the relation of spirostomum ambiguum to the … · spirostomum with a test medium of known hydrogen...

365

THE RELATION OF SPIROSTOMUM AMBIGUUMTO THE HYDROGEN ION CONCENTRATION

(ALKALINE RANGE)

BY PENELOPE M. JENKIN, B.A.

(From the Zoological Laboratory, Cambridge.)

(Received iat.h January 1927.)

(With Three Text-figures.)

INTRODUCTION.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 Spirostomumof the hydrogen ion concentration of any medium. Previous work (Saunders,1924) had shown quite clearly that whereas various solutions of pH 7-3 were harm-less to these animals, decreasing the hydrogen ion concentration to pH 8-o or lessproved fatal.

The question was, whether any more definite relation could be found betweenthe 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 indifferent ways, have so far been employed: (i) a dilute solution of Palitzsch's borax-boric acid buffer mixture, and (ii) Cambridge tap-water containing regulatedquantitiesof carbon dioxide in solution.

Dale (1913) had shown that the time for which Parameciutn survived in a buffersolution bore a definite relation to the pH and it was expected that somewhatsimilar results might be obtained with Spirostomum. According to Dale's results,as shown graphically in her paper, Paramecium can survive indefinitely in solutionsof pH varying between 6-o and 9-0. But the determination by Dale of the survivaltime is open to serious criticisms since she relied on direct observation only, takingthe cessation of movement as indicating the death point. One of the greatestdifficulties 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 butvaries slightly even from one individual to another in the same medium. Cessationof movement is no criterion of death, for if the animals be transferred from the

•dium, in which they have ceased to move, to a medium favourable for survivaly will sometimes recover and sometimes not. In earlier experiments direct

observation was tried but was soon found to be quite unreliable. A method

366 PENELOPE M. JENKIN

described by Packard (1925), for Paramecium, was tried. Packard found thatmecium, if stained pink in "neutral red," could be turned yellow again by ision in a dilute solution of ammonia, the time required being inversely proportionalto the permeability: hence the method could be applied to indicate approachingdeath. Unfortunately in the case of Spirostomum the stain apparently damagedthe animals too severely, for maceration set in before the colour change in ammoniawas completed.

A "recovery" method was finally adopted as being the most satisfactory. Thesurvival time in a medium of given pH. was determined as the longest exposurewhich the animals could withstand in that medium and afterwards recover theirnormal appearance when transferred back to a medium known to be favourablefor prolonged existence. As the survival time of individuals in the same mediumvaried, the average time for a number of individuals was taken in determining thesurvival time for a given concentration of hydrogen ions.

MATERIAL.

Some preliminary experiments with Paramecium, Colpidiuni and Spirostomumled to the choice of the last-named for further work, as being easier to handle andshowing greater susceptibility to variation of the hydrogen ion concentration thanthe other two.

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

These cultures, together with a very prolific stock of the same form receivedfrom Glasgow, were kept and sub-cultured in test-tubes. The culture mediumwas made up with either decaying leaves or wheat grains in water by the methoddescribed by Miss Bishop (1923, p. 402). Some of the tubes were filled up withCambridge tap-water (-0042 N Carbonates) and the others with Manchester tap-water (0-0002 N Carbonates) but I was never able to detect any marked effect uponthe cultures attributable to this difference. If the cultures were left for long un-disturbed marked unhealthiness appeared, but could usually be "cured" bybubbling carbon dioxide through the medium in the culture tubes. One reasonfor this, though there may have been others depending upon the bacterial processesconcerned, was that such tubes often acquired small growths of green algae orflagellates, quite sufficient to lower the hydrogen ion concentration of a hard waterto a harmful degree, by their photosynthesis.

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

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

Relation of Spirostomum to Hydrogen Ions 367

METHODS.

(A) Solutions of variable pH.

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

(i) Palitzsch's borax-boric acid buffer mixture. This medium was made up fromstandard solutions of borax and boric acid, the proportions being varied to givethe required />H, as originally described by Palitzsch (1915). It was then dilutedten 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 thatthe tension of oxygen and carbon dioxide might be similar in all cases.

