the vital staining of normal and malignant cells. i.-vital...

288 It. J. Ludford.

the help of the Government Grants Committee of the Royal Society in purchasing some of the apparatus used in this investigation.

LITERATURE.

(1) Atkins, W. R. G. 4 Some Recent Researches in Plant Physiology,’ London, 1916,pp. 131-2.

(2) Bayliss, W. M. c Principles of General Physiology,’ London, chaps. V, VI, VII,1920.

(3) Blackman, V. H., and Paine, S. G. “ Studies in the Permeability of Mimosa pudica,”4 Ann. of Bot.,’ vol. 32, 1918.

(4) Bose, J. C. 4 Electro-Physiology,’ London, 1906.(5) Bose, J. C., and Das, S. C. 44 On Petiole-Pulvinus Preparation of Mimosa pudica,”

4 Roy, Soc. Proc.,’ B, vol. 89, 1916.(6) Bose, J. C., and Sen Gupta, N. N. 4 Transactions Bose Research Institute,’ vol. 1,

1918.(7) Sen, B. 44 On Relation between Permeability Variation and Plant Movements,”

4 Roy. Soc. Proc.,’ B, vol. 94, 1923.(8) Stiles, W. 4 Permeability,’ London, 1924.(9) Stiles, W., and Jorgensen, I. 44 The Effect of Temperature on the Permeability of

Plant Cells to Hydrogen Ions,” 4 Ann. of Bot.,’ vol. 29, 1915.

The Vital Staining of Normal and Malignant Cells. I .—Staining with Trypan Blue, and the Cytoplasmic Inclusions of Liver and Kidney Cells.

By R. J. Lu d fo r d , Foulerton Student of the Royal Society, and Honorary Lecturer in Cytology, University College, London.

(Communicated by J. A. Murray, F.R.S.—Received June 13, 1928.)

(From the Laboratories of the Imperial Cancer Research Fund.)

1. Introduction.Although intra-vitam staining has now become a routine method in histology,

the application of this technique to the special problems of the cell is comparatively recent. Vital staining has been fully discussed by von Mollendorff (1920, ’21, ’23, ’26). Full references to the literature, and the main results of vital staining, are to be found in his valuable reviews, in which he lays down the fundamental principles of the subject.

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Vital Staining o f Normal and Malignant Cells. 289

2. Principles of Vital Staining.There are two chief methods of vital staining. Either the dye can be injected

into the body of the living animal—intra-vitam staining ; or fragments of living tissue can be removed from the animal and immersed in dilute solutions of the dye—supra-vital staining. With basic dyes the latter method is usually employed owing to the difficulty of retaining the dye in fixed preparations. Basic dyes are also used intra vitam, but most are more toxic than acid dyes. Whichever method is employed, with basic dyes, in general, there is an actual staining of pre-formed structures. Most investigators are in agreement that it is only non-living granules, or droplets, within the cytoplasm that are stained with basic dyes.

Vital staining with acid dyes is usually carried out by injecting a dilute solution of a relatively non-toxic dye into the living animal, either subcutaneously or intravenously, or else intraperitoneally. The rate of diffusibility of dyes determines to a great extent their efficacy for this purpose. Following injection of an appropriate stain, droplets make their appearance in certain of the cells. Those of the reticulo-endothelial system show special propensities for taking up the dye, with which their cytoplasm becomes rapidly filled. Many of the cells of the animal body have not been found to stain, yet the trend of recent research has been to extend the list of cells which do stain with acid dyes.

