QUANTITATIVE STUDIES ON THE CILIATE GLAUCOMA

Transcription

1 422 QUANTITATIVE STUDIES ON THE CILIATE GLAUCOMA I. THE REGULATION OF THE SIZE AND THE FISSION RATE BY THE BACTERIAL FOOD SUPPLY BY J. P. HARDING, PH.D. Zoological Laboratory, Cambridge (Received 2 February 937) (With Three Text-figure) INTRODUCTION IN thi erie of paper an attempt i made to deal with the quantitative and qualitative relation between the ciliate Glaucoma and the quantity of bacteria available for food, other condition of the environment being kept a contant a poible. When uch a et of data ha been completed for particular pecie of ciliate feeding on a certain kind of bacteria, it will be poible to follow with invetigation of the interaction between two or more pecie of ciliate competing for the ame upply of bacteria. The quantity of bacteria in a culture may influence a ciliate in two quite different way. Firt, ince the bacteria are a ource of food their quantity may affect the rate of growth and, through it, the rate of reproduction. Secondly, ince they are living organim producing wate product, the bacteria may have everal phyiological effect on the ciliate according to the nature and concentration of the wate product they give off. The purpoe of thi paper i to tudy the effect of different concentration of the bacterium Peudomona on the rate of feeding, the ize, and the rate of fiion of Glaucoma. MATERIAL The pecie of Glaucoma ued i now being decribed and named by Chatton, it reemble G. pyriformi and wa kindly upplied to me by Dr Muriel Roberton from her tock culture at the Liter Intitute. Glaucoma i eaily cultured and will live for at leat a month without food. WTien well fed the rate of reproduction i very high, about ten generation in 24 hour; o that change in the population may be detected at hort interval of time. All the Glaucoma ued are the decendant of a ingle individual, iolated and wahed free from foreign bacteria by Parpart' (928) technique, and allowed to develop into a culture in a upenion of Peudomona.

2 Quantitative Studie on the Ciliate Glaucoma 423 The Peudomonafluorecenwa obtained from the National Collection of Type Culture at the Liter Intitute a Bacillufluorecennon-liquefacien. Glaucoma appear to be able to grow on Peudomona indefinitely, with great regularity, and without conjugation. When cultured in a uitable way Peudomona grow in the form of hort non-motile rod, which can be haken into a very even upenion in water. The individual bacteria are well eparated from each other and how little or no tendency to ettle o that accurate count can be made. TECHNIQUE All experiment were performed with bacteriological precaution at 25 C, in the dark in a humid atmophere, with terilized pyrex glaware. The Peudomona were grown on olid agar-agar with per cent Lemco for 24 hour. A thick upenion of the bacteria wa then haken up in a terile olution of non-nutrient alt (Peter' olution). After filtering through gla wool the upenion wa diluted to the required concentration by comparion with a erie of "Wellcome" opacity tube. Comparion with haemocytometer count howed that one Wellcome opacity unit correpond very cloely to a upenion of one million Peudomona per cu. mm. The culture of Glaucoma were grown in 0 c.c. of the Peudomona upenion in Erlenmeyer flak. Thi arrangement give a relatively large urface to the fluid o that the oxygen tenion i high, and what i more important, contant throughout the medium. In tudie of the effect of different concentration of bacteria thee concentration mut be prevented from changing during the experiment. Small number of Glaucoma were ued o that relatively few bacteria were eaten, and the bacterial upenion were replenihed at leat once every 3 hour. The Glaucoma were tranferred to freh upenion by centrifuging. Thi had no effect on the number, the ize, or the rate of fiion of the Glaucoma a far a could be detected. Sample were taken by weight, a imple technique being developed which wa found to be both more convenient and more accurate than the uual method uing a volumetric pipette. The organim were fixed with an equal volume of 0 per cent neutral formaldehyde. The bacteria were counted by the method developed by Wilon (922) uing a counting chamber of the haemocytometer type. The >H of the pure mixed culture of Glaucoma and Peudomona remained contant between the limit 67 and 7-0 owing to the buffering action of the two organim. The rate of feeding of the Glaucoma The rate at which Glaucoma feed on Peudomona ha been invetigated in two way: firt by making count of the ciliate and the bacteria at hort interval of time; and econdly by determining the rate of formation of food vacuole by Glaucoma in different concentration of Peudomona. Before proceeding with the firt method it wa neceary to acertain that Peudomona doe not multiply in the medium in which it i upended. Count of

