the effect of partial pressures of inert gases on the ... · it was found that the four inert...

J. exp. Biol. (1976), 64, 615-^37 6 1 5With 6 figures

Printed in Great Britain

THE EFFECT OF PARTIAL PRESSURES OF INERT GASESON THE BEHAVIOUR AND SURVIVAL OF THE

HETEROTRICH CILIATE SPIROSTOMUM AMBIGUUM

BY S. MACDONALD AND J. A. KITCHING

School of Biological Sciences, University of East Anglia, Norwich, England

(Received 9 October 1975)

SUMMARY

A study has been made of the effects of helium, nitrogen and argon atpartial pressures of 10-120 atm on the swimming speed, behaviour andsurvival of the heterotrich ciliate Spirostomum amtnguum. Experiments havealso been carried out with hydrostatic pressure alone. Hydrostatic, heliumand nitrogen pressures reduced the swimming speed approximately equallybut pressure of argon reduced it to an even greater extent. Hydrostatic,helium, nitrogen and argon pressures caused a reduction in the number ofreversals in a given time but the number of reversals/distance travelled didnot change significantly. All the treatments increased the durations ofindividual forward movements. Pressures of helium, nitrogen and argonalso increased the durations of reversals but at high hydrostatic pressuresthe durations of reversals were reduced.

Argon caused cytolysis at 30-40 atm and nitrogen at 80 atm; helium didnot cause any visible cell damage over periods of 1 h at 120 atm. Addition ofsubstantial partial pressures of helium protected Spirostomum againstpartial pressures of argon which would otherwise have caused cytolysis.The increase of hydrostatic pressure may have been responsible.

INTRODUCTION

In a recent study on Echinosphaerium nucleqfilum (Miller, Aidley & Kitching, 1975)it was found that the four inert gases, helium, nitrogen, argon and krypton wereincreasingly potent in causing cell damage or cytolysis at elevated pressures. Treat-ment with helium at 60-100 atm also caused shortening of the axopods, which mustindicate a disintegration of the microtubular axonemes, whereas the other gasescaused leakage or disintegration with little or no shortening of the axopods. Specificeffects of hydrogen and helium on the divisions of Tetrahymena have also beendescribed by Macdonald (1975); these gases sustain cell division at hydrostaticpressures which would otherwise inhibit it. In the case of Echinosphaerium a mixtureof helium and argon produced less axopod shortening than if the same partial pressurehad been used without the addition of argon and less cell damage than if the samepartial pressure of argon had been used without the addition of helium. Themechanism of this antagonism is not yet understood.

In the work to be described we have conducted experiments on the large hetero-

616 S. MACDONALD AND J. A. KITCHING

trich ciliate Spirostomum ambiguum similar to those on Echinosphaerium. Spirostomummoves sufficiently slowly for its behaviour to be followed and recorded. The investiga-tion was designed to ascertain whether helium had specific effects apart from thosewhich could be attributed purely to hydrostatic pressure, whether it differed qualita-tively from other inert gases in its effects and whether it counteracted the effects ofargon when both were given together. Knowledge was already available (Kitching,1969) about the effects of hydrostatic pressure on this ciliate over the range 1000 psi(68 atm) up to 10000 psi (680 atm). The present investigation is concerned withpressures (hydrostatic or gaseous) of up to 120 atm.