I t is generally agreed that an acid dye, such as trypan blue, which enters the living cell, does so in the form of a dilute colloidal solution, and then becomes collected into droplets in certain parts of the cell. This is how von Mollen- dorff (1921) describes the process :—•

“ Zu verschiedenen Zeiten der Farbstoffausbreitung innerhalb des Tierkorpers kann man verfolgen, wie innerhalb der Zellen die Granula an Zahl, Grosse und Farbdichte allmahlich zunehmen; dabei erhalt man den Eindruck, dass zuerst die Granula den Farbstoff in einer sich allmahlich konzentrierenden Losung enthalten. Sie sehen durchscheinend aus. Erst bei weiterer Farbstoffzuf uhr wird der Tropfeninhalt inhomogen, manchmal sind tanzende Kornchen innerhalb der Vakuolen zu beobachten, bis eine vollstandige Ausfallung des Farbstoffes in dem Tropfchen zu- standekommt, wodurch ein Granulum die maximale Farbstoffmenge aufgenommen hat ” (p. 118).

The essential features of vital staining with acid dyes are, therefore,(1) It is a purely physical process.(2) In the living cell there is no staining of pre-formed structures : the dye

droplets are new formations.



290 R. J. Ludford.

If one injects 1 c.c. of a 1 per cent, solution of trypan blue subcutaneously into a mouse, a considerable accumulation of dye can be seen in the cells of the convoluted tubules of the kidney twenty-four hours later. Comparison of a frozen section of such a kidney with mitochondrial and Golgi apparatus preparations will show—

(1) That there is no obviously direct relationship between the mitochondriaand the dye droplets ; .

(2) That there is remarkable similarity between the arrangement and position of the dye droplets, and the form and distribution of the Golgi apparatus.

Jasswoin (1925) was the first- to point out that trypan-blue droplets are formed in kidney cells in that area of the cytoplasm where the Golgi apparatus is situated. He showed that in cells of the convoluted tubules of Amphibians the position of the Golgi apparatus varies in different physiological conditions, and in each case there is complete agreement in the position of the apparatus and the dye droplets. By the methods Jasswoin employed he was not able to obtain either mitochondria or Golgi apparatus in the same preparation as the dye droplets, although in some of his Golgi apparatus preparations he found the latter were represented by brown granules.

I t has been established by recent research work that in gland cells the secretion granules or droplets originate in the cytoplasm in close relationship with the Golgi apparatus. To Nassonov is due the credit for having drawn attention to the essential similarity in the way in which secretion droplets arise in gland cells, and droplets of dye are formed in vital staining with acid dyes. Nassonov (1926) investigated specially the relationship between trypan-blue droplets and the Golgi apparatus in the kidney and liver cells of several vertebrates. He found complete agreement between the position and arrangement of dye droplets and of the Golgi apparatus in the early stages of intra-vitam staining. Furthermore, just as secretion granules of gland cells originate within, or in contact with, the Golgi apparatus, and later move away towards the outer wall of the cell, so do the trypan-blue droplets in the cells of the convoluted tubules. The same relationship between apparatus and dye was also found to hold in liver cells. These observations led Nassonov to attribute a definite physiological function to the Golgi apparatus. He says that—

“ In den normalen Zellen dieses oder jenes Secret im Plasma chemisch vorbereitet wird, wahrend sich die Tatigkeit des Golgi-Apparats auf die elektive Konzentration dieses Secrets und auf die Bildung von Granula oder Vacuolen beschrankt55 (p. 500).

3. Vital Staining with Acid Dyes and the Cell Organs.



I

Nassonov’s paper was published at the same time as a paper by Cramer and the present writer (1926) on the cellular mechanism of bile secretion. We drew attention in this communication to the relation between the Golgi apparatus of the liver cells and the intercellular bile capillaries. From the study of the Golgi apparatus of liver cells under various physiological conditions, we concluded that certain constituents of the bile were formed in relationship with the apparatus in the same way as secretion granules are in gland cells.

The same year a paper was published by Makarov (1926) on this subject. He found in a large number of vertebrates that the Golgi apparatus of the liver cells was arranged around the intercellular bile capillaries. Further, on injecting trypan blue into the animals, dye droplets made their appearance in the region of the Golgi apparatus.

