Effects of Temperature on Tadpole Hearts in Vitro

Home , Hearts Still Beating

/. Embryol. exp. Morph., Vol. 17, 1, pp. 147-159, February 1967 147 With 2 plates Printed in Great Britain Effects of temperature on tadpole hearts in vitro

By ELSIE M. STEPHENSON1 From the School of Biological Sciences, University of New South Wales, Sydney

INTRODUCTION The temperatures currently used for amphibian cell and tissue culture appear to range from 26 °C (Seto, 1964) to 18 °C (Shah, 1964). The experiments of N. G. Stephenson & Tomkins (1964), in which Pseudophryne tadpole humeri and femora transplanted to chick chorioallantoic membranes at 38 °C showed definite growth, suggested the possible advantage of culturing other amphibian tissues at temperatures above 26 °C. The present study has therefore been carried out in an attempt to establish more precisely the optimal and maximal conditions for amphibian cell and organ culture. Whole hearts of tadpoles of the Leptodactylid frog, Limnody- nastes peroni (Dumeril & Bibron) were cultured at a series of temperatures ranging from 5 to 37°C for a period of at least 1 week. Although the short-term behaviour of an isolated, adult frog's heart in Ringer's solution in relation to increased temperature is known (Mitchell, 1956), only incidental observations have been recorded with regard to temperature effects on rate of beat of cultured amphibian heart fragments (Johnson, 1915; Morosow, 1929) or embryonic heart rudiments (Stohr, 1924). By using whole tadpole hearts in culture it has been possible to observe the effects of different temperatures on organ function and maintenance, as indicated by heart beat, as well as on cell outgrowth.

MATERIAL AND METHODS The tadpoles were identified by their labial teeth arrangement, lateral-line patterns and other features recorded by Phillis (1958), but some specimens were kept alive until metamorphosis as a further taxonomic check. Four main experimental series of hearts were cultured (Table 1), each series containing thirty-six hearts. Additional hearts, including those of an incomplete series C, were grown for special purposes such as transfers from one temperature to another. All tadpoles except those of series E came from eggs collected from a single locality at the same time. Each tadpole was rinsed in tap water and immobilized in MS 222. The rate

1 Author's address: School of Biological Sciences, The University of New South Wales, P.P. Box 1, Kensington, New South Wales, Australia. 148 E. M. STEPHENSON of heart beat per minute in vivo of every fourth or fifth animal was recorded. Each tadpole was then washed in detergent (7 X) and immersed briefly in 95 % alcohol before being passed through two changes of Pannett and Compton's saline containing antibiotics. For series A and B, only penicillin sodium G (40 i.u./ml) and streptomycin sulphate (50/*g/ml) were used. As one culture in series A and two in series B later showed fungal contamination, mycostatin (20/tg/ml) was added to all subsequent series and effectively prevented any further infection.

Table 1. Identification of main experimental series

Mean room temperature during ex- Mean heart perimental Approximate age rates/min Experimental temperatures periods Series of tadpoles in vivo used (°C) (°C) A 3 weeks 97 37, 30, 25, 20, 15, 5 23 B 7 weeks 96 As above, but 8° sub- 23 stituted for 5° D 4^- months 84 As for series B 19 E 18-19 days 105 As for series A 23