METHODS

Two pressure vessels were used - the hydrostatic pressure vessel described byKitching (1954, 1957) and the gas pressure vessel described by Miller, Aidley &Kitching (1975). For experiments on survival eight animals were used at a time butsingle animals were used in experiments on behaviour. The animals were mountedin a drop of culture medium on the upper window of the appropriate pressure vesseland the window was then inverted and screwed into place so that the drop was sus-pended. Observations were carried out for an initial period (normally 30 min) withoutapplication of pressure, for an experimental period (60 min unless otherwise specified)at pressure, and for a final period (normally 30 min) without pressure to test recovery.For experiments with gases, humidified air containing 0-5 % COa was passed con-tinually through the pressure vessel during the initial and final periods. At thebeginning of the experimental period helium, nitrogen or argon was added to the1 atm of treated air already in the vessel taking not less than 45 3 to reach the requiredpressure. At the end of the experimental period the pressure was released very slowly(taking 20 min after a treatment at 120 atm) and treated air was then introduced atatmospheric pressure. In experiments with hydrostatic pressure the cavity of thepressure vessel was filled with medicinal oil ('liquid paraffin') which was used ashydraulic fluid. Control experiments were carried out with the gas and hydrostaticvessels in which there was no increase in pressure during the ' experimental' period.

The normal swimming pattern of Spirostomum ambiguum consists of many forwardmovements alternating with reversals. When suspended in a hanging drop theanimal appears to ' explore' the whole of the drop by means of these alternate forwardmovements and reversals. Healthy animals never remain stationary. The averagenumber of forward movements (or reversals) in a 5 min period (based on 20 suchperiods) was 32. Throughout each experiment on behaviour, observations were carriedout in a continuous series of 5 min periods. Each alternate 5 min period (1st, 3rd, 5th,etc.) was used for recording manually but continuously the swimming behaviour of theSpirostomum. A pen recorder controlled through a reversible potential box was usedto register the number and duration of each forward or backward movement of theanimal. During each intervening 5 min period (2nd, 4th, 6th, etc.) six estimates weremade of the swimming speed of the Spirostomum by timing it over a measureddistance as seen with an eyepiece graticule.

The temperature of the two pressure vessels was controlled at 21 °C by the methodalready described (Kitching, 1957; Miller et al. 1975).

Effect of partial pressures of inert gases on S. ambiguum 617

1

1-2100-80-60-40-2

1-2100-80-60-40-2

1-2100-80-60-40-2

1-2100-80-60-40-2

1 1

1

1

1

1 1 1 1 T^T— i , , i i .

• 120 atm

05 15 25 35 45 55 65 75 85 95 105 115

100 atm

80 atm

60 atm

1-210080-60-40-2

40 atm

1-2100-80-60-40-2

20 atm

Time (min)

Fig. i. Swimming speed of Spiroitomum ambiguum under various hydrostatic pressures(each point is a mean of six readings). Period under pressure shown in stipple.

RESULTS

Hydrostatic pressure

Single animals were used. Two control experiments were carried out withoutchange of pressure. In 13 experiments the hydrostatic pressure was raised over aperiod of 45-120 s to a final level ranging from 20 to 120 atm. In two other experi-ments the pressure was raised to 100 and 120 atm by 20 atm steps, with a 1 min

6i8 S. MACDONALD AND J. A. KITCHING

40

30

20

10

Hydrostatic pressure

40

30

20

10

,o

Helium

I 5

3C

nNitrogen

:-S--

0 20i

40 60

Argon

i

80 1001

120

40

30

20

10

Pressure (atm)

Fig. 2. The number of forward movements (mean ± 2 8.B.) separated by reversals made bySpirostomum amiriguum in a standard time at various gas and hydrostatic pressures (replicateexperiments combined).

pause after each increase. A single 2-months old culture was used for all these experi-ments. Its pH was T'l~l"h-

Swimming speed (Fig. i) was not significantly affected at hydrostatic pressures ofup to 6o atm. From 80 atm upwards it was progressively reduced, and averaged onlyabout a third of its normal value at 120 atm.

