Woodruff and other workers claim on the basis of experiment that a single protozoan can be cultured indefinitely without conjugation. Other observers, chiefly centred around Calkins, claim also with experimental support that unless conjugation takes place the protozoa finally die. A review of this controversy and its historical setting is given in Wilson’s The Cell, and nothing is to be gained by its repetition. Less disinterested reviews have been made by Woodruff (1925) and Calkins (1926). Woodruff called attention to the very important fact that, as culture methods have improved, the length of life of the protozoa has been increased. This significant observation is completely omitted by Calkins and the evidence for it will be briefly reviewed here.
While Calkins (1904) reported the death of Paramecium caudatum in two years, Woodruff (1914) by using natural pond water was able to preserve the culture for several years. Metalnikow (1922) has gone further with this organism and cultured it for ten years. Likewise, Didinium nasutum died after a few hundred generations (Calkins, 1916), but in the hands of Mast (1917) it attained the 1646th generation before theculture was discontinued. Maupas(i888) cultured Stylonichia pustulata for 316 generations. This was extended to 572 generations by Baitsell (1912), and Darby (1928) reared 500 generations without any decrease in division rate. The statement, therefore, that the senescence and death of protozoa are due to the culture conditions is hardly unwarranted. The weakness of the criticism, however, lies in the inability of the critics to name the specific conditions that need to be remedied. This paper will attempt to fill the gap, and thus remove the negative support on which the life cycle theory still stands.
The methods of culture have been reported in a previous paper (Darby, 1928) and will be repeated only when germane to the present discussion.
I. Paramecium caudatum
The organism selected for the first part of this study was Paramecium caudatum,, this species being one of those whose cultures had in some cases died out after a typical life cycle; on the other hand, under different conditions it has been successfully cultured for a much longer period. Wherein lay the difference in culture conditions? Could the life cycle be a result of the culture medium?
Owing to its great physiological importance, the role of the H-ion concentration was first investigated. A number of sister Paramecium were isolated into culture media differing only in pH value, and the division rate over a ten-day period was ascertained. The results showed that pH had a marked effect on the division rate and that the optimum was at pH 7·0 ± 0·05 (Darby, 1928). With this information, a series of cultures was continued at pH 6·95 ± 0·05 and these exhibited an almost constant division rate. These cultures were continued for seventy days to ascertain thoroughly the degree of variation in division rate to be expected. The variation proved to be so low (one or two per ten-day period), that the division rate could be considered constant, especially when the time factor of the daily isolation was taken into account. This establishment of a constant division rate was the necessary starting-point for such an investigation.
Sister cells to those kept at the optimum were now isolated in a medium of pH 7·6 ± 0·05. The division rate of the new cultures at once fell to 17·5 on the first ten-day count, as compared with 21 + of the controls. The cultures at 7·6 were run concurrently with those at the optimum, and whereas the latter maintained their steady division rate, the Paramecia at pH 7·6 very gradually decreased their rate of fission. Every twenty days, sub-cultures were transferred from the medium at 7·6 into the one at 6·95, and in each case the cultures resumed their former high division rate. These results are expressed graphically in Fig. 1.
The selection of pH 7·6 as possibly unfavourable to the organism was not fortuitous, since hay infusions made up by the methods used by Calkins and his co-workers generally give a reaction of pH 7· 4–7·8 (See also Austin, 1927.) This pH value is the result of two factors, (i) The culture glasses used are of such a type as to produce a pH of 7·8 in the unbuffered medium, owing to the solubility of the glass. (2) The boiling and concentration incident to the preparation of the medium result in an alkaline pH, since the calcium content of the Great Bear water is originally sufficient to produce a pH of 7·2–7·4 when in equilibrium with the CO2 of the atmosphere. Therefore the lowest pH obtainable under these-conditions would be approximately 7·6.
