The Living Cell in vitro as shown by Darkground Illumination and the changes induced in such Cells by fixing Reagents.

Transcription

1 The Living Cell in vitro as shown by Darkground Illumination and the changes induced in such Cells by fixing Reagents. By T. S. P. Strangeways * and R. G. Canti. (From the Laboratories of the Research Hospital, Cambridge.) With Plates 1-5 and 2 Text-figures. INTRODUCTION. TISSUE growing upon a coverslip in vitro provides peculiarly favourable material for a microscopical investigation of the structure and behaviour of the living cell. Some of the changes which take place in the living cell during growth and division, as seen by direct illumination, have already been described by one of the writers (Strangeways, 1922). By employing dark-ground illumination, however, a much more intimate study of the cell growing in vitro can be made than is possible with the aid of the usual direct methed, and with this technique various internal structures appear as sharply denned, conspicuous objects which by the ordinary method are so indistinct as to be hardly visible. Dark-ground illumination, which has seldom been used for the examination of tissue cultures, has been employed by W. H. Lewis (1923), who records some interesting results obtained from a study of the living cell kept at room temperature. The present writers, by maintaining the cultures at body temperature throughout the period of examination, have been able to extend Lewis's observations, and by the development of a special technique are also able to give some 1 We regret to have to record the death of T. S. P. Strangeways, which took place since this paper was received for publication. NO. 281 B

2 1 T. S. P. STRANGEWAYS AND R. G. CANTI account of the changes wrought in the individual living cell by the application of some of the more common fixing reagents. The writers are indebted to Dr. H. Fell for the beautiful drawings which illustrate the paper, and to the Medical Eesearch Council for a grant towards the expenses connected with this study. TECHNIQUE. Cultures of the choroid and sclerotic, heart, kidney, intestine, and skin of the embryonic fowl.were employed, but in order to render the results comparable, examples of cells from the choroid and sclerotic only, were selected for illustrations in the plates. The tissues were cultivated in plasma and saline, plasma and embryo extract, serum and saline or serum and embryo extract. A tissue fragment was placed in a small drop of medium in the centre of a No. 1 coverslip. Instead of inverting the coversquare over a hollow-ground slide it was inverted over a No. 2, 2 by 1 coverslip, and allowed to rest on two thin, narrow bands of sterile paraffin wax which had been painted along the two long borders of the large coverslip. In those preparations which were required for prolonged observations the coverslip was completely sealed down on to the underlying glass and incubated as usual. Cultures intended for the study of fixation effects were put up somewhat differently. In such cases the two outer borders of the small coversquare were sealed to the larger coverslip with melted paramn wax, whilst the two remaining sides were left open. By means of a pipette the narrow space between the coverslip was then exactly filled with either embryo extract or saline. Finally, a semicircle of melted paramn wax was painted on the surface of the larger coverslip at one end, and at the same time a narrow band of wax was painted on the surface of the small coversquare along the border in such a way as to connect the two ends of the semicircle on the larger glass (Text-fig. 1). By this means the watery film between the two coverglasses was sufficiently thin to allow of critical illumina-

3 LIVING CELL IN VITRO 3 tion of the cells and the total thickness of the preparation was well within the limits required by the condenser. The cultures were placed in trays in an air-tight glass vessel containing a small quantity of distilled water, and the vessel with the enclosed cultures was incubated at 38 C. A moist chamber was thus obtained and evaporation prevented. It was found that cultures treated in this way grew readily and, with the more favourable of the media employed, showed abundant mitosis. The microscope on which the cultures were observed was contained in a special biological thermostat-apparatus kept at TEXT-PIG C. The examination of tissue cultures with dark-ground illumination requires great care and accuracy of manipulation, and particular attention must be paid to the centring of the objectives and the adjustment of the source of light. A full description of the technique and apparatus used by the writers has been published elsewhere (Strangeways and Canti, 1926). In carrying out these observations the authors found it necessary to be very careful that the culture was not subjected to pressure by the objective, as the least compression brings about peculiar and abnormal changes in the structure of the cells. The action of fixing reagents on the cells was investigated as follows. The cell selected for study was carefully drawn in the living condition with the aid of an Abbe camera lucida. A few drops of the fixing fluids were then placed in the semicircle of wax on the larger coverslip, the fluid being prevented from spreading over the outer surface of the small coverslip by the narroav band of wax described above. By means of a small strip of filter paper applied to the opposite end of the B2 I