(ii) Cambridge tap-water. The Cambridge tap-water is a "hard" water, havinga carbonate concentration of -0042 N, which remains constant throughout the year.The pli is about 7-45 as it leaves the tap. On exposure to the air the alkalinityrises to pH 8-6, owing to the diffusion of carbon dioxide into the air, until thatremaining in the water is in equilibrium with the atmospheric carbon dioxide.The solution remains practically constant at this />H although it is slightly belowthe 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. Aftera few hours the absorption of carbon dioxide by the plant, in the process of photo-synthesis, increased the alkalinity of the solution often to as much as >H 9-6. Atthe 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 reductionin quantity of salts in solution will not be serious in its effect upon the hydrogenion concentration. In practice no water was used for experiment which had beenfor more than 30 hours in the Elodea tubes. The water was always filtered beforebeing used.

Solutions of lesser alkalinity thanpH 9-6 were obtained by taking a small quantityof the alkaline solution produced by photosynthesis and breathing alveolar airinto it, in a test-tube, until the increase of carbon dioxide caused a fall in alkalinityto about pH 7-6. This latter solution was then slowly added, with shaking, to morewater of pH 9-6 until the desired hydrogen ion concentration was reached. Althoughit was comparatively easy to prepare these solutions, it was found that only atemporary stability of hydrogen ion concentration was attained, lasting at mostfor 24 hours.

In all solutions of carbon dioxide in Cambridge tap-water, however they wereprepared and despite the utmost efforts to establish an equilibrium at the requiredhydrogen ion concentration, the value of the latter was found to increase slowly,^ n practice this difficulty has so far prevented any very reliable measurementofthe survival time between />H 7-5 and 85 as in this region the time is compara-tively long and the increase in hydrogen ion concentration is large in consequence.


Water can be kept constant at />H 8*6 on exposure to the atmosphere; thismade use of by placing Spirostomum in water, the carbon dioxide content ofwas 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 con-taining 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 resultswere obtainable, since the survival time was shorter and the increase in hydrogenion concentration of the solution, during the experiment, consequently was not sogreat (i.e. at most 0-15-0-2 pH).

The hydrogen ion concentration of these solutions can be conveniently measuredby the colorimetric method of Clark (1922). The indicators used throughout thiswork were thymol blue, cresol red and phenol red, none of which showed signsof having any harmful effect upon the Spirostomum in the dilutions used. Cor-rection has been made for the "salt error" of the indicators, throughout.

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

(B) Apparatus.

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

-Cover-glass

Tj IP Wall of cell• • Marine glue joint

• SlideFig. 1. Diagram showing the construction of the glass cells, above in plan, below in elevation.

These could be observed under the microscope with a § inch objective ^No. 2 (x 6) eyepiece, speed and completeness of observation being facilitated bythe use of a mechanical stage. There is one disadvantage of these cells. The marine


*

, being opaque and black, darkens the walls of the cell so that they cast a heavyow 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 phototropicresponse (negative) is much more marked in solutions of low hydrogen ion con-centration. This leads, not only to the animals escaping from observation, butalso from such illumination as may be intended for them by the experimenter.Canada balsam, though having the advantage of greater transparency, was other-wise unsuitable, apparently having a slightly harmful effect upon the animals asit dissolved in the water and also cracked off the slide very easily.

(C) Conditions of the Experiments.

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

For temperature control a shallow tin bath was used, having a false bottomallowing of a water circulation. The whole was painted dead black inside andcovered with two sheets of glass, placed side by side. A maximum and minimumthermometer showed that the temperature variations within this arrangement wereconsiderably 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 inchesabove the cells in a dark room. This amount of light was certainly not harmful tothe animals and probably had only a very small effect upon them, though theytended to avoid it as far as possible by remaining in the shadow of the marine gluein the cells.