At all pressures used hydrostatic pressure significantly decreased the number ofreversals (Fig. 2) in a given time; the greater the pressure, the less frequently didthe animals reverse. Hydrostatic pressure also increased the durations of forwardmovements but reduced that of backward movements at the higher pressures. Thefrequency of various durations was tabulated against pressure for each pressure used.Extracts for this and other treatments are given in Tables 1 and 2. Different categories


Table 1. Effects of hydrostatic pressure and gas pressure on the frequency (%) distributionof the durations of forward movements made by Spirostomum ambiguum

Duration of movement (s)Treatment

Hydrostatic

Helium

Nitrogen

Table 2. Effects of hydrostatic pressure and gas pressure on the frequency (%) distributionof the durations of reversals made by Spirostomum ambiguum

Duration of movement (a)

Pressure(atm)

0

601 2 0

060

IZO

0

601 2 0

I-IO

78-466-639-6

8383 9 147-390-053-33 6 8

11-20

19-428-830-41493»-o198

9-32 8 624-8

21-30

1 9

4-311-2

1-2

1 3 78 70-79-1

1 7 9

31-40

0 3

0 310-4

0

5-16 30

4 83-4

>4O

0

0

8-50

IO-21 7 9

O

4 317-1

n

1081347

260

17681972 0 7

" 4 52 1 0

117

Treatment

Hydrostatic

Helium

Nitrogen

Argon

Pressure(atm)

060

120

060

120

060

120

040

o-o-s

49963467-7

58-3IS'635757-S24625961 7180

o-6-i-o

34-822-527722-3341297

24929-622'2

22-219-8

I-I-I-5

10-78 14-2

8-996

i i - i

8-i12-38-37-1

137

1-6-2-0

424-20

371634-5

3'910-32-8

3'48-4

2-1-2-5

2-1i-80-4

3-0593-o273 946288-7

>2-5

i-300

4-018-5161

29192

36-i

2931-4

n

1048333238

1593135199

979203108

790344

of duration are used for forward and backward movements because forward move-ments last much longer. The changes in distribution of these frequencies withpressure are highly significant; for duration of forward movement^;2 = 3 8 7 ^ < o-ooiand for duration of backward movement x2 — i n , P < o-ooi. Chi-squared testsbetween groups of experiments at adjacent pressures have indicated that the changein durations of movements is distributed throughout the range of pressures usedand that there is no critical level at which the change suddenly occurs.

In the two experiments in which the pressure was raised by 20 atm steps, theresults were indistinguishable from those obtained by the standard experimentalmethod.

When frequency of reversal was expressed in terms of distance travelled insteadof time, hydrostatic pressure made no significant difference. At 120 atm both speedand number of reversals in unit time were reduced but the number of reversals/distance travelled remained the same.

Other aspects of the behaviour of Spirostomum were also changed by the applicationof hydrostatic pressure. The animals tended, under pressure, to swim in continuouscircles; on reaching the edge of the drop they swam around it instead of reversing


1-2100-80-60-40-2

1-2100-80-60-40-2

1-2100-80-60-40-2

120 atm

I05 15 25 35 45 55 65 75 85 95 105 115

100 atm

GO

"es

80 atm

1-2100-80-60-40-2

60 atm

1-2 T -100-80-60-40-2

40 atm

1-2100-8.0-60-40-2

20 atm

Time (min)

Fig. 3. Swimming speed of Spirostomum ambiguum under various pressures of helium (eachpoint is a mean of six readings). Period under pressure shown in stipple.

This behaviour may be associated with a loss of sensitivity. At 100 and 120 atmespecially it was noted that on reaching the edge of the drop the animals frequentlycollided with the paraffin/water interface, so that the leading portion of the body bentaround along the curvature of the drop.

After release of pressure, normal swimming speed and behaviour were resumedwithin 30 min in all experiments and in many experiments immediately. No changewas found in swimming speed or behaviour in the control experiments in which no


g

i

60-,40-20-

60-40 -20-60-40 -20-

60-40-20 -

80-60-40-20 -

80-60-40-20-

80-60-40-20-

60-40-20-

60-40-20-60-40-20-

80-60-40-20-

60-40-20-

(

- . A

\ _ _

\

\

I I I 1 1 1 1 1 1 1) 1 2 3 4 5 (

\

-0,

\ _

\1 1 1 1 1 1 1 1 1 1 1 1 1 i 1 1 1 1 1 1 1 1 1 1 1

1 1 2 3 4 5 6 7 8 9 10 11 12

Duration (s)

0atm

120atm

0atm

Fig. 4. The percentage frequency of reversals of diflferent durations (replicates combined) at120 atm of (A) hydrostatic pressure and (B) helium. Three pre-pressure 5 min readings arefollowed by six 5 min readings under pressure and a final three 5 min readings after pressurerelease.

pressure was exerted. No obvious damage occurred in any of the experiments inwhich hydrostatic pressure was used.