Each animal was handled individually with a perforated metal spoon which prevented breakage of skin surface. Removal of the hearts was carried out in saline under a binocular dissecting microscope, using iridectomy scissors and jeweller's forceps. Each heart was later checked for signs of damage and any adhering tissue was removed. When all hearts for a complete series had been prepared, clots of equal quantities of cockerel plasma and chick embryo extract (50 % in Hanks saline) were made on small coverslips adhering to larger ones, according to the Maximow technique (see Paul, 1960). While each clot was still semi-liquid, a heart was dropped lightly on to its surface so that it was held in position when the clot set. A drop of liquid medium was added. When sealed to cavity slides the coverslips were inverted to produce lying-drop cultures. The liquid medium consisted of Medium 199 (single strength), 7 parts; Hanks BSS, 7 parts; cockerel serum, 4 parts; chick embryo extract (50 % in Hanks BSS), 1 part. Antibiotics were added in the same concentrations as for the washing saline. The medium was adjusted to a pH of 7-4. The cultures in each series were allocated at random in batches of six to each of six different temperatures (Table 1). The hearts in each batch were numbered in sequence and their rates of beat were recorded before transfer to the incubation temperatures took place. The incubators used varied in type but were all thermostatically controlled and fitted with accurate thermometers. In general, Tadpole hearts in vitro 149 the amount of temperature fluctuation was negligible. The 15 °C incubator used for series A and B was found to be faulty and was replaced for all later experiments. Although most cultures were maintained for at least 9-10 days, the main experimental period was taken as 1 week. The cultures were usually removed daily from their incubators and examined for a very brief period at room temperature (Table 1). Recording of heart-beat rates was carried out with maximum speed and always preceded any examination and estimation of cell outgrowth. Counting of beats followed a standardized routine which made use of a stop-watch and telecounter. The process was simplified by the fact that, although variation in intensity of beat occurred, relatively little irregularity of pulsation or asynchrony of different regions were evident. So-called subculturing took place usually every second day. This was un- necessarily frequent for hearts at lower temperatures but was standardized to meet the probable demands of cultures in the upper part of the temperature range. The small coverslips and cultures were washed in saline and were then provided with fresh liquid medium and sealed in a new culture chamber. The clots were not usually added to or changed but in the rare cases where a heart became loose a very small drop of fresh plasma was provided. All hearts were washed individually in watch-glasses to prevent any possible cross transfer of dissolved substances or of contaminants. Heart-beat records were made after each subculture. When harvested, the cultures were either fixed in formol-saline and stained in Mallory's aqueous haematoxylin, or fixed in absolute methanol and stained in Jenner-Giemsa (see Paul, 1960). In some cases, treatment with silver nitrate (1:400) preceded fixation. Selected cultures were treated with colchicine and hypotonic saline for karyotype analysis (Robinson & Stephenson, 1967). All photographs were taken with a Zeiss photomicroscope. For series A-D, estimations of outgrowth were made visually in relation to a standard microscope field. For series E, drawings of all cultures were made at regular intervals, using a Zeiss projection drawing apparatus and paper of standard weight. Mean weights of explants and outgrowths at each temperature were later obtained (see Paul, 1960).

RESULTS (1) Maintenance of heart beat In general, allowing for occasional temporary cessation, total maintenance of heart beat occurred at all temperatures from 30 to 5 °C inclusive. The only exceptions to this occurred in the first two series A and B in which at 15 and 20 °C not more than one-third of the hearts in culture were beating at the end of a week. The 15 °C results were apparently caused by an incubator fault but those at 20 °C, though obviously anomalous, are less easy to explain. However, 150 E. M. STEPHENSON in all subsequent series and in additional cultures kept at 15 and 20 °C, 100 % maintenance of beat was recorded. At 37 °C all hearts continued to beat for at least 1-2 days. In series B, D and E all hearts had stopped permanently by the fifth day in culture but in series A three out of six hearts were still beating on the sixth day. Only one long-term observation was made on heart-beat maintenance. This is discussed under section 5 below. The immediate effects of subculturing on heart-beat rate were unpredictable. In most cases the beat was maintained but occasionally it stopped for a temporary period. At times, where a beat had stopped before subculturing took place, it began again immediately afterwards.

Series D

1 2 3 4 5 6 7 Days in culture Text-fig. 1. Series D. Graphical illustration of the influence of different temperatures on rate of heart beat during 1 week in culture. Except at 37 °C, where figures in parentheses indicate the number of hearts still beating, each point indicates the mean rate/minute for six hearts. Means have been calculated only from hearts actually beating. (2) Rate of heart beat Comparison of mean rates of heart beat per minute in vivo (Table 1) with recordings made before the extirpated hearts in culture chambers were transferred to their incubation temperatures showed that a very marked drop in rate occurred in each series. The factors causing this must have included general operational effects and reactions to a new environmental medium. Throughout the period of culture, mean heart-beat rates in all series and at all temperatures remained well below the mean rate in vivo, except in series E at 30 °C, where by the end of a week the hearts had almost regained their in vivo average. In order to compare information relating to heart-beat maintenance, heart- beat rate and cell outgrowth, it was necessary to use the same hearts throughout the full term of any one experimental series. If heart-beat rate alone had been under investigation, a different type of experimental model, using successions of Tadpole hearts in vitro 151 different hearts, might have been desirable so that analysis of variance could be carried out. Although this has not been possible under the present experimental conditions, the recordings obtained still supply a picture of relative heart-beat rates at different temperatures over a reasonably long period. The graphs supplied (Text-figs. 1-3) should be examined primarily from this point of view. It is clear that, although the absolute figures for any given temperature could vary widely, the relative patterns were much more stable.

