, so that there resulted the apparent anomaly of recovery taking place in one cell after a longer exposure than that followed by non-recovery in another cell. In these cases a mean value was taken. Though the degree of accuracy, therefore, which can be claimed for these values is not very high, the relative values obtained for solutions of different hydrogen ion concentration should be comparable and of some interest.

Experiments carried out by the above method showed clearly that the survival time of Spirostomum ambiguum decreased as the hydrogen ion concentration of the medium was decreased, by whichever method this latter factor may have been controlled.

The values obtained with the Glasgow cultures of Spirostomum in Palitzsch’s buffer mixture, diluted, are given in Table II. For these experiments the cells were illuminated throughout; the temperature varied between 15° C. and 19·5° C., the mean value being 17° C.

The results obtained with Cambridge tap-water and Miss Bishop’s culture of Spirostomum, under similar lighting conditions to the foregoing and at a temperature of 16° C. are given in Table III.

The values given in Tables II and III are plotted graphically in Fig. 3.

The value of t and t′ indicates the toxicity of the solution, or medium, in which the animal is placed. In the normal environment, represented, perhaps not quite exactly but very nearly, by the Cambridge tap-water, the only harmful factor is the hydrogen ion concentration. In the buffer media the value of t′ is greater than that of t because there is present an additional harmful factor, in the salts forming the buffer mixture. We are able therefore to separate the toxic effects of the salts of the buffer from the toxic effects of the diminished hydrogen ion concentration.

It will be observed that, at points B and D in Fig. 3, where the change in the hydrogen ion concentration ceases to be proportional to the change in the survival time, a very small change in the hydrogen ion concentration will produce a very considerable, and quite disproportionate, alteration in the survival time. This disproportionate effect is important in explaining the appearance and disappearance of the Spirostomum in ponds. Their appearance and disappearance is almost always sudden: they are seen in enormous numbers, they exist for a time and then in two or three days they all vanish. Whereas formerly they may have populated the water to the number of over 100,000 a litre, they rapidly become so scarce that only one or two individuals will be found in several litres of water. When Saunders (1924) found them existing in great numbers in a pond he records the pH of the water as being 7·4. This is very near the limiting value for indefinite survival and a slight increase in alkalinity will overstep the threshold value of the hydrogen ion concentration and the crowds of Spirostomum will disappear with remarkable suddenness in consequence.

The difference in the values of t and t′ indicates a difference in the toxicity of the two solutions. There is moreover a distinct difference in appearance in the dying animals in the two solutions. In the buffer mixture after a period of swimming in circles backwards as much as forwards, as if seeking a way of escape, the animals almost invariably sink to the bottom, become motionless, contract slightly in length, appear more opaque and finally, if left sufficiently long, undergo maceration, the process starting suddenly at the posterior end and passing forward till the whole animal is disintegrated.

In tap-water of low hydrogen ion concentration, on the other hand, the process is apt to be more variable; but, in general, the animals sink to the bottom, after backing and circling as before, and there continue to creep slowly about, instead of becoming still. Gradual swelling ensues, in contrast to the preceding case; this starts posteriorly and the animals pass through the typical pear-shaped stage of Piitter’s (1903, p. 347) description to a final rounded blob of protoplasm. Maceration then begins at what was the posterior border while in many cases the oral cilia may be seen to continue to beat till there is practically no unmacerated tissue left.

In spite, however, of these differences in appearance death undoubtedly occurs after exposure to low hydrogen ion concentrations in either solution. In both cases, moreover, death proceeds by slow stages, from the earlier of which recovery is possible, by an apparent reversal of the above processes; and it is upon this latter fact that the present experimental method for determining the end-point is based.

Further, it may be pointed out, that the difference in appearance of the dying animals in the two solutions is capable of explanation on osmotic grounds. For Whereas the salt-concentration in Cambridge tap-water is of the order of 0·0042 N that in the diluted buffer mixture is 0·0125 N. This latter figure corresponds with that (0·013 N) for such a physiological solution as 0·75 per cent NaCl. Hence if the cell wall of Spirostomum normally has only a restricted permeability to water, then it is reasonable to assume that if this permeability were increased, in any way, greater swelling would subsequently occur in animals placed in tap-water than in buffer solution, owing to osmotic diffusion. The occurrence of marked swelling in the tap-water solutions of low hydrogen ion concentration and of death, with little or no swelling, in the corresponding buffer mixtures points to a change in permeability taking place, in the cell wall of the Spirostomum, in these solutions.