Helium

Sixteen experiments on single animals were carried out with the standard experi-mental procedure already described. Pressures of 10-120 atm of helium were usedin the experimental period. One culture was used throughout, 2 months old andwith a pH of


Swimming speed was not significantly affected up to 60 atm of helium but wasincreasingly reduced at higher pressures (Fig. 3).

The frequency of reversal (or return to forward swimming) was also reduced verysignificantly by pressures of 40 atm upwards. Pressures of helium caused a highlysignificant change in the frequency distribution of the durations both of forwardmovements (x2 = 1136, P < o-ooi) and of reversals (x* = 299, P < o-ooi). Thedurations both of forward movements (Table 1) and of reversals (Table 2) increasedwith pressure of helium. As with hydrostatic pressure the change in duration ofmovements is distributed throughout the range of pressures used. There was nocritical level at which the duration of forward movements or reversals changedsuddenly.

At 120 atm there are highly significant differences between the effects of heliumand those of hydrostatic pressure alone. High pressure of helium caused a greaterincrease in the duration of forward movements than did corresponding high hydro-static pressures (x3 = 82, P < o-ooi) (Table 1). For reversals, the two treatmentsacted in opposite directions (Fig. 4).

With helium as with hydrostatic pressure, when frequency of reversal is expressedin terms of distance travelled instead of time, treatment at 120 atm is not found tohave produced any significant change.

At high pressures of helium the animals tended to swim in continuous circles oraround the edge of the drop. At pressures of over 60 atm they frequently collidedwith the gas/water interface at the edge of the drop and suffered a temporary bendingof the body.

Recovery was often incomplete within 30 min of release of pressure and sometimesnormal behaviour was not resumed even 2 h after release from 60-120 atm helium.This is not apparent from the figures because, when the number and duration ofmovements was returned to normal, the animals no longer swam around the wholedrop but moved about within a small and limited part of it. The wider 'exploratory'behaviour typical of untreated animals had been lost. One animal became sphericaland disintegrated during the final (recovery) period. No other animals showed anycell damage during the experiments.

Nitrogen

Nine experiments were carried out on single animals to test behaviour and 14experiments on groups of eight animals to test survival. A single 2-month-old culturewas used in the former but several cultures ranging from 14 to 125 days in age andfrom pH 7-3 to 7-5 were used to test survival at 80-120 atm nitrogen.

Survival varied between cultures. All animals tested survived 60 atm nitrogen.One animal out of 16 survived 80 atm and two out of 16 from a different culturesurvived 120 atm but none survived 100 or n o atm.

Swimming speed was reduced in the animals which survived exposure to 120 and80 atm nitrogen but there was little or no effect from 60 atm downwards (Fig. 5).

The frequency of reversal (or return to forward movement) declined with increasein nitrogen pressure (Fig. 2, with an aberrant reading at 40 atm). The change infrequency dsitribution of the duration both of forward (xz = 517, P < o-ooi) andbackward movements (xi = 305, P < o-ooi) is highly significant. As seen in Tables


120 atm

05 15 25 35 45 55 65 75 85 95 105 115

1-2-T-

10- -0-8- -0-6- -0-4- -0-2- -

80 atm

1-2'

100-80-60-40-2

1-21-0'0-80-60-4'0-2-

60 atm

40 atm

1 - 2 T -

1 0 - -0-8--0-6--0-4--0 - 2 -

20 atm

Time (min)

Fig. 5. Swimming speed of Spirostomum ambiguum under various pressures of nitrogen(each point is a mean of six readings). Period under pressure shown in stipple.

i and 2, both forms of movement tend to last longer at higher nitrogen pressures.Chi-squared tests on groups of experiments at adjacent pressure showed that therewas no critical pressure at which the duration of forward movements or reversalssuddenly changed; the effect increased progressively up to 6o atm but further increaseof nitrogen pressure had little additional effect.