The latest contribution to this problem has been made by Glasunow (1928). He injected trypan blue into guinea pigs over relatively long intervals of time. By this method he was able to stain cells which previously had been considered unstainable intra-vitally. He also found that after a short period of injecting the dye, it appeared in certain of the cells, such as those of the liver and kidney, in the form of granules. After longer periods of injecting, such granules anastomosed to form filaments, and finally assumed the typical reticulate form of the Golgi apparatus.

The position of this problem up to the present time is, therefore,

(1) Injection of trypan blue for relatively prolonged periods brings about a deposition of the dye in certain cells identical in appearance with the Golgi apparatus.

(2) Injection of trypan blue for shorter periods results in a deposition of the dye as droplets in definite areas of many cells. The general arrangement and distribution of such droplets are identical with the Golgi apparatus.

The next step in the solution of this problem would therefore seem to be to attempt to demonstrate both Golgi apparatus and dye droplets in the same preparation. The technique by which this can be accomplished, and the results obtained by it, are described in this paper.


4. Cytological Technique for Intra-Vitam Staining with Trypan Blue.

Following the advice of von Mollendorff, I have injected mice with 1 per cent, or 0-5 per cent, trypan blue. The 0*5 per cent, solution has been found to be more satisfactory than a 1 per cent, solution with experiments conducted over relatively long periods. In my latter experiments, half a cubic centimetre of a



292 R. J. Ludford.

0*5 per cent, solution of the dye was injected on alternate days over various lengths of time. After three consecutive injections, an interval of two days was allowed before the next injection. All injections were made subcutaneously. I t is desirable to inject as deeply as possible, and, as von Mollendorff has recommended, to employ gentle massage so as to spread the dye as much as possible under the skin.

The cells of the convoluted tubules of the kidney are deeply stained after one or two injections. Most of our observations have been carried out on the cells of the liver and kidney of mice which have received five or six injections over a period of about a fortnight. Tissues have been fixed from 6 to 24 hours after the last injection.

The cytological technique to be employed with tissues stained intra vitam depends upon the structure it is desired to demonstrate in addition to the dye droplets. Material can either be fixed in formalin and then cut frozen, or can be embedded in paraffin and sections cut in the usual way. Von Mollendorff recommends as the best fixatives with trypan-blue stained material, firstly, formalin, 5 to 10 per cent. ; secondly, concentrated sublimate solution. He also adds that the potassium bichromate method of Altmann’s for mitochondria can be used.

The technique we have employed is as follows :—

(i) For Dye Droplets and Nuclear Structures. Frozen Section.—Tissues have been fixed either in 40 per cent, formalin for two or three hours, or in 10 per cent, formalin in normal saline overnight. Sections cut with a C02 freezing microtome were stained with neutral red, as previously described for modified Kopsch sections.

Paraffin Section.—Material has been fixed in formol-sublimate (8-5 parts of saturated corrosive sublimate to 1 part of formol). I t is desirable to bring the fixed material as rapidly as possible through the alcohols. Neutral red has been employed for counter staining the sections.

(ii) For Dye Droplets and Mitochondria.—Frozen sections of tissues fixed in formol, and stained with Hollande’s iron carmine, have given good results with the liver and kidney. Sections have been stained a minute, or less, in the carmine solution, rinsed, then blackened with iron alum and rapidly differentiated. After washing, they have been dipped in pyridin, then washed in running tap water before mounting. In successful preparations the mitochondria are dark brown, and the trypan-blue droplets bluish black to pale blue.



(iii) For Dye Droplets and Golgi Apparatus.—Previous investigators have failed to obtain the Golgi apparatus and the dye in the same preparation. Both Jasswoin and Nassonov worked with the Kolatschev method. Nassonov points out that he based his conclusions upon a comparison of vitally stained preparations with sections demonstrating only the Golgi apparatus, “ weil alle meine Miihe die Vitalfarbung an impragnierten Praparaten zu erhalten, keine positive Resultate ergaben ” (p. 481).