The above is further borne out by Calkins’ chart for P. caudatum (Fig. 2). A comparison of Figs. 1 and 2 shows that the division rate is decidedly lower in the latter. The maximum shown in Fig. 2 is 17, and that rarely; the usual figure is under 13. This fact supports the thesis that the cultures were kept at a pH of 7·6 or higher. Division rate has been taken by all protozoologists as a good indication of the health of a culture. Therefore, it is not surprising that on turning to the work of Metalnikow (1922), who cultured these organisms for ten years, we find a division rate of 20 and over frequently recorded. More favourable culture conditions are expressed not only in the lengthening of the life of the culture, but also in higher division rates.
Three different aspects of Fig. 2 suggest that the medium was even further away from the optimum pH than 7·6. (1) The division rate of 17 is high in comparison with the usual 12 per ten days. (2) The decreases in division rate are of greater magnitude than those shown in my culture kept at 7·6. (3) Whereas it takes 90–110 days for Calkins’ culture to pass from its maximum (17 divisions) to death, my cultures at 7·6 at the end of 130 days were still dividing at the rate of 13 per ten days. A still higher pH was therefore tried. Twenty sister cultures were isolated from the control and placed in pH 8·1 ± 0·1. The new division rate was 13 per ten-day period in contrast to 21 ± of the optimum, and the result of continuing these cultures at this pH is shown in Fig. 3. The further cultures were kept from the optimum,' the quicker was their decline. To test their ability to recover from these conditions, sister cells were sub-cultured at the optimum pH in the early stages of the experiment; these cultures resumed division at a rate characteristic of optimum conditions. In fact, a culture thus returned often tended to show a slightly higher division rate than the controls. No attempt is made to show that Calkins’ cultures were kept at a constant pH, because the medium varies slightly from time to time with the number of bacteria present. It is, however, intended to show that at its best his culture medium could not have been nearer to the optimum pH than 7·6, and that it varied somewhere between 7·6 and 8·1.
The changes in division rate to be seen in every protozoan culture’s life graph are of a sudden nature ; and I have reconstructed a similar history for P. caudatum, cultured in a hay infusion (Fig. 4). The pH of the medium alone was changed from time to time, and the resulting picture closely approximates to the usual protozoan life graph. Such changes in pH could have occurred under natural conditions.
Interest has been so far centred on decreases in division rate, rather than on the factors effecting an increase. The latter will now be dealt with and an attempt made to analyse the methods previously used to revive a failing culture. In general there have been two, namely, external changes of medium and conjugation.
Previous workers have obtained a suitable culture medium by more or less empirical methods, so that the precise nature of the environment of actively growing cultures is really unknown. Dawson (1926) helps to clarify the position by recording the nature of unsuccessful as well as of successful media. The preparation of synthetic or natural media must, however, be carefully carried out, and the assumption that all natural waters are alike and can contain no harmful factor is entirely unwarranted. I have recently collected samples from four ponds around Plymouth, whose pH values were 6·1, 7·0, 8·4 and 9·3.
Another phase of the variability of water is that a single sample can alter with time owing to the solubility of the glass container. This possibility Austin (1927) failed to recognise, for she kept her pond water for months in soft glass containers. Although her cultures in the early months in this medium show an almost constant division rate for Uroleptus mobilis, they finally declined and died. It is regrettable that with pH measurement in constant use she neglected to watch this source of error.
Another method of altering the environment has been to add different food stuffs and salts to the medium. This would necessarily tend to change the pH of the original fluid. It is not meant here to imply that the organism might not need new food material, but that the addition of such introduces more than one variable.
The increase of division rate by conjugation has been seriously challenged by many workers, especially Jennings (1913). But without doubt Calkins’ recent cultures of Uroleptus mobilis have been extended time after time by this method. The method, however, remains open to one serious criticism. To obtain conjugation the organisms are left in great numbers in a dish with more than the usual amount of infusion, so that mass culture conditions are approached (Austin, 1927). They remain in this dish for four or five days before conjugation takes place. Now Bodine (1921) and several others have shown that hay infusions go through marked pH changes in these first five days. Is it not possible that the reinvigoration of the organism is due to these new culture conditions and not to conjugation? I have shown in the previous graphs that removal to pH 6·95 from 7·6 or 8·1 is attended by an increase in division rate. Now the hay infusion, originally alkaline, becomes acid on the fourth or fifth day due to bacterial action ; and the organism may thus be carried to the optimum point for its division rate. This would result in an enhanced division rate even in the absence of conjugation after the return to the usual medium. The gradual decline would of course follow.