With the three survivors at 120 and 8o atm reversals lasted longer than at the samepressures of helium (Table 2). However, the number of reversals/distance travelledremained the same at 120 atm as during the initial period before admission of nitrogen.

All survivors from all experiments resumed normal behaviour during the final(recovery) period.


1-2100-80-60-40-2

40atm

05 15 25 35 45 55 65 75 85 95 105 115

1-2T-1 0 -

00 0 8 ".S 0-6- -1 0 - 4 -

•1 0-2- -c8

20atm

1-2-p10- -0-8- -0-6 -0-4 -0-2 -

lOatm

Time (rain)

Fig. 6. Swimming speed of Spirostomum ambiguwn under various pressures of argon (each pointis a mean of six readings). Period under pressure shown in stipple.

Argon

Six experiments were carried out on groups of eight animals to test survival andsix experiments on single animals to test behaviour. Two cultures were used, each 2months old and at pH 7-2-7-3.

In the first culture all animals subjected to 50 atm or over burst within 2 min ofthe introduction of the gas. Specimens subjected to 10-40 atm argon survived.

In the second culture all animals burst at 30 atm argon or over under experimentalconditions seemingly identical to those used with the first culture. Some of thespecimens tested at 30-41 atm argon burst almost immediately while others survivedfor up to 40 min. Animals which burst within the first 2-3 min after the introductionof the gas were destroyed by the sudden and vigorous bursting of the cell. Thosewhich lasted for up to 40 min gradually became short and swollen and finally roundedup and disintegrated.

There was no detectable change in swimming speed at 10 or 20 atm argon but at40 atm the speed decreased (Fig. 6).

Argon decreased the number of reversals made in a given time (although not interms of distance travelled). It significantly changed the frequency distribution ofthe durations of forward movements ( ^ = 34, P < o-ooi) and of backward move-ments (x2 = 313, P < o-ooi). As with other treatments, there was no critical level.The durations of forward movements increased slightly, and of backward movementsvery considerably, especially at 40 atm argon (Table 2). While under argon pressure


Table 3. The effects of argon and of mixtures of helium and argon on the survivalof Spirostomum ambiguum after 1 h of exposure to the gases

Treatment*

Culture iA28A30

A28 + He72Heio + A28 + He62Aio + Heio + Aio + HeaoHeio + A3o + He2O

Culture 2 aA40

A45He8o + A40He6o + A6oHeyo + Aso

Culture 2 bA30A25A2o + He4S+Aio + He25

No.tested

5624242488

488

168

16

338

16

Elongate,normal

2

516

1957

——16—

1

—

813

Damagedpyriform

3 '31

S3*1

———

—

7

——3t

Spherical E

381

—

—

—

—

—

—

—

>isintegi

2 0

86+

———

488

88

32

The susceptibility of culture 2 changed over a period of 5 days, i.e. culture 2 b is the same cultureas 2 a but with a lower threshold of susceptibility.

The partial pressure in atm. is given after the symbol for the gas.• When mixtures of gas have been used, the entry under 'treatment' shows increments of each gas

(A, argon; He, helium) in the order given.t Animals damaged by argon before the helium had entered the vessel.

the animals frequently contracted suddenly to one half or one third of their normallength. Contractions averaged i/min at 20 atm and 3/min at 40 atm argon. Many ofthe longer forward movements were made while the animals were in the contractedstate.

All surviving animals recovered their speed and normal behaviour within 30 minof release of pressure.