3 424 J. P. HARDING the bacteria in upenion in the alt olution ued were made and no change greater than the experimental error could be detected in their number (Table I). Similar reult were obtained when the Peudomona upenion wa rich in the metabolic wate product of Glaucoma. Table I. Count of Peudomona in the non-nutrient medium ued howing that there i no multiplication Hour o Thouand bacteria percu. mm In the feeding experiment ample were taken ufficiently frequently for there to be no appreciable increae in the number of Glaucoma between ucceive count. The concentration of Glaucoma ued were high, o that the bacteria were eaten ufficiently rapidly for the decreae in their number to be detected. The reult of uch an experiment may be put in the form of a erie of curve by plotting the initial number of bacteria againt the number that remain after hour (Fig. ). By joining the point for each concentration of Glaucoma a erie of contour i obtained. Since the bacteria do not multiply in the medium a contour may be drawn for the cae when there are no Glaucoma a a traight line paing through the origin at an angle of Contour for the different concentration of Glaucoma follow in order, becoming more teep a the concentration of Glaucoma increae. When the concentration of bacteria i high the Glaucoma are able to capture a many bacteria a they are able to take in: o that the number of bacteria eaten in a given time tend to become independent of the bacterial concentration, and the contour become aymptotically parallel to the 45 line. The higher the concentration of Glaucoma the higher i the concentration of bacteria at which the contour fall to thi lope. From the part of the curve for 20 Glaucoma per cu. mm. where the lope ha fallen to 45 0, the number of bacteria eaten i found to be about 0,000 per Glaucoma per hour. Table II. The number of bacteria eaten in hour by different concentration of Glaucoma, and with varying concentration of bacteria. The number of bacteria initially preent i compared with the number left after hour No. of Glaucoma per cu. mm. Thouand of bacteria per cu. mm. Initially After hour i "4 42 2'

4 Quantitative Studie on the Ciliate Glaucoma 425 With concentration of bacteria higher than 500,000 per cu. mm. it wa difficult to obtain ignificant change in their number and the rate of feeding wa invetigated by the econd method of attack. The rate of formation of food vacuole wa tudied by the method of Mill (93). The Glaucoma were left in variou concentration of Peudomona for 9 hour, the latter being replenihed every 3 hour. An equal mall quantity of non-toxic indian ink wa now added to each culture and after hour ample were taken. The number of black food vacuole wa then counted 500,000 - f S. 400,000 - *X),000 o 200,000 >20 t ^ ,000!' 0 I 0 00, , , ,000 Number of bacteria per cu. mm. left after i hour Fig. i. The relation between the number of bacteria eaten and the number of Glaucoma and bacteria preent. (The number near each point give the number of Glaucoma per cu. mm.) in ioo individual from each culture. It wa found that, even with the highet concentration of bacteria ued, every increae in the concentration allowed more food vacuole to be formed by a Glaucoma in a given time (Table III). Even in thee high concentration of bacteria the Glaucoma eemed to take up fewer bacteria than the maximum they were capable of; o that the number of bacteria eaten in a given time i never quite independent of the concentration. The highet concentration of bacteria ued, 8,000,000 per cu. mm., wa a opaque a a very thick tarch pate.