4 4 T. S. P. STEANGEWAYS AND R. G. CANTI small coversquare the fixing fluid was easily and rapidly drawn across the preparation. The cell was kept under observation during the entire procedure, and fixation in the great majority of cases was seen to take place instantaneously. The results of slow or imperfect penetration were disregarded. After an interval (2-20 minutes after fixation) the cell was again drawn to the same scale with the aid of the camera lucida and the changes produced by the fixative noted. THE STRUCTURE AND BEHAVIOUR OF THE LIVING CELL. The outline and internal structure of the vegetative cell are very clearly shown by the technique described above (figs. \a- 15a,Pl. 1-5). When growing upon the surface of the coverslip the cell is greatly flattened, has an irregular feather-like shape, and shows no true cell-wall, the outline being apparently caused by the reflection from the interface between the cytoplasm and the surrounding medium. This outline is unceasingly changing if the culture is being observed in the warm incubator, and the whole cell wanders over the svirface of the coverslip by slow amoeboid movement. The cytoplasm is clear, transparent, and homogeneous up to the limits of resolution of high-power objectives. The most conspicuous bodies which it contains are small, highly retractile, spherical droplets of fat which are more or less numerous in practically all cells cultivated in vitro. The droplets move about the cell either singly or in groups. This motion is purely passive and appears to be due to the amoeboid movement of the cell. The fat-globules vary much in size in different cells, but are largs and more numerous in the cells of an old or badly growing culture. A variable number of very small granules are also present in the cytoplasm, and are distinguishable from the small fatglobules by their lower refractive index. They show active irregular movement: some dart backwards and forwards in the cytoplasm and then come suddenly to rest, others move slowly to and fro. The rapidity of movement of the granules varies ]

5 LIVING CELL IN VITRO 0 considerably. It is largely dependent upon the temperature at which the culture is growing, and does not occur at all in the cold. Its cause is uncertain, but it is probably due, in part at least, to the cytoplasmic currents. The mitochondria are very clearly seen as fine threads which TEXT-FIG. 2. Mitoohondrium in a cell process drawn by the aid of the camera lucida over a period of approximately five minutes. vary greatly in length. They show a slow writhing snake-like motion most fascinating to watch, and move backwards and forwards in the cytoplasm. Sometimes a mitochondrium may be seen to break across (Text-fig. 2), or two will fuse end to end forming a single long filament. The mitochondria are usually more numerous in the advancing processes of the cell; in many cells the majority are situated on one side of the nucleus in the region of the centrosphere. If the culture is for some reason \

6 6 T. S. P. STRANGEWAYS AND E. G. CANTI not growing well the mitochondria are greatly thickened, and may appear as oval or club-shaped bodies which tend to break up into fragments. The centrosphere is seen as a distinct area lying as a cap over one side or end of the nucleus and contains a number of faint granules and filaments. The mitochondria appear to be formed in this region and can be seen wandering out from it into the clear cytoplasm. No trace of any structure which resembles Golgi apparatus can be seen. If pigment-rods are present in the cell they are clearly seen as short rods which are bright and refractile. They often show rapid darting movements. The nucleus, which appears as a clear oval or rounded body with a sharp outline, shows no nuclear wall, the limiting surface being apparently the interface between the cytoplasm and the nucleoplasm. The nucleoli, which are embedded in the clear, viscid nucleoplasm, are usually two in number, and are seen as distinct, slightly opaque, delicate-looking bodies of irregular contour ; they are continually changing in size, shape, and position. A cell undergoing mitosis is not well shown by dark-ground illumination, as it assumes a more or less spherical shape, and thus, having greater depth, is less suitable for critical study by this method than is the flat vegetative cell. The fat-globules, some of the small granules, and a number of mitochondria can be seen lying around a clear, fusiform area of protoplasm which represents the spindle (fig. 14 a, PI. 4). The chromosomes can be faintly distinguished in the clear, spindle-shaped region. The most careful scrutiny failed to show any sign of fibres in the spindle of the unfixed cell. The characteristic foaming movements of the cytoplasm, which have been described elsewhere (Strangeways, 1922), are clearly visible. THE EFFECT OF FIXING KEAGENTS UPON THE LIVING CELL. The action of the following fixing reagents was investigated : osmic acid 2 per cent., chromic acid 0-5 per cent., Plemming's solution with and without acetic acid (strong formula), Flem- (