That normal culture conditions and those under which conjugation take place are not identical is shown by the fact that at no time during my experiments did conjugation take place, not even in buffered cultures many days old. Nevertheless the Paramecium were capable of conjugation, as epidemics occurred when they were placed in plain Great Bear water. The factor here involved was not investigated.
How the changes of division rate are produced by altering the H-ion concentration of the medium is entirely unknown. Evidence up to the present indicates that the H-ion as such does not penetrate normal cells. In this connection it is.suggestive to note the difference in behaviour of an organism at the extremes of its pH range. At a very acid pH the organism continues to divide but increases very little in size. The result is that each succeeding generation is smaller than the last, but fission is maintained until death, and the organisms are capable of complete recovery when removed to the optimum. At the alkaline extreme the organism becomes sluggish in movement and somewhat larger than normal, but no division takes place. An individual may live in this condition of suspended reproduction for 15 to 20 days before dying. Whether this is due to the actual intake of the medium or to changes in permeability has still to be ascertained. Shapiro (1927) has already shown that the pH of the food vacuoles is influenced by the external H-ion concentration.
The exponents of the life cycle theory demand proofs of the well-being of the organisms throughout the experiment. These consist in the ability to conjugate and to encyst. Encystment was unknown in P. caudatum at the time in question, and attempts to induce it resulted in the loss of the organism. Conjugation, however, was obtained from time to time as explained earlier in this paper, and showed that the culture had not degenerated. Of equal importance is the fact that although the graphs record a period of only 200 days, the Paramecium were kept after this in isolation dishes and observed every two or three days. An occasional count showed that they continued to divide at their maximum rate, 21 +. These cultures were used as stocks for some experiments on “Bios,” and a record was thus kept of them for nearly three years.
The evidence thus far presented shows that if the medium used in the cultivation of Paramecium caudatum had been made up according to Calkins’ technique, the organism would have passed through a typical life cycle and would finally have died. The length of the life cycle is definitely correlated with the suitability of the culture medium, and under optimum conditions the cycle disappears completely. Changes in the H-ion concentration of the medium can produce changes in division rate such as are found in the typical life history graph of a protozoan.
II. Stylonichia pustulata
A possible target for criticism of the foregoing work is the organism chosen for the investigation. Ever since Paramecium caudatum has been shown to have endomictic periods, it has been set apart as an organism of dubious character. It was therefore thought necessary to repeat the experiment on some other form. For this purpose Stylonichia pustulata was selected. No suspicion of endomictic changes can be entertained against it, for Fermor (1913) has shown that this phenomenon takes place during encystment. In addition, it was one of the forms used by Maupas in his classical experiments, which have been the subject of much controversy. Repetition was undertaken with a view to the clarification of this longstanding phase of the life cycle discussion.
In a previous paper (Darby 1928) I have shown that Stylonichia can be carried for 500 generations at a constant division rate at pH 7·6. These cultures were carried for about two years more under isolation culture conditions. But the time necessary for counting the organisms when they go through as many as five divisions per day was not thought to be justifiably spent on carrying the culture any longer, especially in the face of other evidence already presented by Woodruff, Bělár (1924) and others. One significant detail was not included in my previous paper; the cultures were tested from time to time for their ability both to conjugate and to encyst. Up to the last day of strict isolation counts, encystment and conjugation were obtained. I was therefore dealing with normal organisms.
