tration at which an effect on the oxidative processes is noticeable. Much greater accuracy in the methods for quantitative studies on intracellular oxidation in multicellular animals is necessary before a satisfactory statement in detail of the conditions for these forms can be made.

SUMMARY

1. Paramecium serves as an ideal organism for accurate quantitative studies on intracellular oxidations.

2. In Paramecium the oxidations stop when the cell is killed by too high oxygen concentration.

3. In a pure line of Paramecium from the same culture different cells differ in respect to the minimal oxygen concentration in which they can continue to live.

4. The rate of intracellular oxidation in Paramecium is independent of the concentration of oxygen. The concentration of oxygen may vary from a minimum of about 0.04 cc. of O2 at N.T.P. per 137 cc. to 2.2 cc. O2 at N.T.P. per 137 cc. or fifty-five times the minimal concentration, without affecting the rate of oxidations. This is true for a temperature of 13.5° as well as 25°C.

BIBLIOGRAPHY

(1) VERNON: Journ. Physiol., 1895, xix, 18.

(2) BARRATT: Zeitschr. f. allg. Physiol., 1905, v, 66.

(3) HARGITT AND FRAY: Journ. Exper. Zoöl., 1917, xxii, 421.

(4) TREADWELL AND HALL: Volumetric analysis.

(5) HEILBRUNN: Sci. N. S., 1915, xxxxii, 615.

(6) THUNBERG: Skand. Arch. f. Physiol., 1905, xvii, 133.

(7) HENZE: Biochem. Zeitschr., 1910, xxvi, 255.

QUANTITATIVE STUDIES ON INTRACELLULAR
RESPIRATION

II. THE RATE OF OXIDATIONS IN PARAMECIUM CAUDATUM AND ITS INDEPENDENCE OF THE TOXIC ACTION OF KNC

E. J. LUND

From the Department of Animal Biology, University of Minnesota

Received for publication January 9, 191

Most of the recent work on the problem of the nature of the effects of anesthetics and narcotics on cells seems quite definitely to support the conclusion that these substances do not exert their distinctive effects upon cells by primarily inhibiting intracellular oxidations. Where a lowered rate of oxygen consumption or carbon dioxide production as the result of injection or immersion in solutions of alcohol, ether, chloral hydrate and the urethanes has been observed, it can usually be interpreted as an indirect effect, due for example to lowered muscular tone or death of cells (1), (2), (3), (4).

In striking contrast to these experimental results stand those from the cyanides. It is very generally agreed that the toxic action of the latter is due to their specific power of inhibiting intracellular oxidations. Any cell which can be isolated and subjected to direct observation of its structure and visible processes under experimental conditions has often, for obvious reasons, many advantages over complex cell aggregates; consequently some of the most direct and convincing evidence for this specific inhibitory action by the cyanides has been obtained from experiments on' sea urchin eggs (5), (6).

On the basis of this and other evidence for specific inhibitory action by cyanides on intracellular oxidations, hypotheses and conclusions have often been arrived at by others which if proven to be correct are of importance. Potassium cyanide has been used in studies on the action of the respiratory center in mammals, its effects being attributed to its inhibitory action on the oxidations in the nerve cells of the respiratory center (7). Child (8) and his colleagues have made extensive use of the susceptibility of organisms to the toxic action of the cyanides

in studies on morphogenesis. They assume that the rate of oxidations serves as the most satisfactory available measure of the rate of (total?) metabolism, and that the rate of metabolism determines the course of morphogenetic processes, such for example as localization of body axis in regenerating pieces of planaria and various coelenterates. If the cyanides do have a specific inhibitory effect on oxidations in general they naturally offer interesting possibilities for experiment.

The purpose of the present paper is to show that in Paramecium caudatum the rate of intracellular oxidations is entirely independent of the action of KNC even in concentrations which injure and finally kill the cell by cytolysis.