7 LIVING CEIiL IN VITRO 7 ming's chromo-acetic acid, Champy's fluid, Mann's fluid, bichromate of potash 2 per cent., bichloride of mercury (saturated solution), Gilson's mixture, Zenker's mixture, acetic acid 5 per cent., picric acid (saturated solution), absolute alcohol, acetone, neutral formalin 4 per cent., and Bouin's fluid. Certain general effects upon the individual living cell are common to all fixing reagents (figs. 1 b-15 b, PL 1-5), although each type of fixative gives various characteristic reactions. The principal change observed in a cell over which the fixing fluid has flowed rapidly is a precipitation of fine granules in the cytoplasm and nucleoplasm. The precipitation in the cytoplasm takes place almost instantaneously throughout the cell, but the amount of precipitate formed and the size of the individual particles varies in different cells and with different reagents. With a few fixing fluids, as for example with chromic acid, absolute alcohol, and acetone (fig. 2, PI. 1, and figs. 10 and 11, PI. 4), a dense precipitate appears throughout, but with the majority of the fixatives tested, the precipitation is more pronounced in the neighbourhood of the. nucleus and centrosphere, becoming fainter towards the margin of the cytoplasm. This localization of the precipitate is especially marked after tho application of 2 per cent, osmic acid (fig. 1, PI. 1), a saturated solution of picric acid (fig. 9, PI. 3), or 10 per cent, neutral formalin (fig. 12, PL 4). If the fixing reagent does not reach the cell quickly, owing to a slow flow beneath the coverslip or to the presence of dense plasma, the precipitation is markedly delayed and often much less pronounced than in a properly fixed cell. All the fixatives used caused a certain amount of shrinkage of the cell, but some reagents, e.g. chromic acid (fig. 2, PL 1), absolute alcohol (fig. 10, PL 4), and acetone (fig. 11, PI. 4), produce more shrinkage than others. Osmic acid 2 per cent. (fig. 1, PL 1) is the only reagent among those tested which is found to produce practically no contraction. In the writers' investigations no attempt was made to determine the degree of shrinkage caused by prolonged contact with the fixing'fluid. The shrinkage described in the present paper and shown in the drawings I

8 8 T. S. P. STRANGEWAYS AND R. G. CANTI is that which takes place after contact with the fixing solution for 2-10 minutes, although observations for longer periods of minutes show little further contraction. It is probable that shrinkage is often less pronounced in cells growing upon a coverslip in vitro than in ordinary tissues, since such cells are flattened with their upper surface more or less adherent to the glass. Comparatively little distortion of the main contour of the cell is observed, but the delicate processes sometimes show considerable change, being either withdrawn owing to contraction of the cell or broken up and transformed into detached globules (figs. 8 and 9, PI. 3). The general effect of fixation, therefore, is to give an artificially smooth outline to the cell. Osmic acid (fig. 1, PI. 1) alone of the reagents used preserves the cytoplasmic processes almost perfectly. The effect of various fixatives upon fat is very readily studied by the present method. Many fixatives, among which may be mentioned Zenker's fluid (fig. 7, PL 3), Gilson's fluid (fig. 6, PI. 3), corrosive sublimate (fig. 5, PI. 2), and saturated solution of picric acid (fig. 9, PI. 3), cause the droplets lying in groups to fuse with one another, forming large, often irregularly shaped, refractile globules which in size and arrangement bear no resemblance to the original globules in the living cell. Pat solvents such as acetone (fig. 11, PI. 4) and absolute alcohol (fig. 10, PL 4) cause first a fusion and then a complete solution of the fat-globules, which are represented by dark non-refractile areas. Osmic acid (fig. 1, PL 1) and solutions containing osmic acid (fig. 3, PL 2, and fig. 15, PL 5) cause little or no alteration in the shape and distribution of the fat-droplets, which gradually assume a brownish tinge owing to osmication. The behaviour of the mitochondria during fixation is often difficult to observe, as these bodies tend to become obscured by the precipitate forming in the cytoplasm. Broadly speaking, however, the present writers' results confirm what is already known of the fixation of the mitochondria. Osmic acid (fig. 1, PL 1) and Flemming's solution without acetic acid (fig. 3, PL 2) preserve the mitochondria almost perfectly. Neutral formalin