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30- Mea n - o -o 20- / \ .>* 10- V- -o- ~-^~^5 °c 1 1 \ I I 1 I 1 2 3 4 : 5 Days in culture Text-fig. 2. Series E. Explanation as for Text-fig. 1.

At 37 °C in all series, heart-beat rate rose sharply to a comparatively high peak (Text-fig. 3, day 1), which was followed by a decline until all hearts at this temperature had stopped beating. Drop in rate was accompanied by irregularity and progressive weakening of beat. In general, the optimal temperature for heart-beat rate in vivo was 30 °C. Throughout the greater part of the experimental period, each series showed a descending scale of rates associated with progressively lower temperatures from 30 to 5°C. The differences were particularly marked in series E (Text-fig. 2). In other series, absolute differences in rate were less obvious while temporary overlaps (e.g. Text-fig. 1, 20 and 15 °C; Text-fig. 3, series B, 30 and 25 °C) sometimes occurred. During subculturing, changes in concentrations of oxygen, glucose, serum, potassium and calcium ions, temporary changes of temperature and probably other factors undoubtedly exerted a complex of effects. In most cases an im- 152 E. M. STEPHENSON mediate, marked rise in heart-beat rate was apparent but occasionally a decrease or even complete but temporary cessation of heart beat was noted. No attempt was made to analyse these variations in response.

(3) Relative extent of outgrowth At 37 and 5 °C no definitive outgrowth of cells was observed. At 37 °C, however, some temporary outpushing of the superficial cells was usually initiated quite early but the cells in question were not able to expand in a normal fashion and quickly became rounded off. Degeneration eventually occurred.

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5 8 20 25 30 37 5 8 15 20 25 30 37 Degrees Centigrade Text-fig. 3. Comparison of initial heart-beat reaction to incubation temperature (day 1) with rate after a period of adjustment (day 4). Day 1 is shown by broken lines; day 4 by unbroken lines.

Fixed and stained preparations of hearts cultured at 37 °C have a roughened, irregular appearance around the edges where the cells have undergone the changes described above. As indicated below (section 5), viability of cells may be maintained at 37 °C for several days, permitting cell outgrowth to occur when the cultures are transferred to a more favourable temperature. Cell outgrowth occurred at 30, 25, 20, 15 and, to a much lesser extent, at 8 °C. From 30 to 15 °C inclusive the general pattern of outgrowth was quali- tatively similar, although the rapidity and extent of cell migration decreased J. Embryol. exp. Morph., Vol. 17, Part 1 PLATE 1

Magnifications are as for fig. A, except in the case of fig. E. Fig. A. Series A. Heart and outgrowth after 8 days at 30 °C. Fig. B. Series B. Heart and outgrowth after 8 days at 25 °C. The outgrowth is relatively small for this temperature but the general appearance of the culture is typical. Fig. C. Approximately a quarter of outgrowth area after 14 days at 30 °C. A cellular monolayer covers the central clot-free area, ph Original position of dislodged heart; c, region of clot. Fig. D. Series C. Culture after 4 days at 5 °, 2 days at 25 °, 7 days at 5 ° and 3 days at 30 °C. The heart has been displaced to one side during fixation. Fig. E. Series D. Part of outgrowth and edge of relatively large heart after 10 days at 20 °C. Anchoring threads are obvious. Fig. F. Culture after maintenance for 4 days at 37 °C, with no outgrowth, followed by 3 days at 30 °C.