5 426 J. P. HARDING Table III. The rate of formation of food vacuole by Glaucoma in different concentration of bacteria, with tandard deviation of the mean Million of bacteria per cu. mm No. of vacuole formed per hour (mean value) ± "* '42±O-2O THE SIZE AND THE RATE OF FISSION OF GLAUCOMA IN DIFFERENT CONCENTRATIONS OF BACTERIA A erie of concentration of Peudomona were et up, the concentration ranging from 00,000 to 7,000,000 per cu. mm. Glaucoma were grown in thee concentration for 9 hour or more before ample were taken. The influence of the previou hitory of the ciliate wa 5 3 U O i.53 S -0 3 I Concentration of bacteria (million per cu. mm.) Fig. 2. The ize and the rate of reproduction of Glaucoma in relation to the concentration of bacteria. eliminated in thi way and meaurement of the Glaucoma taken after thi time were characteritic of the concentration of bacteria from which they were taken. It ha been found convenient to meaure only adult Glaucoma, i.e. thoe that are jut about to divide. Thi eliminate any danger of error due to difference in

6 Quantitative Studie on the Ciliate Glaucoma 427 the age ditribution of the culture. The volume of the individual Glaucoma have been calculated from the length and breadth of the two daughter cell on the aumption that the latter are prolate pheroid. The rate of fiion of the Glaucoma wa meaured in term of the generation-time calculated from the increae in the number found in ample taken at interval of 3 hour. At leat three determination of the generation-time were made at different time for each concentration of bacteria, each determination wa the reult of counting ix ample, and three or four count were made of each ample. For every concentration of bacteria in which the ize of the Glaucoma ha been meaured at leat ix determination of the average ize were made at different time. The mean generation-time and the mean adult ize of Glaucoma in the different concentration of bacteria, together with their repective tandard error, are given in Table IV, and hown graphically in Fig. 2. Table IV. Size and rate of reproduction of Glaucoma in variou concentration of bacteria Million bacteria per cu. mm O IO O O'l Generation-time 2'39±OO ± ± ±OI3 2'39±o-o7 23 ± ±06 2'33±oi3 a-9o±o-i9 3-+J ±0-3 Adult ize 39,9oo± 00 29,400 ± ,030 ± ,000 ± ,580 ± 930 2, ,870 ± 580 In concentration of le than 600,000 bacteria per cu. mm. the Glaucoma were emi-tarved, and both the ize and the fiion rate were dependent on the concentration of bacteria. A concentration of 600,000 bacteria per cu. mm. caued a maximum rate of fiion of Glaucoma, Higher concentration, although increaing the ize, had no effect on the rate of fiion. Camera lucida drawing of repreentative individual from each concentration have been made. Thoe for concentration of 600,000 and 7,000,000 bacteria per cu. mm. are hown in Fig. 3. The difference in ize i very noticeable; yet the rate of fiion i the ame in both cae. A complete range of ize intermediate between thee two i found in the drawing from the intermediate concentration of bacteria, each in it proper equence. DISCUSSION Johnon (933, 936) ha found that the reproduction of ome ciliate i inhibited and even topped altogether if the concentration of food bacteria i too high. Peudomona, however, ha no inhibitory effect on Glaucoma even when ten time a concentrated a Johnon' figure. An increae in the concentration from

7 428 J. P. HARDING about half a million to about eight million per c.c. reult in a 60 per cent increae in the ize of the Glaucoma. A the rate of reproduction i unchanged preumably the number of bacteria eaten in a given time increae by 60 per cent. The number of food vacuole formed per hour increae by 36 per cent for uch an increae in the concentration of bacteria; o that each food vacuole probably contain more bacteria. Thi i in accordance with the fact that the large Glaucoma from high concentration of bacteria have viibly larger food vacuole than the maller individual from lower concentration. The following table (Table V) i derived by applying the above argument to the data available. 00x Fig- 3- Camera lucida drawing of Glaucoma: upper row, with 7,000,000 bacteria per cu. mm.: lower row, with 600,000 bacteria per cu. mm. Table V. The rate of feeding of Glaucoma in three widely different concentration of bacteria, with other relevant data Million bacteria per cu. mm. Generationtime of Glaucoma in hour Size of adult Glaucoma M* Bacteria eaten per Glaucoma per hour No. of foodvacuole formed per hour No. of bacteria per foodvacuole o-s »"4 25,000 32,000 40,000 0,000 3,000 6, ' '35 400

