ABSTRACT
Glaucoma was cultured in various concentrations of bacteria which were kept as constant as possible.
The rate of disappearance of the bacteria was a function of the concentration of the Glaucoma.
In low concentrations of bacteria the rate of feeding of the Glaucoma was a function of the concentration of bacteria.
In high concentrations of bacteria the Glaucoma were able to find almost as much food as they could take in; so that the rate of feeding tended to become independent of the concentration of bacteria.
Counts of food vacuoles showed that the rate of feeding never became quite independent of the concentration of bacteria. For every increase in the concentration, up to at least 8,000,000 bacteria per cu. mm., there was an increase in the rate of formation of food vacuoles.
High concentrations of bacteria did not inhibit the growth or the reproduction of the Glaucoma.
When the concentration of bacteria was less than 600,000 per cu. mm. the Glaucoma were semi-starved and both their size and their rate of fission was dependent on the concentration.
Between concentrations of 600,000 and 7,000,000 bacteria per cu. mm. the rate of growth in size, but not the rate of fission of Glaucoma, was influenced by the concentration.
The generation-time was constant throughout this range at between 2-3 and 2-4 hours.
INTRODUCTION
In this series of papers an attempt is made to deal with the quantitative and qualitative relations between the ciliate Glaucoma and the quantity of bacteria available for food, other conditions of the environment being kept as constant as possible. When such a set of data has been completed for particular species of ciliate feeding on a certain kind of bacteria, it will be possible to follow with investigations of the interactions between two or more species of ciliate competing for the same supply of bacteria.
The quantity of bacteria in a culture may influence a ciliate in two quite different ways. First, since the bacteria are a source of food their quantity may affect the rate of growth and, through it, the rate of reproduction. Secondly, since they are living organisms producing waste products, the bacteria may have several physiological effects on the ciliates according to the nature and concentration of the waste products they give off. The purpose of this paper is to study the effects of different concentrations of the bacterium Pseudomonas on the rate of feeding, the size, and the rate of fission of Glaucoma.
MATERIAL
The species of Glaucoma used is now being described and named by Chatton, it resembles G. pyriformis and was kindly supplied to me by Dr Muriel Robertson from her stock culture at the Lister Institute. Glaucoma is easily cultured and will live for at least a month without food. When well fed the rate of reproduction is very high, about ten generations in 24 hours; so that changes in the population may be detected at short intervals of time. All the Glaucoma used are the descendants of a single individual, isolated and washed free from foreign bacteria by Parpart’s (1928) technique, and allowed to develop into a culture in a suspension of Pseudomonas.
The Pseudomonas fluorescens was obtained from the National Collection of Type Cultures at the Lister Institute as Bacillus fluorescens non-liquefaciens. Glaucoma appears to be able to grow on Pseudomonas indefinitely, with great regularity, and without conjugation. When cultured in a suitable way Pseudomonas grows in the form of short non-motile rods, which can be shaken into a very even suspension in water. The individual bacteria are well separated from each other and show little or no tendency to settle so that accurate counts can be made.
TECHNIQUE
All experiments were performed with bacteriological precautions at 25° C., in the dark in a humid atmosphere, with sterilized pyrex glassware. The Pseudomonas were grown on solid agar-agar with 1 per cent Lemco for 24 hours. A thick suspension of the bacteria was then shaken up in a sterile solution of non-nutrient salts (Peter’s solution). After filtering through glass wool the suspension was diluted to the required concentration by comparison with a series of “Wellcome” opacity tubes. Comparisons with haemocytometer counts showed that one Wellcome opacity unit corresponds very closely to a suspension of one million Pseudomonas per cu. mm. The cultures of Glaucoma were grown in 10 c.c. of the Pseudomonas suspension in Erlenmeyer flasks. This arrangement gives a relatively large surface to the fluid so that the oxygen tension is high, and what is more important, constant throughout the medium.
In studies of the effects of different concentrations of bacteria these concentrations must be prevented from changing during the experiment. Small numbers of Glaucoma were used so that relatively few bacteria were eaten, and the bacterial suspensions were replenished at least once every 3 hours. The Glaucoma were transferred to fresh suspensions by centrifuging. This had no effect on the number, the size, or the rate of fission of the Glaucoma as far as could be detected.
Samples were taken by weight, a simple technique being developed which was found to be both more convenient and more accurate than the usual method using a volumetric pipette. The organisms were fixed with an equal volume of 10 per cent neutral formaldehyde. The bacteria were counted by the method developed by Wilson (1922) using a counting chamber of the haemocytometer type.