An attempt was made to repeat the experiments in complete darkness, onlyusing a red lamp for the necessary microscopical observations. The results onlydiffered from those obtained in the light by what was clearly a much greater ex-perimental error arising from the conditions of work. It is however hoped toextend some such method as this for examining the effect of more widely varyingintensities of illumination upon the animals.

(D) Procedure.

A number of Spirostomum in about 1 c.c. of fluid were pipetted off from theculture tube into 10 c.c. of glass-distilled water, in a test-tube, and left until theanimals were swimming freely in their new environment. This usually requiredan hour or so, but after that they appeared normal and would live for several daysi^teft untouched, after which they died, apparently of starvation. Once "acclima-! ^ B " no harmful effect was apparent such as might have been expected from aconsideration of Blattner's (1926) work, but the adulteration of the distilled waterwith an appreciable quantity of culture fluid may easily have determined this

BJEB-Iviv 25

37° PENELOPE M.JENKIN

difference, without, I think, being sufficient to render the washing of the animineffective. ^ ^

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

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

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

If, for instance, the survival time was expected to be between two and threehours the first cell would be removed after i\ hours and another after every suc-ceeding 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 themicroscope, and the appearance and number of the Spirostomum noted. Thecoverslip was next removed and washed, and the greater part of the liquid pipettedout of the cell, every care being taken to avoid touching or removing the animals.The cell was quickly refilled with freshly drawn tap-water (pK 7-45) and theprocess repeated before the coverslip was replaced and the whole returned to thewater bath for a further period of 24 hours. This length of time was found to beabout the least which would ensure the animals recovering as far as they could: atthe 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 thecontents noted.

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

I. Animals looking quite healthy and swimming freely, having recoveredcompletely.

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

III. Abnormal animals appearing:(a) Only swollen at the posterior end, which can readily be distinguished at

all stages by the presence of the excretory vacuole. The anterior part inthese cases has alone regained the normal form and they may be regardedas having been unsuccessful in recovering from the considerable swellingwhich they had undergone in the original solution.

1 It was assumed that four specimens were actually safely introduced in each case, but errors wereapt to occur here, especially when working in the " dark." These latter experiments are not of greatimportance however in the present connection.

Relation of Spirostomum to Hydrogen Ions

(b) Much contracted, sometimes distorted and often quite rounded off. Thecontracted and distorted forms were usually found to correspond withanimals which had been severely damaged but did not swell. (This wascommonly the case in the buffer mixtures.) The rounded forms werethose animals which had become even more swollen than I l i a in theoriginal solution.

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

e.iv

Fig. 2. Spirostomum ambiguum after "recovering" for 24 hours fromthe effects of exposure to solutions of low hydrogen ion concentration.

I. Complete recovery, a.c. adoral cilia; e.v. excretory vacuole; g, posi-tion of lower end of gullet.

II. Recovery nearly complete, but the animal is inactive.I l l a and b. Various stages of non-recoveiy.I, II and III correspond to the "recovery" stages described in thetext (p. 37s).

Stages I and II were considered to have recovered, whereas III and IV repre-sented 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 harmfulhydrogen ion concentration and afterwards "recover," as defined above, whentransferred back to a solution, the hydrogen ion concentration of which was knownto be harmless.

An example of an actual result obtained with a set often cells containing the buffersolution is given on the following page (Table I) to show the determination of thisend-point. In this case it is quite clear1, but often the individual variations of theSpirostomum amounted to one-and-a-half or two hours, so that there resulted theapparent anomaly of recovery taking place in one cell after a longer exposure thanthat followed by non-recovery in another cell. In these cases a mean value wastaken. Though the degree of accuracy, therefore, which can be claimed for thesevalues is not very high, the relative values obtained for solutions of different

ion concentration should be comparable and of some interest.

may be explained that though the survival time based on this experiment alone is l£ hours(1 hour 25 mins.) the value i j hours was based on the average of this and two other experiments,and appears in the results.

25-2


Table I. Experiment to determine the survival time of Spirostomum(Glasgow culture) in a solution of Palitzsch's borax-boric acid. Buffer, dilux 10 with glass distilled water, and aerated 5 hours.