9 LIVING CELL IN VITRO 9 4 per cent. (fig. 12, PI. 1) also fixes them well but tends to cause granulation, giving the filaments a somewhat moniliform appearance. Other reagents, notably those containing acetic acid, produce considerable distortion and even complete destruction of the mitochondria. With potassium bichromate (fig. 4, PI. 2) the mitochondria round up into small, fairly refractile globules, whilst after Bouin's fluid (fig. 13, PI. 4), Gilson's mixture (fig. 6, PI. 3), saturated solution of picric acid (fig. 9, PI. 3), acetic acid (fig. 8, PI. 3), and others, the mitochondria, where not destroyed or obscured by the precipitate, are sometimes still distinguishable as very faint, granular threads. The fine, rapidly moving granules in the cytoplasm described above are usually quite obscured by the precipitate formed in the cytoplasm, and their fate is therefore almost impossible to follow. They have been observed, however, in cells fixed by 2 per cent, osmic acid and 2 per cent, potassium bichromate. The most obvious effect of fixation upon the nucleus is the instantaneous precipitation of the nucleoplasm. As described in the case of the cytoplasm the character of the precipitate formed depends upon the reagent employed. Osmic acid (fig. 1, PL 1), Flemming's (fig. 3, PI. 2, and fig. 15, PI. 5), Mann's and Ohampy's solutions, produce a fine precipitate, but with the other fixatives employed comparatively coarse granules are formed. The outline of the nucleus is usually more distinct after fixation, and the nucleus appears to be enclosed by a definite refractile membrane. After absolute alcohol (fig. 10, PI. 4), acetone (fig. 11, PI. 4), neutral formalin 4 per cent. (fig. 12, PI. 1), and Gilson's mixture (fig. 6, PI. 3), on the other hand, the outline of the nucleus is difficult to distinguish. Fixation causes very little distortion of the nucleus, but almost always results in a slight amount of shrinkage. The nucleoli are practically always more distinct in the fixed than in the living cell. A certain amount of shrinkage is always noted, and distortion also occurs to some extent. Occasionally the nucleolus appears to divide into two when fixed (fig. 13, PI. 4), but whether this represents an actual splitting or merely the separation of two superimposed or adjacent bodies it is

10 10 T. 3. P. STRANGEWAYS AND R. G. CANTI impossible to say. In this connexion it may be remarked that true division of nucleoli undoubtedly occurs in the living cell, and has been described elsewhere by one of the writers (Strangeways, 1922). Interesting changes are seen to take place in the spindle of mitotic cells during fixation. As described above, no trace of fibres can be demonstrated in the spindle of the living cell under dark-ground illumination. On fixation with certain reagents, such as Flemming's chromo-acetic acid (fig. 14, PI. 4), Bouin's solution, chromic acid, and Zenker's solution, well-defined spindle-fibres suddenly appear in the spindle, which immediately becomes very distinct and conspicuous; the chromosomes are also seen more clearly. Other fixatives, e.g. osmic acid and strong Flemming's solution without acetic acid, do not show this effect nearly so markedly, and the fibres are sometimes barely seen in the cell fixed by such reagents. This formation of spindle-fibres by coagulation has also been described by LeAvis and Eobertson (1916) and M. E. Lewis (1923). An interesting observation was made upon the effect of strong light upon cells which have been fixed by a reagent containing chromic acid. If the light of the dark-ground condenser is allowed to play upon a cell fixed by such a reagent it is found that the cells and, if these are growing in plasma, the surrounding medium also, rapidly disintegrates. The fibrin and colloidal particles of the medium around the cell begin to dissolve, and the hitherto motionless particles pass into violent Brownian movement. At the same time the outline of the cell becomes hazy (fig. 15 c, PI. 5). This haziness gradually extends towards the nucleus, the particles of precipitate throughout the cell show Brownian movement, and ultimately the entire cytoplasm and precipitate melts away, leaving the fat-globules and nucleus floating freely in the dissolved medium. The nucleus becomes blurred in outline, and after two or three minutes also passes into solution. The nucleoli are slightly more resistant, but they too quickly disappear (fig. 15 d, PI. 5). The same process of dissolution takes place if the cell is in mitosis. If the cell is in metaphase this effect is particularly interesting to watch. The