The pH of the pure mixed culture of Glaucoma and Pseudomonas remained constant between the limits 6·7 and 7·0 owing to the buffering action of the two organisms.
The rate of feeding of the Glaucoma
The rate at which Glaucoma feeds on Pseudomonas has been investigated in two ways: first by making counts of the ciliates and the bacteria at short intervals of time; and secondly by determining the rate of formation of food vacuoles by Glaucoma in different concentrations of Pseudomonas.
Before proceeding with the first method it was necessary to ascertain that Pseudomonas does not multiply in the medium in which it is suspended. Counts of the bacteria in suspension in the salt solution used were made and no change greater than the experimental error could be detected in their numbers (Table I). Similar results were obtained when the Pseudomonas suspension was rich in the metabolic waste products of Glaucoma.
In the feeding experiments samples were taken sufficiently frequently for there to be no appreciable increase in the numbers of Glaucoma between successive counts. The concentrations of Glaucoma used were high, so that the bacteria were eaten sufficiently rapidly for the decrease in their numbers to be detected.
The results of such an experiment may be put in the form of a series of curves by plotting the initial number of bacteria against the number that remain after 1 hour (Fig. 1). By joining the points for each concentration of Glaucoma a series of contours is obtained. Since the bacteria do not multiply in the medium a contour may be drawn for the case when there are no Glaucoma as a straight line passing through the origin at an angle of 45°. Contours for the different concentrations of Glaucoma follow in order, becoming more steep as the concentration of Glaucoma increases. When the concentration of bacteria is high the Glaucoma are able to capture as many bacteria as they are able to take in: so that the number of bacteria eaten in a given time tends to become independent of the bacterial concentration, and the contour becomes asymptotically parallel to the 45° line. The higher the concentration of Glaucoma the higher is the concentration of bacteria at which the contour falls to this slope. From the part of the curve for 20 Glaucoma per cu. mm. where the slope has fallen to 45°, the number of bacteria eaten is found to be about 10,000 per Glaucoma per hour.
With concentrations of bacteria higher than 500,000 per cu. mm. it was difficult to obtain significant changes in their numbers and the rate of feeding was investigated by the second method of attack. The rate of formation of food vacuoles was studied by the method of Mills (1931). The Glaucoma were left in various concentrations of Pseudomonas for 9 hours, the latter being replenished every 3 hours. An equal small quantity of non-toxic indian ink was now added to each culture and after 1 hour samples were taken. The number of black food vacuoles was then counted in 100 individuals from each culture. It was found that, even with the highest concentrations of bacteria used, every increase in the concentration allowed more food vacuoles to be formed by a Glaucoma in a given time (Table III). Even in these high concentrations of bacteria the Glaucoma seemed to take up fewer bacteria than the maximum they were capable of; so that the number of bacteria eaten in a given time is never quite independent of the concentration. The highest concentration of bacteria used, 8,000,000 per cu. mm., was as opaque as a very thick starch paste.
THE SIZE AND THE RATE OF FISSION OF GLAUCOMA IN DIFFERENT CONCENTRATIONS OF BACTERIA
A series of concentrations of Pseudomonas were set up, the concentrations ranging from 100,000 to 7,000,000 per cu. mm.
Glaucoma were grown in these concentrations for 9 hours or more before samples were taken. The influence of the previous history of the ciliates was eliminated in this way and measurements of the Glaucoma taken after this time were characteristic of the concentration of bacteria from which they were taken.
It has been found convenient to measure only adult Glaucoma, i.e. those that are just about to divide. This eliminates any danger of error due to differences in the age distributions of the cultures. The volumes of the individual Glaucoma have been calculated from the lengths and breadths of the two daughter cells on the assumption that the latter are prolate spheroids. The rate of fission of the Glaucoma was measured in terms of the generation-time calculated from the increase in the numbers found in samples taken at intervals of 3 hours.
At least three determinations of the generation-time were made at different times for each concentration of bacteria, each determination was the result of counting six samples, and three or four counts were made of each sample. For every concentration of bacteria in which the size of the Glaucoma has been measured at least six determinations of the average size were made at different times.
The mean generation-time and the mean adult size of Glaucoma in the different concentrations of bacteria, together with their respective standard errors, are given in Table IV, and shown graphically in Fig. 2.
In concentrations of less than 600,000 bacteria per cu. mm. the Glaucoma were semi-starved, and both the size and the fission rate were dependent on the concentration of bacteria. A concentration of 600,000 bacteria per cu. mm. caused a maximum rate of fission of Glaucoma. Higher concentrations, although increasing the size, had no effect on the rate of fission.