Light. Electric "daylight" bulb.Temperature. 15-170 C.pH of solution. 9 • 12

No. ofcell

12345

6

89

10

No. ofanimals

44444

4

4444

Time of transferto pH 7-45

S-35 P-m.5-5O „6.5 .,6.25 „

6-45 ..

6-45 ..7-5O „7-5O „7-5O „7-5O „

Date. 23. vii. 1926.Time of start (Greenwich)5.10 p.m.

No. ofanimals

after 24 hrs.

4444

{i4444

Stage ofrecovery

(see p. 370)

IIIIIIV

fH< Ilia

iivIVIVIVIV

End-Point. Between 4th and sth cell.Survival time (to nearest \ hr.) i j hrs.

RESULTS AND CONCLUSIONS.Experiments carried out by the above method showed clearly that the survival

time of Spirostomum ambiguum decreased as the hydrogen ion concentration ofthe medium was decreased, by whichever method this latter factor may have beencontrolled.

The values obtained with the Glasgow cultures of Spirostomum in Palitzsch'sbuffer mixture, diluted, are given in Table II. For these experiments the cellswere illuminated throughout; the temperature varied between 150 C. and 19-5° C ,the mean value being 17° C.

Table II. Results obtained with Spirostomum ambiguum in Palitzsch's borax-boricacid buffer solution diluted x 10 with glass distilled water. July 1926.

pH of solution

Survival time in hours

Light

"Daylight" electric bulb200 v. 60 w.

737

48

762

7

7-87

3i

8 1 2

3

8-37

2

8 6 2

i f

8 8 2

i i

8 9 2

1

9 1 2

i i

9 2 2

I

Temperature

Maximum195° C.

Minimum15-0° C.

Mean17-0° C

The results obtained with Cambridge tap-water and Miss Bishop's ^ pSpirostomum, under similar lighting conditions to the foregoing and at a tempera-ture of 16° C. are given in Table III.


|Bble III. Results obtained with Spirostomum ambiguum in Cambridge tap-water^*with controlled quantities of carbon dioxide in solution. October 1926.

pH of solution

Survival time in hours

Light

"Daylight" electric bulb200 v. 60 w.

7-35

OC

7-6S.

(20)

7-95

13

8-37

6

8-57

4

878

ai

8-98

1 *

9 1 9

1

9 2 9

i

9'39

i

Temperature

Maximumi8°C.

Minimum12° C.

Meani6°C.

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

- 1 0

-9-0

-8o

-10

0 0

\

1o>g .Survival

}\Time

l'c

e+

in. Hei _

1 2

Ti|> WaterBuffer future

\ D\ \J

'o

9

8;

0 -

0 -

:

Fig. 3. Curve showing the relation between the survival time of Spirostomum ambiguum andthe hydrogen ion concentration of the medium in which the animals are placed.

On looking at the curve in Fig. 3 it is clear that the survival time is proportionalto the hydrogen ion concentration within two limits. The lower limit of the hy-drogen ion concentration is approximately 4-0 x io~10 (/>H 0/4). At this arid alllower concentrations death is practically instantaneous. The upper limit of thehydrogen ion concentration, above which the animal will survive indefinitely, isapproximately 4-0 x io"8 (pH 7-4). Within these limits the survival time is relatedto pH by the equations

tlogT = k (1),'logT' = k' (2).

When the survival time T is very small it is seen from Fig. 3 that the pH. in bothequations is the same, so that the constant is the same for both equations, that is

* = *' (3).t^^ t' measure the slope of the lines AB and CD in Fig. 3. The value of t and t',when the survival time is measured in hours, is I - I I and i-66 respectively.

