Quantitative Studies on the Ciliate Glaucoma: II. The Effects of Starvation

Cultures of Glaucoma were starved by centrifuging them free from the Pseudomonas on which they had been fed, and placing them in a solution of non-nutrient salts. It was impossible to remove all the bacteria in this way, but they were reduced to insignificant numbers and the few that remained were eaten by the ciliates in a very short time.

The length and breadth of an individual were measured with a micrometer eyepiece, and as the transverse section was circular the volume or the surface-area could be calculated with a fair degree of accuracy from the formulae for a prolate spheroid.¹ Twenty individuals were measured from each sample. Other details of technique have been described in a previous paper (Harding, 1937).

The exact course of the changes induced by starvation depends on the initial fate of the ciliates, their size and their content of food vacuoles, which in turn depends on the intensity of feeding previous to starvation. In all the experiments the food vacuoles were digested and disappeared in 4–5 hours. Multiplication of the Glaucoma usually ceased after about 6 hours; but in one case it lasted for 12 hours. The volume of the individuals decreased very rapidly at first because of the multiplication of the ciliates and the disappearance of the food vacuoles. After these influences had ceased the volume decreased only very slowly. When the Glaucoma had been starved for a month the volume was about one-tenth that of the original individuals. Although they were still able to swim actively most of the individuals settled at the bottom after a few days; but none of them died for at least a month. The apparent increase in the numbers between the 12th and the 25th days in Table 1 was probably due to concentration of the culture by evaporation.

The changes in the shape of the Glaucoma were very pronounced, the animals at first became relatively slender, and later tended to return to their normal more rounded shape. The length at first actually increased, the rapid decrease in volume at this time was due to the decrease in breadth which was considerable. After an hour or so of starvation the length began to decrease and continued to do so regularly. The breadth on the other hand ceased to decrease after a time; so that the Glaucoma became more rounded. The comparatively slender shape was found to be characteristic only of slightly starved Glaucoma. The different behaviour of the length and the breadth as starvation proceeded is clearly shown by the changes in the ratio of breadth to length. The higher this ratio, which will be referred to as the “shape-index”, the more round and “plump” are the ciliates regardless of their absolute size. During the early stages of starvation there was a rapid decrease in the shape-index until a minimum was reached at about the time that multiplication ceased; then the index increased again and finally the ciliates regained their original shape.

The changes in shape were not artefacts due to fixation of the ciliates in different physiological states because the changes were sufficiently pronounced to be followed by eye in the living cultures. Nor was the initial decrease in the average shape-index of a starving culture due to the fact that as the ciliates stopped multiplying fewer young individuals were present. This is evident because adult Glaucoma which were undergoing fission were found to have a much higher shape-index when well fed than when slightly starved (Fig. 3)..

For measurements of the mega-nucleus it was necessary to fix the Glaucoma in Schaudinn’s fluid and stain with Feulgen’s nuclear reagent. Schaudinn’s fixative caused some contraction of the cell and of the nucleus; but this was the only combination found that would stain the nucleus so that it could be distinguished from the food vacuoles. The nuclei were considerably more heavily stained than the food vacuoles, although the latter contained Pseudomonas which took up the stain. The approximate volume of the nucleus was calculated from the semi-length, a, and the semi-breadth, b, by the formula ⁠. When the size of the Glaucoma decreased on starvation the nuclear volume decreased in a similar way, in so far as the diminution was rapid at first and slower when multiplication ceased. The decrease in the volume of the nucleus, however, was relatively less than that of the ciliates. The nucleus at first occupied about 2-5 per cent of the volume of the ciliate; but after 14 hours of starvation it occupied nearly 5 per cent (Table III).

Glaucoma which had been starved for 48 hours were fed again by adding a few bacteria to the culture (Table IV, Fig. 4). In about 6 hours most of the bacteria had been eaten, and the ciliates had trebled in volume. The Glaucoma did not begin to multiply until after the bacteria had disappeared, then they did so for 12–15 hours, with the result that there was an immediate decrease in their average volume. By the time the Glaucoma stopped multiplying their volume had been reduced to that of the original starved ciliates. The maximum breadth and the maximum shapeindex coincided in time with the maximum size of the ciliates; but the length did not reach its maximum until about 6 hours later. The Glaucoma at the beginning of the experiment had been starved for 48 hours and those at the end for about 20 hours and it is instructive to compare the two. Although the average volume was about the same the ciliates starved for 20 hours were longer and more slender than those starved for 48 hours, the shape-indices being 39 and 57 per cent respectively. Fig. 6 shows that the dimensions of the Glaucoma at the end of the experiment were approaching the dimens’ons of those that had been starved for longer with which the experiment was started.