Camera lucida drawings of representative individuals from each concentration have been made. Those for concentrations of 600,000 and 7,000,000 bacteria per cu. mm. are shown in Fig. 3. The difference in size is very noticeable; yet the rate of fission is the same in both cases. A complete range of sizes intermediate between these two is found in the drawings from the intermediate concentrations of bacteria, each in its proper sequence.
DISCUSSION
Johnson (1933, 1936) has found that the reproduction of some ciliates is inhibited and even stopped altogether if the concentration of food bacteria is too high. Pseudomonas, however, has no inhibitory effect on Glaucoma even when ten times as concentrated as Johnson’s figures. An increase in the concentration from about half a million to about eight million per c.c. results in a 60 per cent increase in the size of the Glaucoma. As the rate of reproduction is unchanged presumably the number of bacteria eaten in a given time increases by 60 per cent. The number of food vacuoles formed per hour increases by 36 per cent for such an increase in the concentration of bacteria; so that each food vacuole probably contains more bacteria. This is in accordance with the fact that the large Glaucoma from high concentrations of bacteria have visibly larger food vacuoles than the smaller individuals from lower concentrations. The following table (Table V) is derived by applying the above arguments to the data available.
There are many more food vacuoles in the Glaucoma from high concentrations of bacteria than there are in those from low concentrations, as is clearly shown in Fig. 3. The possibility arises that the former Glaucoma are larger because they contain more undigested food, and that the rate of fission is the same because perhaps the quantity of living protoplasm is the same in both cases. The latter is unlikely because the fact that the rate of formation of food vacuoles is greater in the higher concentrations indicates that the rate of formation of living protoplasm is also greater.
Monod (1935), Monod & Tessier (1936) and Phelps (1936) have measured the rate of fission of Glaucoma pyriformis in various concentrations of organic solutions. They also find that there is a maximum rate of reproduction which is independent of the concentration of food; but the maximum rates found differ. Monod & Tessier found the generation time to be 8 hours, Phelps found it to be 3·8 hours. Both of these rates of reproduction are slower than that found for Glaucoma fed on Pseudomonas where a generation takes only 2·3 hours. None of these workers measured the sizes of the Glaucoma. It would be instructive to know whether the various concentrations of peptone or of yeast autolysate used by these workers act in the same way as do high concentrations of bacteria in accelerating growth without influencing fission rate.
This work is, as far as the writer is aware, the first in which the size of a ciliate has been altered at will, between wide limits, with the rate of multiplication kept constant. Other workers, however, have found that the rate of multiplication may be independent of size. An admirable review of such work has been written by Adolph (1931) who comes to the conclusion that “when the processes which have to do with age come to a certain point fission occurs regardless of how much body substance is present”. The Glaucoma in the concentrations between 600,000 and 7,000,000 bacteria per cu. mm. reproduce at the same rate, in spite of the differences in size, probably because this is the maximum rate possible. The rate of fission of the nucleus may be the limiting factor.
SUMMARY
Glaucoma was cultured in various concentrations of bacteria which were kept as constant as possible.
The rate of disappearance of the bacteria was a function of the concentration of the Glaucoma.
In low concentrations of bacteria the rate of feeding of the Glaucoma was a function of the concentration of bacteria.
In high concentrations of bacteria the Glaucoma were able to find almost as much food as they could take in; so that the rate of feeding tended to become independent of the concentration of bacteria.
Counts of food vacuoles showed that the rate of feeding never became quite independent of the concentration of bacteria. For every increase in the concentration, up to at least 8,000,000 bacteria per cu. mm., there was an increase in the rate of formation of food vacuoles.
High concentrations of bacteria did not inhibit the growth or the reproduction of the Glaucoma.
When the concentration of bacteria was less than 600,000 per cu. mm. the Glaucoma were semi-starved and both their size and their rate of fission was dependent on the concentration.
Between concentrations of 600,000 and 7,000,000 bacteria per cu. mm. the rate of growth in size, but not the rate of fission of Glaucoma, was influenced by the concentration.
The generation-time was constant throughout this range at between 2-3 and 2-4 hours.
Acknowledgements
I wish to express my thanks to Dr J. Gray, F.R.S., and Dr G. Salt for their kind help and encouragement at Cambridge, Dr M. Robertson for her generous advice on protozoological methods, and Prof. G. S. Wilson for his helpful advice on the technique for counting bacteria.