11 LIVING CELL IN VITRO 11 outline of the spherical cell becomes vague and the cytoplasm melts, leaving only the spindle with the chromosomes embedded in it. The chromosomes, hitherto not very clearly visible, are now distinct, and as the spindle dissolves they become more and more discrete until, with complete disappearance of the spindle, they are left floating freely in the now dissolved medium. The ultimate fate of the chromosomes is difficult to determine, as they move about rapidly, rising and sinking in the fluid and often floating out of the field ; eventually, however, they seem to dissolve like the rest of the cell. If the beam of light is filtered through 2 cm. of freshly prepared, slightly acid, saturated solution of ferrous sulphate, which eliminates the heat-rays, the same effect is shown, but is somewhat delayed, presumably owing to the loss of light occasioned by the filter. The action can also be considerably delayed by passing the light through coloured glass, e.g. a Wratten green filter. Cells outside the cone of light coming from the condenser remain unaffected until they are brought within the area of its action, when they likewise melt away. SUMMARY AND CONCLUSIONS. 1. A technique is described for the preparation of cultures suitable for the study by dark-ground illumination of the living cell and of the effects of fixation upon such a cell. 2. The structure and behaviour of the living cell and the various forms of motion exhibited by the different cell organs and inclusions were observed at 38 C. by dark-ground illumination. 3. Cells demonstrated by this method showed no cell-wall, nuclear membrane, or Golgi apparatus, and, in the case of dividing cells, no spindle-fibres. The nucleus, nucleoli, mitochondria, fat-globules, granules, and pigment-rods were admirably distinct. 4. The effects upon individual cells of various common fixing reagents were investigated. 5. In successful experiments fixation was in every case instantaneous, but where the penetration of the reagent was slow and imperfect the coagulation took place gradually.

12 12 T. S. P.. STRANGEWAYS AND E. G. CANTI 6. The principal changes induced by fixing reagents were : (1) the formation of more or less dense precipitate in nucleus and cytoplasm, (2) a variable degree of shrinkage of nucleus and cytoplasm, (3) distortion or destruction of the delicate cytoplasmic processes giving an artificially regular outline to the cell, (4) after certain fixatives the fusion of the adjacent fatglobules, (5) after most reagents the rounding up, granulation, or disappearance of the mitochondria, (7) the appearance of spindle-fibres in the dividing cell. 7. A study was made of the destructive effect of light upon cells and plasma which had been fixed with a reagent containing chromic acid. The entire cell, with the exception of the fatglobules, was completely dissolved and the fibrin disintegrated. 8. Of all the reagents tested it was found that osmic acid 2 per cent, produced the least change in the cell. BEFERENCES TO LITERATURE. 1. Lewis, M. R. (1923). " Reversible Gelation in living cells ", ' John Hopkins Hosp. Bull.', vol. 34, p Lewis, M. R., and Robertson, W. R. B. (1916). " The mitochondria and other structures observed by the tissue-culture method in the male germ-cells of Chorthippus curtipennis scudd ", ' Biol. Bull.', vol. 30, p Lewis, W. H. (1923). " Observations on cells in tissue culture with dark-field illumination ", ' Anat. Rec.', vol. 26, No. 1, p Strangeways, T. S. P. (1922). " Observations on the changes seen in living cells during growth and division ", ' Proc. Roy. Soc.', B, vol. 94, p Strangeways, T. S. P., and Canti, R. G. (1926). "Dark-ground illumination of tissue cells cultivated in vitro", ' Brit. Med. Journ.', July 24, p. 155.