RATE OF OXYGEN CONSUMPTION BY PARAMECIUM IN SOLUTIONS OF KNC IN TAP WATER

The procedure for oxygen determination and preparation of Paramecia has been described in a previous paper (9). Table 1 gives the results of a preliminary experiment carried out before the methods described in the first paper of this series had been fully worked out, therefore several sources of error such as that in the filling of the bottles, adsorption of iodine and the drawing of samples of Paramecia from the suspension were not eliminated as fully as in the remaining three experiments. It is given because in spite of some non-uniformity in oxygen concentration in the bottles the results show very clearly that KNC does not inhibit the oxidations to any noticeable degree even in the concentrations which killed some or nearly all of the Paramecia.

It will be seen from column 7 that in those bottles where a large number of Paramecia were dead at the end of fifty-one hours, the total oxygen consumed was in general a little less than in the bottles where all the Paramecia were living. The average quantity of oxygen consumed which is equivalent to 2.32 cc. thiosulfate per 150 cc., was practically the same in the bottles containing Paramecia without KNC (column 4), as in those bottles indicated by the bracket in column 7 where it was equivalent to an average of 2.43 cc. thiosulfate per 150 cc. The lower average oxygen consumption in column 7 is entirely accounted for by the death of Paramecia which occurred during the experiment in the higher concentrations of KNC. In spite of the evident errors due to non-uniformity in conditions for each bottle the fact is clear, from a glance at the figures in columns 2, 4 and 7, that intracellular oxidations were not inhibited to any marked degree by

the cyanide either in the weak or in the strong concentrations, and that an average of almost 3.00 cc. thiosulfate equivalent of oxygen was con

TABLE 1

Preliminary experiment. Bottles were filled from tap by rubber tube. The Paramecia were centrifuged, washed twice in tap water and used immediately without starving. The KNC solution was first added, then 1 cc. Paramecium suspension was added and the bottle stoppered and shaken. Temperature 21 ± 2°C.

sumed by the Paramecia in the KNC solutions. Is the rate of oxidations in Paramecium completely unaffected by cyanide? For an answer to this question greater degree of accuracy is necessary.

In the following experiments the bottles were of equal volume and contained nearly equal amounts of oxygen, the degree of accuracy is greatest in experiments the results of which are given in tables 2 and 3. Controls for determining the average amount of iodine adsorbed by Paramecia, are given in columns 1 and 2. This is equivalent to 0.43 cc. thiosulfate, a relatively large amount due to the large number of Paramecia in each bottle (table 2). The Paramecia in bottles containing 3 and 2.6 cc. N/10 KNC would probably not have lived more than a few hours longer, for a number in each bottle were dead and many were deformed and showed abnormal swimming movements. In bottles containing less than 1.4 cc. м/10 KNC the cells were perfectly normal and active and would undoubtedly have lived much longer. In other experiments similar to this one the Paramecia lived in concentrations of 0.05 cc. up to 0.4 cc. N/10 KNC in 137 cc. tap water for several days, until nearly all the oxygen was consumed or death occurred as a result of starvation. The average amount of oxygen in cubic centimeters of thiosulfate remaining after sixteen and one-half hours in the first six bottles of column 5 is 4.42. That of the last six bottles is 4.54 cc. thiosulfate. No animals had died in any of the bottles although the concentrations of KNC in the first three bottles were sufficient to kill many of the Paramecia had they been left for twentyeight hours as shown in column 7. Evidently a lethal concentration of KNC in this case accelerated the oxidations or else was entirely without effect. The first three bottles (column 7) show a slightly lower oxygen content than the remaining ones. The only way I can account for this is by an accelerating effect or increased oxygen consumption due to stimulation of the organisms by the cyanide or more probably by errors in filling bottles.

A final experiment (table 3) was performed in which the greatest care was taken to avoid errors in manipulation. The range of variation of the cyanide concentrations-5 cc. to 0.05 cc. N/10 KNC-was greater than in previous experiments. It happened that the resistance to KNC of this lot of pure line Paramecia was greater than that of those used in any of the previous experiments so that at the end of twentynine and one-half hours relatively very few Paramecia were dead, even in the bottle containing 5 cc. N/10 KNC. Results of an analysis of the conditions for variability in resistance to KNC by Paramecium will be given elsewhere.

At the end of ten hours all Paramecia were alive in all the bottles. Those in the bottles containing 5 cc. KNC were moving more slowly