Studies in the Respiration of Paramecium Caudatum

With ‘wild’ cultures of Protozoa, the first problem in preparing animals for experiment is one of lowering to an insignificant level the percentage of contaminant bacteria associated with the animals; failure to do this invites criticism of some earlier work in the field. The usual method is washing by repeated centrifugation (Lund, 1918; Pitts, 1932, etc.), but in our hands this procedure was found to damage a large percentage of the animals,-and it became necessary to discover some other means of purifying the suspension. Root (1930) collected Paramecium at the top of a glass cylinder by use of the animals’ negatively geotropic reactions. The method described in this paper is one involving electrically directed migration up a ‘sterile ‘column of salt solution. This treatment leaves the animals intact and also produces a certain degree of physiological segregation. However, any experiments with organisms separated from non-sterile cultures should include bacterial blanks with animals removed (Boell & Woodruff, 1941) or killed thermally (Peters, 1927). This precaution, too, has often been omitted (Leichsenring, 1925).

A more difficult problem is that of measuring the metabolism of the small amount of material which thus becomes available for study. Even though the cultures may be luxuriant, enormous numbers of animals are needed to investigate respiration or enzyme composition by ordinary manometric techniques. An examination of the experimental figures given by various workers who have used Warburg manometers, presumably of normal types (Mast et al. 1936; Pace & Belda, 1944a, b’, Burt, 1945; Pace, 1945, etc.) would indicate that the oxygen uptakes recorded were below the sensitivity of the methods employed. The most successful studies have been carried out with ultra-microrespirometers which demand less protozoan material and which give adequate readings within permissible experimental time.

In some cases the experiments have been performed to demonstrate the utility of a particular instrument (Holter, 1943; Zeuthen, 1943) rather than to obtain information on the metabolism of Protozoa (Boell & Woodruff, 1941; Boell, 1945). In this paper is described a Cartesian diver respirometer of ‘macro’ dimensions which, though lacking the precision of the Carlsberg apparatus, was more practicable and useful in view of the accuracy necessary for the problem under investigation.

For many years the belief that Paramecium respiration was entirely mediated by an unusual route, cyanide-stable, has persisted. The demonstration of the presence of cytochrome (Sato & Tamiya, 1937) and of cytochrome oxidase (Boell, 1945) in Paramecium has shed new light on this. Since succinic dehydrogenase is one of the two animal dehydrogenases known to react directly with cytochrome and cytochrome oxidase, we have studied its occurrence in P. caudatum.

The P. caudatum used in this study was supplied, in admixture with Euglena and Chilomonas, by Prof. Agar of the Department of Zoology, University of Melbourne. A clone free from the other Protozoa was established. The culture medium consisted of 5 ml. of Osterhout solution (Leslie, 1940) and 5 ml. of 20% Vegemite suspension in 1 1. of distilled water. The Vegemite is a yeast concentrate manufactured by the Kraft-Walker Cheese Co. Pty. Ltd., Australia, and served to support a rich bacterial flora upon which the Protozoa fed. The medium was distributed in 150 ml. aliquots in 250 ml. Florence flasks. The inoculum was 10 ml. of suspension from a 2- or 3-day culture; all cultures were grown at 28° C., at which temperature optimum division rate occurred.

The population reached its maximum density of one to two thousand animals per ml. on the fourth day (Fig. 1).’ The organisms used in experiments were always culled from 2- or 3-day cultures, where growth was approximately logarithmic. Counts were by the Sedgewick-Rafter cell (Hall, Johnson & Loefer, 1935), and the number of fields which it was necessary to count to give a precision of 10% was determined statistically. As a routine, thirty fields were counted.

The fact that a slight electric current causes a reversal of ciliary beat (Jennings, 1904) and a migration to the cathode formed the basis of our ‘electromigration’ apparatus (Fig. 2). This consisted of a 100 ml. Erlenmeyer flask, to the top of which was fused 15-20 in. of $34$ in. glass tubing. An electrode was inserted at the bottom of the flask either by fusing in a short length of tungsten wire, or by fixing, by means of sealing wax, a piece of nichrome wire into a short, tapering side-arm of $14$ in. glass tubing. This lower electrode was connected through a mercury pool to the anode of a dry cell. The other electrode, attached to the cathode, was a hook of nichrome wire at the top of the apparatus. The flask was filled with 100 ml. of wild culture, filtered through cotton-wool to remove mould. The column was then filled cautiously with the wash fluid so that mixing did not occur. The wash fluid consisted of 1:100 Osterhout solution, in which dilution the animals were found to survive longest.