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 notexactly but very nearly, by the Cambridge tap-water, the only harmfulthe hydrogen ion concentration. In the buffer media the value of t' is greater thanthat of t because there is present an additional harmful factor, in the salts formingthe buffer mixture. We are able therefore to separate the toxic effects of the saltsof 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 thehydrogen ion concentration ceases to be proportional to the change in the survivaltime, a very small change in the hydrogen ion concentration will produce a veryconsiderable, and quite disproportionate, alteration in the survival time. Thisdisproportionate effect is important in explaining the appearance and disappearanceof the Spirostomum in ponds. Their appearance and disappearance is almostalways sudden: they are seen in enormous numbers, they exist for a time and thenin two or three days they all vanish. Whereas formerly they may have populatedthe water to the number of over 100,000 a litre, they rapidly become so scarcethat only one or two individuals will be found in several litres of water. WhenSaunders (1924) found them existing in great numbers in a pond he records thepH of the water as being 7-4. This is very near the limiting value for indefinitesurvival and a slight increase in alkalinity will overstep the threshold value of thehydrogen ion concentration and the crowds of Spirostomum will disappear withremarkable suddenness in consequence.

The difference in the values of t and t' indicates a difference in the toxicity ofthe two solutions. There is moreover a distinct difference in appearance in thedying animals in the two solutions. In the buffer mixture after a period of swimmingin circles backwards as much as forwards, as if seeking a way of escape, theanimals almost invariably sink to the bottom, become motionless, contract slightlyin length, appear more opaque and finally, if left sufficiently long, undergo macera-tion, the process starting suddenly at the posterior end and passing forward tillthe whole animal is disintegrated.

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

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

Further, it may be pointed out, that the difference in appearance of the dyinganimals in the two solutions is capable of explanation on osmotic grounds. For


the salt-concentration in Cambridge tap-water is of the order of 0-0042 N^ 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 ifthe 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 inbuffer solution, owing to osmotic diffusion. The occurrence of marked swellingin the tap-water solutions of low hydrogen ion concentration and of death, withlittle or no swelling, in the corresponding buffer mixtures points to a change inpermeability taking place, in the cell wall of the Spirostomum, in these solutions.

There is evidence, then, that the decreased hydrogen ion concentration of themedium in which the Spirostomum are placed causes a proportionate increase inpermeability. Let us assume that (1) when Spirostomum is placed in an alkalinemedium the decreased hydrogen ion concentration of the medium causes the exitof 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 ionconcentration 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 itmust do work in order to prevent any change in the "milieu interne" when thehydrogen ion concentration of the medium, in which the animal is living, differsfrom that of the "milieu interne." The most favourable medium will be one inwhich no work of this nature is necessary. This will only be the case when thehydrogen ion concentration both outside and inside the body is the same. In-definite survival occurs in water when the pH is 7-4, so that this medium isobviously favourable. Moreover Saunders (1924) has shown that, if Spirostomumbe placed in a tube of Cambridge tap-water varying from pH 7-2 at one end of thetube to pH 7-8 at the other, the animals will all collect and cluster together at onespot in the tube where the />H 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. ThepH of the interior of the body of Spirostomum is then 7-4, a result which is insubstantial agreement with the observations of Needham (1925) who, by injectingindicators, determined the internal pK of another protozoan, Amoeba, as being"in the very close neighbourhood of 7-6."

The experiments described using two entirely different media for the controlof the hydrogen ion concentration suggest that the hydrogen ion concentration isan extremely important factor in determining the time of survival of Spirostomumambiguum in a given medium. It is clear moreover that in these experiments it isthe hydrogen ion concentration and not the carbon dioxide which is the controllingfactor, 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^jlrbacia eggs, that the factor controlling viscosity was the amount of carbondioxide present and not the hydrogen ion concentration. The most marked effects,however, in his experiments, were produced in acid solution, a/>H of 5-0, produced


by carbon dioxide, causing coagulation of the protoplasm and eventual deajThe present experiments refer to the alkaline range, death being most rapicpH 9-4, not only in solutions with less than the normal concentration of carbondioxide, but also, in the buffer mixture, containing carbon dioxide in equilibriumwith that in the air.