When Glaucoma which had been starved for 27 days were placed in a suspension of 4,000,000 Pseudomonas per cu, mm. they fed on the bacteria and increased in size (Fig. 5). After 9 hours they began to reproduce so that dividing individuals could be found in the samples. There was a considerable lag in the formation of food vacuoles. At the end of the first hour most of the individuals contained no food vacuoles and after 3 hours of feeding there were only three or four food vacuoles per ciliate. Well-fed Glaucoma form about ten food vacuoles under these conditions.

It appeared that the oral ciliary apparatus had degenerated considerably so that few bacteria were captured until it had grown again.

The shape-index of the Glaucoma was higher when they were feeding again after starvation than it was at any other time (i.e. they were more nearly spherical). Compare the ciliates of Fig. 5 with the one that had been well fed for several generations in Fig. 2 (1st drawing). Even the short period of feeding described earlier was sufficient to produce a shape-index of 75 per cent which is higher than that for normal well-fed Glaucoma.

The starvation of mass cultures of Colpidium colpoda was studied by Vieweger (1925). The behaviour of Glaucoma was similar to that of Colpidium in that the ciliates decreased rapidly in volume at first, when the ciliates were still multiplying, and that later the decrease in size was very slow. There were also differences: Colpidium continued to multiply for 8 days while Glaucoma ceased to do so in about as many hours. It is probably for this reason that Colpidium decreased to one-fifteenth of its original volume in less than a week, while Glaucoma took a month to decrease to one-tenth of its volume. Vieweger’s measurements of the nucleus of Colpidium showed that the decrease in its volume did not begin until some days after the food was removed and the volume of the ciliate had greatly decreased. On the other hand the nucleus of Glaucoma decreased rapidly in volume as soon as starvation began and decreased only slowly after the Glaucoma had stopped multiplying.

The interpretation of the changes in shape of starving Glaucoma is by no means simple. It is mathematically true that the surface of the Glaucoma in the early stages of starvation was greater than it would have been if the ciliates had retained their normal shape as they decreased in volume, and it is tempting to speculate further. The interpretation is complicated by the fact that the Glaucoma were still multiplying at this stage. It may be shown mathematically that if the total volume of the ciliates were to remain constant, and the shape to remain the same, the total surface of the ciliates would increase times every generation time. Since the ciliates were observed to become more slender in shape, either the total volume was decreasing or else the total surface increased more than 1·26 times every generation time, or both.

From the data of Table I, where the first generation time was 3 hours, it is found that the total volume decreased in this time from 33,600 μ³ to 31,300 μ³ per cu. mm. and the total surface increased from 7100 μ² to 9560 μ², which is more than 1·26 times (95604÷7100= 1·35); so that both these factors seem to play a part in decreasing the shape-index of the Glaucoma.

The daughter cells of a dividing Glaucoma are almost spherical in shape. This is what would be expected, since a great increase in the surface is necessary to cover the ciliate as it becomes constricted into two, and any deficiency in the surface area would result in an appraoch to the spherical shape. This however is not true for all ciliates. Jennings (1908) found that in Paramecium the length increased rapidly as the constriction of fission deepened and the breadth decreased to some extent until the time of separation. When Paramecium divides the pellicle must increase rapidly in area and instead of there being a deficiency of surface there is actually an excess. The processes which cause fission to take place must also induce a rapid increase of the pellicle in Paramecium ; this is probably to a lesser extent also true for Glaucoma.

The decrease in the shape-index is most pronounced in the early stages of starvation when the ciliates are assimilating the food in the food vacuoles that are still present. The area of the surface pellicle is probably in some way related to the amount of living protoplasm. When the volume of the Glaucoma decreased owing to the disappearance of the food vacuoles, there was not a corresponding decrease in the volume of the living protoplasm (this may have been increasing). The surface was therefore in a sense “too large” for the volume and, as a result, the shape departed farther from the spherical. It is reasonable to suppose that later, when the Glaucoma have used up all the food of the food vacuoles and are dependent on reserves, the substance of the pellicle is used as a source of nourishment. This would result in a decrease in the ratio of surface to volume so that the ciliates would approach the normal, more spherical shape again.

The very rounded shape of the Glaucoma that are being fed after starvation, compared with individuals that have been fed for several generations, suggests that the increase in volume takes place so rapidly that the surface is unable to keep up sufficiently for the shape to be normal. This may be because a higher proportion of the volume of the ciliates consists of undigested food than is normally the case.

1. Glaucoma may be starved for at least a month without any deaths occurring.

2. Starved Glaucoma stop multiplying in from 6 to 12 hours according to the extent of feeding before starvation.

3. The food vacuoles are all eliminated by about 5 hours of starvation.

4. The starving individuals become smaller and smaller, the decrease in size being most rapid at first while the ciliates are still multiplying.

5. The nucleus becomes smaller in the same way as the volume of the ciliate, but to a relatively less extent.

6. During the early stages of starvation the Glaucoma become long and slender, and later they tend to return to their normal more spherical shape.

7. The changes in shape induced by starvation are interpreted in terms of the relative changes in the surface and volume of the ciliates.