E. M. STEPHENSON facing p. 152 /. Embryo], exp. Morph., Vol. 17, Part 1 PLATE 2

E. M. STEPHENSON facing p. 153 Tadpole hearts in vitro 153 successively with decreasing temperature. Cells first grew out at various levels on and through the clot but sooner or later a concave area, mainly free from clot but more or less covered by a cellular monolayer, surrounded the heart itself (Plate 1, figs. A-E). The remaining part of the clot formed a thickened ring around the inner area (Plate 1, figs. A-C; Plate 2, fig. A). In most cases, anchoring threads or sheets of cells (Plate 1, fig.E ; Plate 2, figs. G, H) stretched from the explant into the clot, often at a level well above that of the central monolayer. In all series the optimal temperature for cell outgrowth was 30 °C (Text- fig. 4). At this temperature, cell migration began within 24 h and continued rapidly. At temperatures from 25 to 15 °C progressively greater delays occurred in the initiation of migration, which at 15 °C did not usually begin until the fourth day (Text-fig. 4). Decrease in the mean bulk area of explants was very small compared with the mean increase in outgrowth area (Text-fig. 4). It was greatest at 30° and least at 15 °C. Mitoses occurred at all temperatures from 30 to 15 °C. No quantitative estimations of mitotic rates were made but it is likely that these would agree with the comparative picture of cell outgrowth. (4) Cell types present Different incubation temperatures did not produce any obvious qualitative differences in cell types. The cell population in all cases was composed almost entirely of epithelioid cells. In routinely stained cultures, these cells (Plate 1, fig. E; Plate 2, fig. D) appear to be more or less widely separated, resembling the 'spacy' condition of cells in cultured pulmonary epithelium of newt (Danes, 1949). Yet from cultures treated with silver nitrate before fixation (Plate 2, figs. A, B) it is clear that the cells form a closely fitting sheet and that the amount of intercellular material is small.

PLATE 2 Magnifications are as for Fig. F, except in the case of Fig. A. Fig. A. Series E. Edge of heart (h) and part of outgrowth after 4 days at 30 °C. Culture treated with silver nitrate before fixation and stained with haematoxylin. The central clot-free well (w) is clearly demarcated from the edge of the clot (ec). Fig. B. Series E. General area of epithelioid cells after 9 days at 15 °C. Culture treated with silver nitrate and stained with Jenner-Giemsa. Fig. C. Series D: 30 °C. Giant cell with indented nucleus, surrounded by smaller cells. Fig. D. Series D: 25 °C. Multinucleate giant cell showing budding, surrounded by smaller epithelioid cells. Fig. E. Series D: 20 °C. Multinucleate giant cell with nuclei in horseshoe pattern. Fig. F. Series D: 30 °C. Multinucleate giant cell with nuclei in 'doughnut' pattern. Fig. G. Series E: 25 °C. Anchoring sheets of cells. Fig. H. Series E: 30 °C. Branching threads of anchoring cells, overlying the monolayer seen faintly in the background; h, edge of heart. 154 E. M. STEPHENSON Cells of fibroblastic morphology were absent or very sparse. It is possible that an age factor is involved (Shah, 1964), but no increase in fibroblasts was noted in cultures from the oldest tadpoles used in the present experiments. Scattered pigment cells, presumably derived from aggregations covering the truncus, were usually found. In early stages of most cultures, migrating leu- cocytes were obvious but it was not usually possible to trace their subsequent fates.

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0123456789 Days in culture Text-fig. 4. Series E. Diagram showing relative decrease in bulk area of explant and increase in area of outgrowth of hearts at four different temperatures. The comparison is based on mean weights in grams of drawings on standard paper (10cm2 = 0*045 g) at x 32 magnification.