In a time varying with the voltage applied and the age of culture, the majority of animals in the bottom flask migrated through the clear wash solution and congregated in the top 25 ml. of fluid. It was thought that some physiological segregation occurred as dividing forms did not appear at the top of the column, and in old cultures the number of animals which failed to migrate was greatly increased. Organisms from 2- and 3-day cultures always completed each migration in less than 20 min. Those from 4- and 5-day cultures needed from 20 to 60 min. and gave less dense collections of migrated animals; 5-day cultures took over an hour. The animals in the top 25 ml. of several migrators were withdrawn, combined and subjected to a second migration before being used in an experiment; 22 V. was always used as the potential difference between the two electrodes (Table 1).

The suspension of animals withdrawn from the top of the apparatus after the second migration was centrifuged in 10 ml. tubes for 30 sec. at 1000 r.p.m., and the supernatant liquid removed by suction. This centrifugation did not damage the animals as judged by microscopic examination. The final suspension, containing 2−6 × 10⁵ organisms per ml., was transferred to a ground-glass homogenizer (Humphrey, 1946) and an aliquot withdrawn for counting; in the remainder, cell structure was completely destroyed by homogenizing for 2 min. at o° C. Homogenizing at room temperature, or for 3 min., yielded a preparation which did not respire. Aliquots were placed in small tubes and buffer, substrate, inhibitor, etc., added; 10 µl. of the mixture was then pipetted into the appropriate diver.

The general plan of apparatus followed that of Linderstrom-Lang (1937), Linderstrom-Lang & Glick (1938) and Boell, Needham & Rogers (1939). These were the only papers available until towards the end of the investigation when micro-films were-obtained of the complete papers of the Carlsberg Laboratory (Holter, 1943). The apparatus contained six chambers, with aim. manometer scale, and pressure in the manometer was controlled by means of a 20 ml. and a 2 ml. syringe.

If, on raising the level in the outer limb 10 cm., the inner limb level rises 6-6i cm., the difference between the two levels is 4-39 cm. and obviously, this difference is the effective pressure acting on the surface of the flotation medium. The factor applied should be, therefore, not 0·66i, but 0·439; or, more generally, if x is the rise in the closed limb for every cm. rise in the open limb, the factor to be applied to the constant obtained by the Warburg equation is (1–x). Then multiplied by the manometer discursion in the open limb during the experiment gives the alteration in gas volume within the diver, expressed in µd. CO₂. It seems improbable that this error was really entertained by Boell et al. (1939), and is other than an inadvertent misstatement, but until its formal correction, it must cast some confusion on the results of Boell & Woodruff (1941), Boell (1945), and any others who have ‘Followed closely the descriptions given by Boell, Needham & Rogers (1939)’ (Clark, 1945).

It follows that the larger the gas space between the surface of the flotation medium in the chamber and the water-level in the inner limb of the manometer, the more nearly will the rise in the inner limb of the manometer approximate to that in the outer limb, and the smaller will the difference in levels, and hence the factor, become. The decrease in factor correspondingly occasions a decrease in the constant K, i.e. increases the sensitivity of the instrument, since a 1 cm. change on the manometer scale indicates a smaller gas change within the diver. For this reason, the gas space between the closed limb water-level and the surface of the flotation medium was increased in our apparatus by the insertion of a- 250 ml. Erlenmeyer flask into the closed circuit. This lowered the factor from 0-82 to 0-54 (i.e. without the extra gas space, a rise of 10 cm. on the open limb caused a rise of i-8 cm. in the closed limb; after insertion of the gas space, a rise of 10 cm. in the open limb caused a rise of 4-6 cm. in the closed limb), and for the experiments quoted here, an instrument with a factor of 0-54 was used.

These considerations with regard to the method of calculation do not apply to the methods adopted by the Carlsberg school, since those workers calculate the gas exchanges in a more fundamental manner, i.e. by reducing the gas space between the top of the flotation medium and the manometer fluid to a minimum, and working out a constant based on the specific gravities of the oil seal, the glass of which the diver is made, the flotation medium, etc. In this case, the applied pressure is fully effective since the gas space is small, i.e. the factor is unity.