Moreover it has been pointed out that the degree of swelling which the animalsundergo in the two experimental media can be explained on the assumption ofincreased permeability of their cell walls. That various external factors can alterthe 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 variousunbalanced mixtures of electrolytes in solutions, among other workers. Loeb(1921) has also shown that the rate at which osmotic diffusion proceeds throughartificial (collodion and gelatin) membranes can be modified by changes of pH inacid solutions. It is also well known that if abnormally increased permeability isnot again reduced to the normal state by restoration of the cells to some favourablemedium within a limited time (Lillie 1915), the power of reversibility, of the pro-cesses involved is lost, and death ensues.

It is therefore suggested that increased permeability of the cell wall causes thedestruction of Spirostomum ambiguum and that, in the present experiments, thisincrease in permeability is dependent upon the decrease of hydrogen (or increaseof hydroxyl) ions in the harmful solutions, since it is found that the time requiredfor this destruction of the animals is inversely proportional to the concentrationof 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 workwas carried out in the Zoological Laboratory, Cambridge.

SUMMARY.

1. A recovery method is described for determining the survival time of Spiro-stomum ambiguum (Ehrenberg) major in solutions of different hydrogen ion con-centration. The survival time is defined as the longest time for which the animalscan remain in the solution of harmful hydrogen ion concentration and afterwardsrecover their normal appearance when transferred back to a solution, the hydrogenion concentration of which is known to be harmless.

2. The results are given of experiments carried out in two solutions both ofwhich provided media of known hydrogen ion concentration, but allowed thisfactor to be controlled by entirely different substances. The solutions were:

(i) Palitzsch's borax-boric acid buffer mixture diluted ten times with glassdistilled water.

(ii) Cambridge tap-water (-0042 iV carbonates) containing regulatedcarbon dioxide in solution.

3. These experiments showed that the hydrogen ion concentration was a very


important factor in determining the survival time of Spirostomum in the medium.

f media of pH 7-4 the Spirostomum survive indefinitely but in solutions of9-4 they rapidly die after greater or less swelling, according to the osmotic

pressure of the medium. The time of survival decreases with increasing alkalinityand between pH 9-0 and 7-6 it is inversely proportional to the hydrogen ionconcentration. Between pH. 7-6 and 7-4 the time of survival is not proportionalto the hydrogen ion concentration and a very slight decrease in the hydrogen ionconcentration at this point will cause* a very considerable, and quite dispropor-tionate, decrease in the time of survival. It is suggested that such a slight decreasein the hydrogen ion concentration of the water is the possible explanation of thevery sudden disappearance of Spirostomum from ponds, where it was existing invery large numbers.

The destruction of the Spirostomum in media of greater alkalinity than pH 7*4is probably due to the increase in alkalinity affecting the body wall in such a waythat it becomes more permeable to water, the result, as observed, being that theanimals swell up and eventually burst.

REFERENCES.BISHOP, ANN. (1923). Quart. Journ. Micr. Sci. N.S. 67, 391.BLATTNER, H. (1926). Arch.f. Protistenkunde. 53, 253.CLARK, W. M. (1922). The Determination of Hydrogen-ions p. 48. Baltimore.DALE, DOROTHY. (1913). Journ. of Physiol.46, 129.'JACOBS, M. H. (1922). Biol. Bull. 42, 14.LILLIE, R. S. (1915). Biol. Bull. 28, 260.

(1918). Amer. Journ. Physiol. 45, 406.LOEB, J. (1921). Journ. of Gen. Physiol. 4, 213.NEEDHAM, J. and D. M. (1925). Proc. Roy. Soc. B, 98, 259.OSTERHOUT, W. J. V. (1922). Injury, Recovery and Death in relation to Conductivity and Permeability.

Philadelphia.PACKARD, J. (1925). Journ. of Gen. Physiol. 7, 363.PALITZSCH, C. (1915). Biochem. Zeitschrift. 70, 333.POTTER, A. (1903). Zeit.f. allg. Physiol. 3, 363.SAUNDERS, J. T. (1924). Proc. Camb. Philos. Soc, Biol. Sci. 1, 189.

(1926). Brit. Journ. Exper. Biology. 4, i, 46.

the relation of spirostomum ambiguum to the … · spirostomum with a test medium of known hydrogen...

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