Mixtures of helium and argon

Experiments on survival were carried out with argon alone and with mixtures ofargon and helium, close together in time and on the same cultures. As already ex-plained, cultures may differ in susceptibility to argon and they may also change withtime. Details of the experiments are summarized in Table 3. Animals in culture 1either burst or were severely damaged at 28 atm of argon (or over), while in culture 2the critical argon pressure for damage or bursting was initially 40 atm (reported inTable 3 as culture za) but fell after a week to 30 atm (26). For all three of theseculture phases (1, 2a, zb) a series of alternate experiments was carried out, in whichthe treatment consisted either of a critical pressure of argon, just sufficient to causesubstantial damage, or of a mixture of argon at the same partial pressure with asubstantial additional pressure of helium. Culture 2 a was also used to test mixtureswith a still higher partial pressure of argon. The order of entry of gases is shownin Table 3 and was varied. In some experiments on cultures 1 and zb the argon andhelium were admitted by alternate steps, with a 6 min pause between the first and

40 KXB 64


second admissions of argon; there was also a 6 min interval between the two admis-sions of argon in the alternate experiments using argon alone.

The addition of a substantial partial pressure of helium greatly improved theresistance to damage and the survival of Spirostomum at partial pressures of argonwhich would otherwise have proved damaging or lethal. If the argon was admittedfirst to its full partial pressure, a few animals were damaged or destroyed before thehelium could be admitted, but the subsequent admission of helium gave significantprotection. If the argon was admitted either last or by steps, alternating with helium,better protection was obtained. Admissions of argon alone by steps were as damagingas admissions in one increment. Helium (60 atm) failed to protect against 60 atm ofargon, and helium (70 atm) gave only slight protection against 50 atm argon with aculture (2 a) for which 40 atm of argon alone was critical.

DISCUSSION

Gas pressure inevitably involves a component of hydrostatic pressure. Only thoseresults which are obtained by gas pressure and not by an equivalent hydrostaticpressure can be attributed to the action of the gas. Hydrostatic pressure alone, withinthe appropriate range, caused no detectable cell damage, so that our observations ofcell damage and cytolysis under the influence of ' inert' gases imply a special actionby these gases.

Our experiments on the survival of Spirostomum ambiguum under different pressuresof these inert gases demonstrate a sequence of toxicity (helium < nitrogen < argon)consistent with generally accepted conclusions for most other material (Schreiner,1968). In respect of the levels of pressure necessary to cause cell damage or cytolysisover periods of an hour, they are rather closely in agreement with the results obtainedby Miller et al. (1975) for Echinosphaerium nucleofilum. On the other hand, Amoebaproteus, Paramecium aurelia and Tetrahymena pyriformis have been found by us tosurvive for at least 12 h in n o atm of argon in similarly conducted experiments.Stentor coeruleus survived for similar periods in argon pressures of up to 80 atm. Athigher pressures cytolysis occurred within minutes of the admission of the gas.

It seems possible that these differences are to be interpreted in terms of structuraldifferences in the cortical or pellicular layer of these various Protozoa. The cortex ofEchinosphaerium probably derives mechanical stability from the closely packedsystem of vacuoles of which it is composed and which might be weakened under theinfluence of the gas. On the other hand the cortex of Amoeba proteus consists of a morecontinuous protoplasmic phase, continually changing and not dependent on vacuolesfor its mechanical properties. Spirostomum ambiguum differs in cortical structure fromParamecium and Tetrahymena in that, like Stentor, it is designed for contractabilityand lacks the stabilizing system of closely packed alveoli and associated microtubulesand fibres found in holotrichs. Spirostomum is also notably unstable in the presenceof a slight alkalinity (pH > 7-4) of the medium (Jenkin, 1927). Its susceptibility tothe inert gases may result from this difference in surface structure.