8 Quantitative Studie on the Ciliate Glaucoma 429 There are many more food vacuole in the Glaucoma from high concentration of bacteria than there are in thoe from low concentration, a i clearly hown in Fig. 3. The poibility arie that the former Glaucoma are larger becaue they contain more undigeted food, and that the rate of fiion i the ame becaue perhap the quantity of living protoplam i the ame in both cae. The latter i unlikely becaue the fact that the rate of formation of food vacuole i greater in the higher concentration indicate that the rate of formation of living protoplam i alo greater. Monod (935), Monod & Teier (936) and Phelp (936) have meaured the rate of fiion of Glaucoma pyrifor mi in variou concentration of organic olution. They alo find that there i a maximum rate of reproduction which i independent of the concentration of food; but the maximum rate found differ. Monod & Teier found the generation time to be 8 hour, Phelp found it to be 3-8 hour. Both of thee rate of reproduction are lower than that found for Glaucoma fed on Peudomona where a generation take only 2-3 hour. None of thee worker meaured the ize of the Glaucoma. It would be intructive to know whether the variou concentration of peptone or of yeat autolyate ued by thee worker act in the ame way a do high concentration of bacteria in accelerating growth without influencing fiion rate. Thi work i, a far a the writer i aware, the firt in which the ize of a ciliate ha been altered at will, between wide limit, with the rate of multiplication kept contant. Other worker, however, have found that the rate of multiplication may be independent of ize. An admirable review of uch work ha been written by Adolph (93) who come to the concluion that "when the procee which have to do with age come to a certain point fiion occur regardle of how much body ubtance i preent". The Glaucoma in the concentration between 600,000 and 7,000,000 bacteria per cu. mm. reproduce at the ame rate, in pite of the difference in ize, probably becaue thi i the maximum rate poible. The rate offiionof the nucleu may be the limiting factor. SUMMARY. Glaucoma wa cultured in variou concentration of bacteria which were kept a contant a poible. 2. The rate of diappearance of the bacteria wa a function of the concentration of the Glaucoma. 3. In low concentration of bacteria the rate of feeding of the Glaucoma wa a function of the concentration of bacteria. 4. In high concentration of bacteria the Glaucoma were able to find almot a much food a they could take in; o that the rate of feeding tended to become independent of the concentration of bacteria. 5. Count of food vacuole howed that the rate of feeding never became quite independent of the concentration of bacteria. For every increae in the concentration, up to at leat 8,000,000 bacteria per cu. mm., there wa an increae in the rate of formation of food vacuole.

9 43 J. P. HARDING 6. High concentration of bacteria did not inhibit the growth or the reproduction of the Glaucoma. 7. When the concentration of bacteria wa le than 600,000 per cu. mm. the Glaucoma were emi-tarved and both their ize and their rate of fiion wa dependent on the concentration. 8. Between concentration of 600,000 and 7,000,000 bacteria per cu. mm. the rate of growth in ize, but not the rate of fiion of Glaucoma, wa influenced by the concentration. 9. The generation-time wa contant throughout thi range at between 2-3 and 2-4 hour. ACKNOWLEDGEMENTS I wih to expre my thank to Dr J. Gray, F.R.S., and Dr G. Salt for their kind help and encouragement at Cambridge, Dr M. Roberton for her generou advice on protozoological method, and Prof. G. S. Wilon for hi helpful advice on the technique for counting bacteria. REFERENCES ADOLPH, E. F. (93). The Regulation of Size a Illutrated in Unicellular Organim. Springfield, U.S.A. JOHNSON, W. H. (933). Phyiol. Zool. 6, (936). Phyiol. Z06L 9, -4. MILLS, S. M. (93). J. exp. Biol. 8, MONOD, J. (935). C. R. Acad. Sci., Pan, 20, MONOD, J. & TESSIER, G. (936). C. R. Acad. Sci., Pari, 202, PARPART, A. K. (928). Biol. Bull. Wood' Hole, 58, PHELPS, A. (936). J. exp. Zool. 72, WILSON, G. S. (922). J. Bad. 7,