Since, according to von Mollendorff, corrosive sublimate is one of the best fixatives for trypan-blue stained tissues, it seemed probable that by employing the modified Kopsch method I have used previously, successful preparations might be obtained. After having tried a number of variations of this technique, the following has given the most satisfactory results :—

Very small pieces of tissue were cut up with a safety-razor blade and fixed overnight in corrosive-osmic solution (equal parts of 6 per cent, corrosive sublimate and 2 per cent, osmic acid). After fixation the fragments were washed for one-half to one hour in distilled water, which was repeatedly changed. The material was then transferred to 2 per cent, osmic acid, and kept in an incubator at 35° C. for days. At the end of this time the osmic acid was poured off and the fragments of tissue were washed with distilled water at 35° C. The w^ater was continually changed until no smell of osmic acid remained ; then the material, still in distilled water, was put back in the incubator for another 24 hours. Finally, it was brought up through the alcohols during the course of seven or eight hours, left overnight in soft wax (45° C. melting point), and embedded in hard wax the next morning.

Sections were cut 3 (ji in thickness, and either mounted directly in balsam, or else counterstained lightly with neutral red. This method has given excellent results with the kidney. In the liver cells, however, there is a less intense intra-vital staining with trypan blue, and the dye droplets are not so clear. I t was, therefore, found convenient to counterstain the trypan-blue droplets with the basic dye, neutral red. This method has also been employed with sections of the kidney.


5. Relation of the Dye Droplets to the Cytoplasmic Structures in the Kidney.

The general arrangement of the dye droplets in cells of the convoluted tubules of the kidney, 24 hours after subcutaneous injection of 1 c.c. of 1 per cent, trypan blue, is shown in fig. 1. In the next figure is represented the arrangement of the Golgi apparatus (G) in the cells of a convoluted tubule of



21)4 li. J. Ludford.

F igs. 1-4.—Cells of the convoluted tubules of the mouse—all, except fig. 2, stained intra-vitally with trypan blue.

Fig. 1.—Typical arrangement of trypan-blue droplets (B) 24 hours after a subcutaneous injection of 1 c.c. of 1 per cent, trypan blue.

Fig. 2.—Golgi apparatus (G) in cells of a normal mouse.Fig. 3.—Mitochondria (M) and dye droplets (B).Fig. 4.—Golgi apparatus and dye droplets.

(Protocol of experiment of which figs. 3 and 4 represent the result: 1st day— 0-5 c.c. of 1 per cent, trypan blue injected. 4th day—0*5 c.c. of 1 per cent, trypan blue injected. 7th day—0*5 c.c. of 1 per cent, trypan blue injected. 8th day—tissues fixed.)



a normal mouse. These two figures illustrate the essential features of vital staining of such cells with trypan blue, namely :—

(i) That there is no diffuse staining of the living cell;

(ii) That the dye accumulates in the cell as droplets in a definite area of the cytoplasm ; and

(iii) That the general arrangement of the dye droplets coincides with that of the Golgi apparatus.

It was by comparison of such figures that Nassonov arrived at his conclusion as to the function of the Golgi apparatus. There have been workers who have attributed to the mitochondria a role in the staining process. Such a view has not been sustained by recent researches. Fig. 3 shows the mitochondria (M) and dye (B) in a frozen section stained by Hollande’s carmine method. The mitochondria (M) present the typical filamentous form of such cells. At the inner extremities of the long filaments, granular mitochondria occur. They appear to be mixed up with the dye droplets. I t is impossible to tell whether such granular mitochondria may take up the dye. One can, however, definitely conclude that there is no accumulation of the dye by the mitochondria, as a whole, in the living cell.