Our divers were much larger than any others reported in the literature, having a total volume of 40-60µl. The neck diameter was 1-1·5 mm. and the neck length 5-7 mm. Pipettes were graduated opsonic pipettes, drawn out by the technique of Holter (1943) to capillary tips. The bottom drop in the diver was 10/J., and alkali, oil and neck seals about 1 µ1., though these were not measured. The volume occupied by these was estimated by observing the fraction of the neck occupied by liquid at the equilibrium position. The use of large divers and large volume of experimental fluid are undoubtedly undesirable, since the first lowers the sensitivity, and the second introduces the possibility of a diffusion effect through the liquid. That a diffusion lag did exist was indicated by the fact that divers containing only 5µl. of suspension generally exhibited a rate of gas consumption 10-15% higher than a corresponding diver containing lOµl. at the end of the first hour, though the results more nearly coincided for the second hour. However, through greater ease of reading large manometer discursions, and higher accuracy of pipetting io/d., it was found that greater consistency of duplicates was obtained using the larger fluid volume. Therefore, this was adopted as a standard procedure. Results may be regarded as precise to 10%.

Decinormal sodium hydroxide was used as an alkali seal (Linderstrom-Lang, 1943), and kerosene-paraffln mixture as an oil seal (Linderstrom-Lang & Glick, 1938). The flotation medium was, in early experiments, saturated ammonium sulphate, but when the paper of Holter (1943) finally became available, a change was made to the medium recommended by this author.

In the method of electromigration described above for collecting the organisms, it was stated that migration was carried out with a potential difference of 22 V.; the adoption of this figure followed a consideration of the effect of voltage on the time of migration (Table 1).

In the absence of electrical stimulation, animals failed to achieve a second migration, whereas a high voltage killed the animals. The voltage which we adopted combines speed’ of migration and lack of physical injury to the animals (which were capable of living and dividing indefinitely after the treatment).

The effect of pH on the endogenous respiration of the homogenate was studied in the presence of phosphate over the range 3·-8·5-As Fig. 3 shows, there is an optimum in the range 6·6–7·6 with the rate falling off very rapidly in more alkaline reactions. On the acid side of the optimum, however, respiration shows more stability, falling off only gradually to pH 3·7. This may be correlated with the wide pH tolerance of the animals in culture medium.

The experiments reported in this paper were subsequently carried out at a pH of 7-2.

Table 2 shows the effect of cyanide, azide and methylene blue on the endogenous respiration of the homogenate.

Inhibition by cyanide was 56%, and by azide 44%. Azide might be expected to be less effective at pH 7-2, but attempts to demonstrate its effect at pH 4-2, in which region it has been shown to have its maximum effect on the oxygen consumption of yeast cells (Keilin, 1936), were unsuccessful, owing, probably, to the volatilization of the free acid. It might be noted here that no attempt was made to add suitable concentrations of cyanide to the alkali seal of the diver neck, as is the custom in manometric experiments involving cyanide (cf. Boell, 1945; Clark, 1945). These workers state that they used the mixtures recommended by Krebs (1935). However, since the mixtures recommended by Krebs for use in Warburg manometers were of one to two molar strength, compared with the decinormal alkali recommended by LinderstrØm-Lang (1943), there is the possibility of. error due to the difference in osmotic strength between alkali seal and the experimental drop. Also, in view of the work of Riggs (1945), emphasizing the inadequacy of any simple theoretical analysis to predict conditions required in experimental assemblies to prevent alteration in cyanide concentrations due to distillation into alkali, it seems doubtful whether the adoption of the mixtures suggested by Krebs would contribute effectively to the maintenance of a given cyanide concentration. It was tentatively decided not to add cyanide to the alkali at all, especially in consideration of the low alkali concentration, the temperature, and the experimental period which we employed. This procedure was justified by the observation that the degree of inhibition did not alter during the first and second hour periods.

Methylene blue did not increase the endogenous respiration, a finding which might be attributed to a sufficiency of carriers already present in the system for the low respiratory rate, or perhaps a lack of substrate.

Table 3 presents the effect of inhibitors on oxygen consumption in the presence of succinic acid, with and without the addition of methylene blue.

Succinic acid caused a large increase in oxygen uptake which was sensitive to cyanide and azide, as would be expected if succinate oxidation were proceeding through the succinoxidase system. In the presence of methylene blue, cyanide did not inhibit the succinate oxidation, while methylene blue alone caused a higher rate of succinate oxidation. Further evidence that a succinic dehydrogenase similar to that found in other animal tissues was present in the homogenate is given by the action of malonate. This inhibitor completely abolished the increase in respiration due to succinate.