It is not known whether the epithelioid cells all had the same origin or whether they were initially heterogeneous. The difficulty of distinguishing endocardial from mesenchymal cells in chick heart cultures has already been pointed out (Lewis, 1926; Nishibe, 1929). No pulsations of epithelioid cells as seen by Lewis (1926) were noted. It is possible that some of the anchoring sheets or threads (Plate 1, fig. E; Plate 2, figs.G , H) linking the explants with the epithelioid sheets may have included muscle cells, but this has not so far been convincingly demonstrated. These anchoring structures, though sufficiently strong and flexible to withstand the strain imposed by vigorous heart pulsations, were not observed to have individual rhythms. Their cells, which were frequently seen in Tadpole hearts in vitro 155 division, graded imperceptibly into the epithelioid outgrowth and successively formed part of it as migration continued. Particularly noticeable among the epithelioid cells on the peripheral ring of the clot but also occurring in pockets in the central clot-free region were giant cells of a specific type. Their general morphology was epithelioid but they were larger in size and their nuclear condition varied from mononucleate (Plate 2, fig. C) to multinucleate. Characteristic nuclear arrangements included horseshoe formations (Plate 2, fig. E) and 'doughnut' patterns (Plate 2, fig. F), as well as examples of budding and fragmentation (Plate 2, fig. D). Cells of this type are common also in cultures of newt pulmonary epithelia (Danes, 1949) and tadpole kidney epithelia (Stephenson & Walton, unpublished). A second type of giant cell, which is apparently formed from coalescence of mononucleate cells (Lewis, 1927; Weiss & Fawcett, 1953 and Robinson & Stephenson, 1966), was comparatively rare in tadpole heart cultures. It took the form of a syncytial mass of cytoplasm containing variable numbers of small, round to oval nuclei.

(5) Transfers from one temperature to another (a) Transfers from 37 °C Six hearts were cultured at 37 °C for 6 days, after which time three hearts were still beating. All hearts were then transferred to 30 °C for 3 days and were then fixed. No outgrowth of cells occurred and only one heart was beating immediately before fixation. One of the hearts was embedded, sectioned and stained, and its tissues showed clear evidence of degeneration. Three hearts were cultured at 37 °C for 4 days. All were then beating but showed no outgrowth. After transfer to 30 °C for 3 days, all hearts were still beating and all had produced at least some outgrowth (Plate 1, fig. F). Apparently, tadpole heart tissue of Limnodynastes peroni may remain viable for at least several days at 37 °C but eventually reaches a moribund condition where not even transfer to an optimal temperature will activate cell proliferation. (b) Transfers from 5 °C Six hearts were cultured at 5 °C for 7 days. All continued beating but showed no outgrowth. After transfer to 30 °C, outgrowth began in all six cultures within 24 h, while in the same period the mean rate of heart beat per minute rose from 16 to 32. Cultures were fixed after 3 days. One, which was treated with colchicine and hypotonic saline, though showing only a limited area of outgrowth, showed thirty-five sets of chromosomes around the rim of the clot. Six hearts were maintained at 5 °C for 4 days and then transferred to 25 °C for 2 days. At 25 °C cell outgrowth began in four cultures, which were then returned to 5 °C. The migrated cells remained in an expanded condition during 7 days at this low temperature but further outgrowth did not occur. After transfer to 30 °C, migration began again and continued for 3 days before fixation (Plate 1, fig. D). 156 E. M. STEPHENSON Two hearts were maintained at 5 °C and subcultured normally for 2 weeks. Subculturing then became very irregular, sometimes at intervals of over a fortnight. After 30 days, they were transferred to 25 °C for 3 days and slight migration occurred in one culture. After return to 5 °C, the migrated cells of this culture remained in an expanded condition for approximately a week but gradually rounded up and appeared to degenerate. The second heart stopped beating after 44 days and was fixed. The first continued to beat regularly for 106 days. It was then transferred to 30 °C where it continued to beat and produced a further very slight amount of epithelioid outgrowth within the 8 days before it was fixed.