13 LIVING CELL IN VITRO 13 DESCRIPTION OP PLATES. PLATE 1. Fig. 1. a, cell before, 6, after fixation with 2 per cent, osmic acid. Note in 6 the almost perfect preservation of the cell and its internal structures, and the relatively slight precipitation in the nucleus and cytoplasm. Fig. 2. a, cell before, 6, after fixation with 0-5 per cent, chromic acid. The fixed cell shows considerable shrinkage, heavy and coarse precipitation in nucleus and cytoplasm, and fusion of fat-globules. PLATE 2. Fig. 3. a, cell before, b, after fixation with strong Flemming's solution without acetic acid. The mitochondria and fat-globules are unaltered in 6 ; the outline of the cell is somewhat distorted. Fig. 4. a, cell before, 6, after fixation with 2 per cent, potassium bichromate. Note the partial fusion of the fat-globules in b, the rounding of the mitochondria (m), and shrinkage of the nucleoli. Fig. 5. a, cell before, 6, after fixation with bichloride of mercury. The small fat-globules seen in a have coalesced in 6 into large, irregular masses and the cytoplasm shows a coarse precipitate. PLATE 3. Fig. 6. a, cell before, 6, after fixation with Gilson's mixture. The fatglobules in the fixed cell show partial fusion, the mitochondria appear as faint granular threads, and the nuclear outline is almost invisible. Fig. 7. a, cell before, 6, after fixation with Zenker's fluid. The cytoplasm in 6 shows a coarse precipitate and many of the fat-globules have run together. Fig. 8. a, cell before, 6, after fixation with 5 per cent, acetic acid. The fixed cell shows considerable shrinkage ; the fine processes have contracted into rows of small globules ; the nucleolus is distorted ; the remains of the mitochondria are in places distinguishable as faint lines of granules. Fig. 9. a, cell before, 6, after fixation with a saturated solution of picric acid. Note the fusion of the fat-globules in 6, the moniliform mitochondria, and the contraction into globules of some of the cytoplasmic processes. PLATE 4. Fig. 10. a, cell before, b, after fixation by absolute alcohol. In b the fat is seen to have been dissolved after preliminary fusion, the precipitate is coarse, the mitochondria are not seen, and the cell shows considerable shrinkage. Fig. 11. a, cell before, b, after fixation with acetone. The fixed cell shows pronounced shrinkage, heavy precipitate in both nucleus and cytoplasm, solution of fat, faint nuclear outline, and shrinkage of nucleolus.

14 14 T. S. P. STRANGBWAYS AND R. G. OANTI Fig. 12. a, cell before, b, after fixation with 4 per cent, neutral formalin. Note in 6 the well-marked but slightly granular mitochondria and the somewhat fribillar appearance of the cytoplasm at one end of the cell. Fig. 13. a, cell before, b, after fixation with Bouin's fluid. The cell after fixation shows considerable shrinkage, faint indications of the mitochondria, and apparent splitting of the nucleolus. Fig. 14. Cell in metaphase a, before, and b, after fixation with Flemming's chromo-acetic acid. Compare the absence of spindle-fibres in a and their sharp definition in b. The chromosomes are more distinct in b. PLATE 5. Fig. 15. Series of drawings showing the effect of light upon a cell fixed with strong Flemming's solution with acetic acid. This effect is due to the presence of chromic acid in the fixing fluid. The light was filtered through 2 cm. of freshly prepared saturated solution of ferrous sulphate made slightly acid, a, cell before fixation; 6, immediately after ; c, after 45 minutes ; d, 2 hours later. This cell was growing closely applied to the coverslip, to which most of the fat-globules have adhered after the complete solution of the rest of the cell. N.B. It will be noted that there are small differences in the disposition of the mitochondria and fat-droplets shown in the drawings of the living and fixed cells respectively. These differences are of no importance and are due to the movements of the cell contents which occurred between the time of drawing the living cell and the application of the fixative.