Jennings (1904) has described the reactions of Protozoa to electric current and studied the polarizing effect on the cilia of the infusorian body. However, the cathodic migration of Paramecium has not previously been used with a view to concentrating the animals for metabolic studies. In the method described here, this migration is supplemented by the negative geotropism of Paramecium, a property which Glaser & Coria (1930) have utilized in the preparation of bacteria-free animals. The animals in our suspension were not prepared under sterile conditions, or considered sterile, for this was not necessary in view of the fact that our bacterial blanks, which consisted of determinations of the oxygen consumption of suspensions of animals killed by thermal treatment for 5 min. at 45° C., and of the last wash fluid (the supernatant from centrifugation), gave insignificant values. It seems probable that the reversal of ciliary beat caused by the electric stimulus might serve to dislodge the adherent bacteria during migration through the column of wash fluid. It is also suggested, in view of the difference in speed of migration of young and old cultures, the increasing number of animals which fail to migrate as the culture ages, and the absence of dividing forms from the final suspension, that some degree of biological segregation occurs during the washing. This is, indeed, desirable, since the physiological state of the organism is known to affect the respiration rate significantly (Boell & Woodruff, 1941; Hutchens, 1939).

Little information is available on the effect of pH on protozoan respiration, but the wide pH tolerance of some types in culture has long been noted. Phelps (1931) found the division rate of P. aurelia unaffected over the range 59–7·7-Loefer (1938) showed that the limits of growth for P; bursaria were 4.0–8.0 with an optimum at 6·8. A similarly wide pH tolerance is reported (Loefer, 1935) for the growth of Chilomonas paramecium and Chlorogonium elongatum. Mast (1931) found that Amoeba proteus, in non-nutrient salt solution, withstood pH 3·8–8·3 for 7 days. Our experiments showed that Paramecium caudatum survived and divided in Vegemite-Osterhout solution over the range 4·7–8·5.

It seems that a corresponding pH tolerance of respiration occurs in Protozoa. Von Dach (1942), using Astasia klebsii in an inorganic medium, found no variation in respiration at pH 4·5, 5·8 and 7·9, although in the presence of acetate, pH 4·5 depressed the increase in respiration which this substance normally caused. On the other hand, Hall (1941) found an optimum for Colpidium campylum at pH 5·5, the rate declining rapidly on the acid side but slowly on the alkaline side. Root (1930) found no effect on the respiratory rate of Paramecium caudatum with solutions as acid as 4·5. The results reported here indicate a considerable tolerance on the acid side of the optimum though alkaline conditions decidedly inhibit.

The early work giving rise to the supposition that the respiration of Protozoa is peculiar in being largely or entirely cyanide-stable has been regarded more critically in recent years, and the technical faults which gave rise to misapprehension on this point, such as lack of control of cyanide distillation into alkali, the presence of substrates, lack of control experiments and bacterial blanks, have been discussed. Gradually the uncritical acceptance of work such as that of Lund (1918) who, with the Winkler technique, often omitted control experiments and was severely criticized by Hyman (1919), and also of Shoup & Boykin (1931), whose technique and logic are both most questionable, is being withdrawn and workers now talk of the cyanide-stable respiration of Protozoa with more caution. Future investigations will probably elucidate the respiratory mechanisms linked to the cytochromes and cytochrome oxidase.

With improved techniques, recent workers have found decided inhibition of the respiration of Paramecium spp., and other Protozoa formerly reported as cyanide-stable. Thus Pace (1945) found 60% inhibition with young Paramecium in o-ooi M-cyanide. Boell (1945) reports about 60% inhibition with cyanide and azide when present in a concentration of o-oi M. Our results show about 60% inhibition of endogenous respiration by this concentration of cyanide, while azide caused only a 40% inhibition. The effects of azide reported by Boell (1946) in a short communication, are not readily understood. In these experiments, azide at pH 6-02 caused 70% inhibition, whereas at 6-59, a stimulation of 138% occurred, this increase being inhibited by cyanide. Thus some accord seems to have been reached on the cyanide sensitivity of this particular genus and also a recognition that the nutritional state of the organism affects the degree of inhibition; e.g. Pace (1945) states that the percentage inhibition depends on the saturation of the enzyme systems with substrate. This conclusion corresponds to the work of Commoner, on yeast (1939), who reached the same conclusion, the oxygen uptake of starved yeast showing a higher degree of cyanide stability.