DISCUSSION By the use of entire hearts with readily countable beats it has been possible to obtain a comprehensive picture of temperature effects on whole organ function and maintenance as well as on cell outgrowth. While the optimal temperature is the same for heart beat as for cell outgrowth, the latter has a generally more restricted range and does not occur at all at 37 and 5 °C. It is clear that 37 °C must be regarded as an ultimately lethal temperature for tissues of Limnodynastes peroni. However, results from the present study, which indicate variable viability of up to 6 days at this temperature, are intermediate between those of Drew (1913) who found 12 h exposure at 37 °C lethal to tissues of Rana temporaria, and N. G. Stephenson & Tomkins (1964) who obtained actual growth of cartilage of Pseudophryne bibroni on chick chorioallantoic membranes at 38 °C. As the genera, tissues and experimental conditions differed in all three cases, questions of species specificity and tissue specificity in relation to maximum temperature tolerance remain unanswered. Of the temperatures selected for testing, the optimum for rate of beat and for cell outgrowth of heart cultures of L. peroni has clearly been 30 °C. This is not necessarily the optimal temperature for organ viability for, other environmental factors being satisfactory, total maintenance of beat of hearts in vitro can be achieved throughout the range of at least 30-5 °C. However, considering organ function and cell outgrowth together, 30 °C obviously produced the best results. This agrees with Paul's observation (1960) that the optimal temperature for cell function is only slightly below that which is maximal. In experiments using whole kidneys and chopped kidney explants of L. peroni (Stephenson & Walton, unpublished), 30 °C again appears to be optimal for cell outgrowth. If the results for this species are found to be generally applicable to amphibian tissues, they probably help to explain the comparative slowness of growth found in many cultures of amphibian tissues maintained at relatively low temperatures by early workers. The medium used throughout contained no homologous fluids but was a combination of a synthetic medium with natural avian fluids and extracts. Except for a reduction in the total quantity of embryo extract it was identical Tadpole hearts in vitro 157 with that used for avian and mammalian fibroblasts (Abercrombie, Lamont & Stephenson, unpublished) and was also identical with that used for reptilian, avian and mammalian epithelia (author, unpublished). The fact that such a medium has been used successfully for amphibian tissues is in agreement with the results of Wolf, Quimby, Pyle & Dexter (1960) and with Paul's predictions (1960) but does not appear to supportShah's contention (1964) that the provision of media resembling amphibian body fluids is a necessity. Of particular interest is the reaction to temperature of migrating cells and of those still retained within the heart. At 5 °C, regular pulsations occur indefinitely and heart muscle cells in situ are undoubtedly active. Yet no migration of any cell types occurs from the heart at this temperature and cells which have migrated at higher temperatures, if transferred to 5 °C, do not remain expanded for long and gradually degenerate. These are additional illustrations of differential responses of cells to temperature emphasized by N. G. Stephenson & Tomkins (1964) and further illustrated by experiments with reptilian cells (N. G. Stephenson, 1966). SUMMARY 1. Whole hearts of tadpoles ofLimnodynastes peroniv/Qie cultured successfully in a combined avian-synthetic medium identical with that used for mammalian, avian and reptilian tissues. 2. The extirpated hearts were cultured from 37 to 5 °C. Temperature effects on organ viability and function and on cell outgrowth were studied comparatively. 3. Although viability could be maintained for up to 6 days at 37 °C, this temperature was ultimately lethal. From 30 to 5 °C inclusive total viability or maintenance was the general rule. 4. Rate of heart beat was at first accelerated at 37 °C but dropped as moribund conditions developed. For the other temperatures used, though absolute values varied considerably, a more or less constant relative pattern emerged, with the optimal rate at 30 °C and successive decreases occurring from 25 to 5 °C. 5. Cell migration did not occur at 37 or 5 °C. The optimal temperature for outgrowth was 30 °C, while progressively slower migration occurred from 25 to 15 °C. At 8 °C, outgrowth was confined to a few cells in a limited number of cultures. 6. Although these results apply at present to only one species, the establish- ment of 30 °C as an optimal incubation temperature may be important in view of the fact that almost all previous amphibian cultures have been grown at temperatures not exceeding 26 °C and often considerably lower.