The first point of interest was a comparison of the division rate obtained by me with those by previous workers. The maximum obtained by Maupas (1888) was 37 divisions per ten days, at a temperature of 25-2° C. Baitsell (1912) reports a maximum of 32 divisions per ten days at 24° C. I have obtained 45 divisions per ten days at 25·0° C. and pH 8·0. The usual division rates obtained by both Maupas and Baitsell are much lower than the figures I have just quoted, and show that the Stylonichia were not being kept anywhere near their optimum. Moreover, these lower division rates could be produced in my cultures by lowering the pH of the medium, especially below 7·0.
The next question was how many generations the organism could pass through when cultured at this lower pH and division rate. This was important because both Baitsell and Maupas had succeeded in culturing Stylonichia for some time. A series of cultures at pH 6·1 and 25° C. was kept under isolation conditions and passed through 310 divisions before death. This figure compares favourably with 316 generations passed through by Maupas’ cultures at an equally low division rate.
The third feature of interest was the investigation of an apparent inconsistency in Maupas’ work, namely, that degeneration followed long culturing, although a high division rate was maintained up to the end of the culture. Repetition of his experiments completely confirmed his results. Stylonichia (at pH 6·1) continued to divide but became smaller and smaller, giving exact counterparts of the degenerate forms drawn and described by Maupas. The size of the degenerate form was about that of Colpidium colpoda (St.). The division rates, however, for the last four ten-day periods were 35,37,41 and 39 (average of ten lines). In this connection I may recall that P. caudatum also keeps up its division rate on the acid side of its optimum until death supervenes. Hence degeneration and high division rate need not be incompatible.
Maupas’ medium was a hay infusion, but differed from the one now in general use by being allowed to stand many days before inoculation. By the time the Stylonichia were isolated into it, the hay infusion had become acid. This acidity was favourable to the organism on which the Stylonichia were feeding ; so much so that when the cultures showed signs of falling off, Maupas revived them by making them slightly acid again. The Stylonichia were thus kept well on the acid side of their optimum, in contrast to the alkalinity of the usual hay infusion of to-day.
The fourth feature of interest was a comparison of the division rates of normally dividing forms and ex-conjugants. With this in view, 12 ex-conjugants were isolated in a medium at pH 7·6, and compared with 10 control lines at the same pH. The following table gives the results.
The division rates of both lines were equal and showed no effect of conjugation. Perhaps the organisms were cultured so near to the optimum that no marked increase was possible. Similar experiments were therefore run at pH 6·1 and 6·95, but no differences in division rate came to light. It must be remembered that at these lower pH values an increase of nearly 40 per cent, was possible before the maximum rate was reached. Conjugation per se did not increase the rate of division when the organisms were kept continuously in the same H-ion concentration.
The ex-conjugants bore out one of Maupas’ observations very clearly. He recorded that an isolated ex-conjugant gave no divisions for the first day and only one on the second. My cultures likewise gave no divisions for the first two days following conjugation. The data in Table I begin with the third day after isolation. I am aware that had these days been averaged with the next eight, the division rate for the first ten-day period would have been low and in agreement with Jennings’ (1913) findings, that conjugation generally causes a decrease in division rate. The reason for my exclusion of these first days lies in the striking difference in appearance of the organisms at this time. They became almost black and shrank in size. These visible changes were doubtless correlated with the cytological reorganisation that we know to occur. Enough time must be allowed for these phenomena to be completed before the resumption of fission can be expected. Some forms will require less time than others.
The last point concerns the health of the culture. It has been maintained by Calkins (1926) that Uroleptus mobilis is capable of encysting only in the early stages of the life cycle. He also held this to be the case with Didinium nasutum ; but Beers (1927) has presented unchallengeable evidence to show that Didinium can encyst at any time of its life, if the culture conditions are such that the organisms are normal and healthy. I therefore tried to obtain encystment at various times in the life of Stylonichia. At the 167th, 314th, 354th and 500th generation encystment was obtained by the technique described in a previous paper (Darby, 1928). After a year of isolation cultures with occasional counts, I again succeeded in inducing encystment in approximately the 1460th generation. The failure of Calkins to obtain encystment in the later stages of his cultures seems only further evidence of the unsuitability of his medium.