There are, too, more precise indications that the cytochrome-cytochrome oxidase system functions in Paramecium. The enzyme cytochrome oxidase has been demonstrated quite conclusively in P. calkinsii by Boell (1945), using ascorbic acid as a substrate. Cytochrome has been detected spectroscopically in P. caudatum by Sato & Tamiya (1937); these workers demonstrated that cyanide prevented the reoxidation of reduced cytochrome. They also report the presence of haemoglobin, though the significance of this pigment in the animal’s economy cannot be assessed.

The nature of the mechanisms supplying electrons to the cytochromes is still obscure; there is no knowledge of flavoprotein or co-enzymes in Paramecium, though the synthesis of co-enzyme 1 has been demonstrated in Chilomonas (Hutchens, Jandorf & Hastings, 1941). Further, nutritional studies which have shown that riboflavin and nicotinic acid are growth factors (Kidder & Dewey, 1946) might indicate that these compounds were functioning in cell metabolism. Succinic dehydrogenase has been reported (Humphrey & Humphrey, 1947), and.this enzyme seems to complete a succinoxidase system similar to that found in other animal tissues; this may be compared with the finding of Leichsenring (1925), that succinic acid increased the oxygen uptake of Paramecium.

However, it also seems certain that quite a large part of the endogenous respiration of Paramecium is stable to cyanide. Indeed, the respiration of few tissues is entirely inhibited by cyanide, but the nature of this cyanide-stable respiration can be considered only in speculation. It is possible that the tissues are functioning on a so-called ‘oxygen-debt’ and are continuing to produce carbon dioxide. This would be mirrored by a higher respiratory quotient in the presence of cyanide. There are no such R.Q. studies on Paramecium, though Pitts (1932), using Colpidium campylum, found an increase in R.Q. from 0-65 to 0-90 in the presence of cyanide. The amount of flavoprotein has often been associated with cyanide-stable respiration in other tissues (Groen & Schuyl, 1938; Commoner, 1940), but in Protozoa there is no evidence on this possibility. It is also suggested that cyanide-stable respiration is associated with non-carbohydrate substrates (Commoner, 1940), and certainly in Protozoa there are indications that protein is a more prominent cell substrate than carbohydrate. Thus Leichsenring (1925) showed that protein and amino-acid substrates always increased Paramecium respiration more than carbohydrates. Emery (1928) found that the rate of ammonia production by Paramecium was proportional to the rate of utilization of some twelve amino-acids. Specht (1935) also reports ammonia production during the endogenous respiration of Spirostomum, while, recently, Boell (1946) has claimed that 75 % of the respiration of Paramecium calkinsii is due to the use of protein as a substrate; here, too, ammonia production was observed. It is tempting, though hardly justified, to link the protein respiration with the cyanide stability of the flavoprotein D-amino-acid oxidase; but with the scant information available and our ignorance of a possible physiological function of this enzyme, the question must certainly be left open.

1. A method is described for reducing the numbers of bacteria in a suspension of Paramecium caudatum by an electrically directed migration through a sterile column of liquid. The resulting suspension was suitable for metabolic experiments.

2. Details are given of a Cartesian diver respirometer of ‘macro’ dimensions; this apparatus has a precision of about 10%.

3. The effect of pH on the endogenous respiration of a homogenate of P. caudatum showed an optimum in the region 7·0–7·3, with a wide tolerance on the acid side of the optimum but low tolerance on the alkaline side.

4. The endogenous oxygen consumption had a value of 1·9µ1. per 10⁴ animals per hr. and was inhibited 60% by 001 M-cyanide and 40% by 0·01 M-azide. Methylene blue did not increase the endogenous oxygen uptake.

5. Succinic acid doubled the oxygen consumption, this increase being inhibited by malonate. Methylene blue increased oxygen consumption in the presence of succinate still further, and also abolished the inhibition of this extra respiration by cyanide and azide.

6. It is concluded that P. caudatum resembles other animal tissue in possessing an active succinic dehydrogenase.

Our thanks are due to Mr I. M. Thomas and Mr B. R, O’Brien of Sydney University, for helpful advice.