RESUME 1. Des coeurs entiers de retards de Lymnodynastes peroni ont ete cultives avec succes sur un milieu semi-synthetique identique a celui qui est utilise pour la culture de tissus de mammiferes, d'oiseaux et de reptiles. 158 E. M. STEPHENSON 2. Les coeurs explantes sont cultives a des temperatures variees, de 37 a 5 °C. Les effets de la temperature sur la survie et le fonctionnement de l'organe ainsi que sur la migration cellulaire font l'objet d'une etude comparative. 3. La temperature de 37 °C provoque la mort de l'explant, la survie ne depassant pas 6 jours. En regie generate les explants survivent bien aux temperatures comprises entre 30 et 5 °C inclusivement. 4. Le rythme des battements est tout d'abord accelere a 37 °C, mais baisse lorsque la culture periclite. Aux autres temperatures utilisees, les valeurs absolues du rythme varient considerablement d'un cas a l'autre. Neanmoins une relation plus ou moins constante peut etre mise en evidence, la temperature optimum etant 30 °C et une diminution progressive du rythme s'observant de 25 a 5 °C. 5. La migration cellulaire ne se produit ni a 37 ni a 5 °C. La temperature optimum est 30 °C tandis que la migration diminue progressivement de 25 a 15 °C. A la temperature de 8 °C la migration ne s'observe que dans un nombre limite de cultures et ne concerne que quelques cellules. 6. Bien que ces resultats ne concernent actuellement qu'une seule espece, le fait que la temperature de 30 °C soit la meilleure pour l'incubation des explants peut etre important etant donne que la plupart des cultures de tissus d'amphibiens ont ete faites jusqu'ici a des temperatures n'excedant pas 26 °C et etant souvent considerablement plus basses. I thank my husband, Dr N. G. Stephenson, for his criticism and encouragement and for providing the tadpoles used in this work. I am deeply grateful to Miss Shirley Walton, B.Sc, for technical assistance. Incubator space was very kindly made available by Dr E. Shipp of the Department of Entomology and Mr G. Barbour of the Department of Microbiology. A number of items of equipment used in this project were provided by the New South Wales State Cancer Council.

REFERENCES DANES, B. (1949). Pulmonary epithelium of the newt, Triturus viridescens, studied in living cultures with cinematographic apparatus and phase contrast technique. /. exp. Zool. 112, 417^8. DREW, G. H. (1913). On the culture in vitro of some tissues of the adult frog. /. Path. Bact. 17, 581-92. JOHNSON, J. C. (1915). The cultivation of tissues from amphibians. Univ. Calif. Publs Zool. 16, 55-62. LEWIS, W. H. (1926). The cultivation of embryonic heart muscle. Carnegie Inst. Wash. Contr. Embryol. 18, 1-21. LEWIS, W. H. (1927). The formation of giant cells in tissue cultures and their similarity to those in tuberculous lesions. Am. Rev. Tuberc. pulm. Dis. 15, 616-28. MITCHELL, P. H. (1956). A Textbook of General Physiology, 5th ed. New York: McGraw-Hill. MOROSOW, B. D. (1929). Explantationsversuche mit getrockneten Amphibienherzen. Arch. exp. Zellforsch. 7, 213-20. NISHIBE, M. (1929). Growth of endocardial cells from the chick embryo heart in vitro. Arch. exp. Zellforsch. 7, 333^3. PAUL, J. (1960). Cell and Tissue Culture, 2nd ed. Edinburgh and London: Livingstone. PHILLIS, E. (1958). A study of the structure and pattern of the lateral line organs in some Australian anurans. University of Sydney B.Sc. Honours Thesis. Tadpole hearts in vitro 159 ROBINSON, E. S. & STEPHENSON, E. M. (1967). A karyological study of cultured cells of Limnodynastes peroni (Anura: Leptodactylidae). (In the Press.) SHAH, V. C. (1964). Comparative growth characteristics and nuclear morphology of amphibian tissues cultured in vitro. Cellule 64, 381-96. STEPHENSON, N. G. (1966). Effects of temperature on reptilian and other cells. (In the Press.) STEPHENSON, N. G. & TOMKINS, J. K. N. (1964). Transplantation of embryonic cartilage and bone to the chorioallantois of the chick. /. Embryol. exp. Morph. 12, 825-39. STOHR, P. (1924). Ober Explanation embryonaler Amphibienherzen. Wilhelm Roux Arch. EnhvMech. Org. 102, 426-51. WEISS, L. P. & FAWCETT, D. W. (1953). Cytochemical observations on chicken monocytes, macrophages and giant cells in tissue culture. /. Histochem. Cytochem. 1, 47-65. WOLF, K., QUIMBY, M. C, PYLE, E. A. & DEXTER, R. P. (1960). Preparation of monolayer cell cultures from tissues of some lower vertebrates. Science, N.Y. 132,1890-1.

(Manuscript received 8 March 1966, revised 25 July 1966)