III. Paramecium aurelia
The experiments on P. caudatum have proved that division rate can be altered by changes in the H-ion concentration. This was substantiated on Stylonichia pustulata. The experiments on the latter form went further and showed that conjugation does not affect the division rate when the H-ion concentration is controlled. In view of the similarity of endomixis and conjugation, it seemed advisable to run some parallel cultures on P. aurelia, a form in which the rhythms of endomictic activity occur with regularity (Woodruff, 1917). Through the courtesy of Prof. L. L. Woodruff I was able to procure some of his pedigreed stock. The medium used was the usual hay infusion, although towards the end of the experiment it was changed to Woodruff’s beef extract. The latter medium is undoubtedly a better one for this organism.
The theory under investigation is that endomixis is comparable to conjugation in revitalising the organism, and therefore accounts for Woodruff’s long continued cultures. If this view is true, as claimed by Calkins, there should be periods of high division rate following each period of endomixis. A review of Woodruff’s (1917) work on several strains of P. aurelia shows that the division rate after endomixis may be either higher or lower than before. Of course the average for the period in which endomixis occurs is usually low, owing to the fact that the intracellular reorganisation takes time. A comparison to be valid must be made of the division rate before endomixis and after, and not between the period after and the period including the phenomenon. A series of cultures was run at pH 6·95, and on being averaged for ten-day periods gave no indication of periodicity. The cultures continued to divide at a fairly constant rate for 130 days. When, however, the daily rate of division was plotted, a distinct cessation of fission was observed every 34 ± 2 days. The divisions were constant up to the time of endomixis. Then came a pause, followed by another period of constant division at the same rate as before. No enhancement of division rate by endomixis was observed.
A study of the relative frequency of endomictic periods in different H-ion concentrations was undertaken with a view to explaining the variation in length of period observed by Woodruff (1917). The cultures at pH 6·95 showed signs of endomixis at constant intervals of 34 ± 2 days. Five separate lines behaved consistently in this respect. At pH 7·6 the period was reduced to 22 days in ten separate lines. In a medium in which the pH was not controlled, it is to be expected that the endomictic period would vary somewhat in length. It will be interesting to see how short the period can be made by placing the organism in an unfavourable environment, and whether the response is identical on both sides of the optimum pH.
The exponents of the life cycle theory have drawn their concept from the Sporozoa. In this class of protozoa elaborate changes in behaviour and appearance are observed as the parasite moves from one host to another. It is not conceived by these workers that such changes are responses to different environmental conditions. The H-ion concentration, temperature and salt balance met with in the human blood by a Plasmodium are totally different from those in the mosquito. It is open to experimentation to alter the medium in one of these respects and attempt to produce at will the various forms displayed in the life history of a parasite. I have had some slight success in producing encystment in Endamoeba histolytica by merely changing the H-ion concentration to that found in the lower intestine.
The work of Carrel on his long continued tissue cultures has removed any possibility of doubting the immortality of cells if the proper environment is found for them. It is significant that Carrel’s success lay in using the fluid medium in which the cells were normally bathed, and Woodruff was likewise successful when using the pond water in which the organism occurred naturally. The problem in work of this kind is to differentiate the elements of a complex medium and evaluate the relative importance of each factor concerned. We now know that temperature is a feature to be carefully governed ; and I hope that I have presented sufficient evidence for the necessity of controlling the H-ion concentration.
Constant division rate in ciliates can be maintained by keeping the culture medium at constant optimum H-ion concentration.
The variations in division rate found in the typical protozoan life history, including gradual decline and death, can be reproduced experimentally by altering the pH of the medium.
When cultures are maintained under optimum conditions, encystment and conjugation can take place at any age; the life cycle disappears.
An explanation based on experiment is given for the apparently contradictory findings of Maupas.
Neither conjugation nor endomixis has any effect on the division rate under constant conditions.
The length of the endomictic period is affected by the H-ion concentration of the medium.