There is evidence, then, that the decreased hydrogen ion concentration of the medium in which the Spirostomum are placed causes a proportionate increase in permeability. Let us assume that (1) when Spirostomum is placed in an alkaline medium the decreased hydrogen ion concentration of the medium causes the exit of hydrogen ions from, or the entry of hydroxyl ions into, the body of the animal, and that (2), as is very probable, there is a certain optimum value of hydrogen ion concentration of the body fluids in the interior of the animal, the “milieu interne” of Claud Bernard. If these assumptions are correct, the animal or some part of it must do work in order to prevent any change in the “milieu interne” when the hydrogen ion concentration of the medium, in which the animal is living, differs from that of the “milieu interne.” The most favourable medium will be one in which no work of this nature is necessary. This will only be the case when the hydrogen ion concentration both outside and inside the body is the same. Indefinite survival occurs in water when the pH is 7·4, so that this medium is obviously favourable. Moreover Saunders (1924) has shown that, if Spirostomum be placed in a tube of Cambridge tap-water varying from pH 7·2 at one end of the tube to pH 7·8 at the other, the animals will all collect and cluster together at one spot in the tube where the pH is 7·4. This pH, where the animals all collect, represents the pH of minimum permeability or, if our assumptions are correct, the hydrogen ion concentration of the interior of the body of the animal. The pH of the interior of the body of Spirostomum is then 7·4, a result which is in substantial agreement with the observations of Needham (1925) who, by injecting indicators, determined the internal pH of another protozoan, Amoeba, as being “in the very close neighbourhood of 7·6.”

The experiments described using two entirely different media for the control of the hydrogen ion concentration suggest that the hydrogen ion concentration is an extremely important factor in determining the time of survival of Spirostomum ambiguum in a given medium. It is clear moreover that in these experiments it is the hydrogen ion concentration and not the carbon dioxide which is the controlling factor, and this is particularly interesting in contrast to the results of Jacobs (1922) who came to the conclusion, from experiments on the viscosity of the protoplasm of Arbacia eggs, that the factor controlling viscosity was the amount of carbon dioxide present and not the hydrogen ion concentration. The most marked effects, however, in his experiments, were produced in acid solution, a pH of 5·0, produced by carbon dioxide, causing coagulation of the protoplasm and eventual death. The present experiments refer to the alkaline range, death being most rapid at pH 9·4, not only in solutions with less than the normal concentration of carbon dioxide, but also, in the buffer mixture, containing carbon dioxide in equilibrium with that in the air.

Moreover it has been pointed out that the degree of swelling which the animals undergo in the two experimental media can be explained on the assumption of increased permeability of their cell walls. That various external factors can alter the permeability of the living protoplasmic cell wall has been shown by Lillie (1915 and 1918) for heat, acids and anaesthetics, and by Osterhout (1922) for various unbalanced mixtures of electrolytes in solutions, among other workers. Loeb (1921) has also shown that the rate at which osmotic diffusion proceeds through artificial (collodion and gelatin) membranes can be modified by changes of pH in acid solutions. It is also well known that if abnormally increased permeability is not again reduced to the normal state by restoration of the cells to some favourable medium within a limited time (Lillie 1915), the power of reversibility, of the processes involved is lost, and death ensues.

It is therefore suggested that increased permeability of the cell wall causes the destruction of Spirostomum ambiguum and that, in the present experiments, this increase in permeability is dependent upon the decrease of hydrogen (or increase of hydroxyl) ions in the harmful solutions, since it is found that the time required for this destruction of the animals is inversely proportional to the concentration of hydrogen ions in the experimental medium.

I am indebted to Mr J. T. Saunders for his help and advice throughout the work. Miss Bishop very kindly supplied me with cultures of Spirostomum. The work was carried out in the Zoological Laboratory, Cambridge.