Hydrostatic pressures of 260 atm or more have already been found to inhibit theavoiding reactions of Spirostomum (Kitching, 1957). Our results show that thisinhibition starts at much lower pressures (20 atm) and it seems likely that the reduc-tion in the frequency of reversals is also due to a depression of sensitivity. It is not


known (Kitching, 1969) whether this results from an impairment of ciliary mechano-reception associated with the decrease in swimming speed found under hydrostaticpressure or from some effect of pressure on the properties of the plasma membrane.In any case, similar effects were obtained in the present work with gas as with hydro-static pressure.

The mechanism by which a state of reversal is brought about and maintainedis still not understood (Jones, 1974), although movements of calcium are believed tobe involved. Reversal may express a state of excitation, originating in the cell mem-brane. The duration of reversals is depressed by high hydrostatic pressure but in-creased by corresponding pressures of helium, nitrogen, or argon with potencyincreasing in that order. The simplest comprehensive explanation is that the inertgases influence the structure of the plasma membrane in such a way as to promote astate of excitation and ultimately of physiological breakdown and disintegration.Nitrogen and argon have been found to penetrate the lipoid phase of plasma mem-branes at high pressures much more readily than helium (Bennett, Papahadjopoulos& Bangham, 1967).

Our experiments on the effect of gas mixtures confirm the findings of Miller et al.(1975) on Echinosphaerium, in that a critical pressure of argon is less toxic toSpirostomum if helium is also added. This reversal of toxicity has not yet been satis-factorily explained. The added helium or the associated hydrostatic pressure mayaffect the solubility of argon in the external aqueous solution, the solubility or argonin the hydrophilic regions of cell membranes, or the reaction between argon andreactive sites within the Spirostomum. Although the solubility of a gas per atmosphereof partial pressure is reduced by increase of pressure (Schroder, 1969) it is notpossible to predict the effect on critical toxic levels of argon for a living organism.However, the beneficial effects of a substantial addition of helium are limited to anarrow range of argon pressure around the critical level. Thus it is possible that theeffect is simply one of elevated hydrostatic pressure upon the sites of biologicalreactivity similar to 'pressure reversal' of anaesthesia. There is as yet no need toinvoke any competition for specific molecular sites between the gases involved.

This work was generously supported by the Science Research Council.

REFERENCES

BENNETT, P. B., PAPAHADJOPOULOS, D. & BANCHAM, A. D. (1967). The effect of raised pressure ofinert gases on phospholipid membranes. Life Sci. 6, 2527-33.

JENKIN, P. M. (1927). The relation of Spirostomum ambiguum to the hydrogen ion concentration(alkaline range). J. exp. Biol. 4, 365-77.

JONES, A. R. (1974). The CiliaUs. London: Hutchinson University Library.KITCHINO, J. A. (1954). The effects of high hydrostatic pressure on a suctorian. J. exp. Biol. 31, 56-67.KITCHING, J. A. (1957). Effects of high hydrostatic pressure on the activity of flagellates and ciliates.

J. exp. Biol. 34, 494-510-KITCHING, J. A. (1969). Effects of high hydrostatic pressures on the activity and behaviour of the

ciliate Spirostomum. J. exp. Biol. 51, 319-24.MACDONALD, A. G. (1975). The effect of helium and of hydrogen at high pressure on the cell division

of Tetrahymena pyriformis W.J. Cell. Physiol. 85, 511-28.MILLER, J. B., AIDLEY, J. S. & KITCHING, J. A. (1975). Effects of helium and other inert gases on

Echinosphaerium nucleopltilum (Protozoa, Heliozoa). J. exp. Biol. 63, 467—81.SCHREINER, H. R. (1968). General biological effects of the helium-xenon series of elements. Fedn

Proc. Am. Socs exp. Biol. 37, 872-8.SCHRODER, W. (1969). Beobachtungen an Losungen von Gasen in Flussigkeiten. Z. Naturf. 24b, 500-8.

40-2

the effect of partial pressures of inert gases on the ... · it was found that the four inert...

Documents