The intimate relationship existing between the dye and the Golgi apparatus is shown in fig. 4, which represents a section of a tubule from the same animal as fig. 3. Since by the method already described both dye and Golgi apparatus have been demonstrated in the same preparation, it has been possible to figure in detail the relationship between the two. This is shown in figs. 5 to 10, and these figures serve as a confirmation of Nassonov’s view. They show that the dye droplets (B) arise in association with the Golgi apparatus (G)> and as with secretion granules in gland cells, the formed droplets move out away from the apparatus into the cytoplasm. Figs. 5 to 10 are arranged as a progressive series to illustrate what may be regarded as successive stages in the formation and accumulation of dye droplets (B) within the cell.

I t has been noticed occasionally in examining sections that part of the apparatus has reduced the osmic acid and appears black, while other parts seemed to be stained blue. This is shown in figs. 6 and 7. Such an appearance is probably due to the dye accumulating at the surface of the apparatus and opposing a barrier to the penetration of the osmic acid.




296 R. J. Ludford.

F igs. 5-10.—Golgi apparatus (G) and dye droplets (B) in cells of convoluted tubules of the mouse kidney. These figures are arranged as a progressive series to illustrate what are regarded as successive stages in the deposition of the dye.

(Protocol of experiment—same as figs. 3 and 4.)

6. Relation of Dye Droplets to Cytoplasmic Structures in Liver Cells.

Essentially the same relationship has been found to exist between dye droplets and the cytoplasmic structures in parenchymatous cells of the liver, as in the cells of the tubules of the kidney. Fig. 11 shows vitally stained liver cells ; fig. 12 the normal form of the Golgi apparatus (G) in such cells ; and fig. 13 the intimate relationship between the dye (B) and the apparatus (G) when both are demonstrated in the same preparation. The absence of any clear relationship between the dye (B) and mitochondria (M) is illustrated in fig. 15.

I t has been clearly established that during mitosis the Golgi apparatus fragments, and becomes scattered in the cytoplasm. There is a similar dispersal of trypan-blue droplets (B) in liver cells during mitosis, as is seen in fig. 14.




C 11

13

F igs. 11-13.—Parenchymatous cells of the liver of the mouse.

Fig. 11.—Typical arrangement of dye droplets (B) after three injections of 0*5 c.c. of 1 per cent, trypan blue.

F ig. 12.—Peri-nuclear arrangement of the Golgi apparatus (G) in a normal mouse.

Fig. 13.—Golgi apparatus (G) and dye droplets (B), after three injections of 0*5 c.c. of 1 per cent, trypan blue.

To facilitate the study of the liver cells it has been found convenient to stain sections of modified Kopsch material with neutral red. The trypan- blue droplets are by this means counterstained red. Figs. 16 to 19 represent the result of the application of this technique. Figs. 16 and 17 are two cells cut at a plane at right angles to the longitudinal axes of two intercellular bile capillaries (C). The cells of fig. 18 are cut along the plane x—y



298 R. J. Ludford.

14

F igs. 14 and 15.—Parenchymatous cells of the liver of the mouse stained intra vitam with trypan blue.

Fig. 14.—Metaphase of mitosis showing scattered dye droplets (B), and chromosomes (N).

(Protocol of experiment—1st day, injected subcutaneously 0*5 c.c. of J per cent, trypan blue. The same repeated on 3rd, 5th, 8th and 10th days. The tissues fixed on the 12th day.)

Fig. 15.—Mitochondria (M) and dye droplets (B).(Protocol of experiment—Mouse received seven injections of 1 per cent, trypan

blue over a period of 27 days.)

of fig. 16, and are looked at from the direction m or n ; while the cell shown in fig. 19 has been sectioned along the plane m—n (fig. 16), and is looked at from the direction x or y. The Golgi apparatus (G) in all these cells, therefore, occupies a peri-nuclear position, and is directed




F igs. 16-19.—Golgi apparatus (G) and dye droplets (B) in parenchymatous cells of the liver of a mouse stained intra vitam with trypan blue. The dye droplets have been counterstained with neutral red in the sections. Figs. 16 and 17 are cells cut along a plane at right angles to two intercellular bile capillaries (C). Fig. 18 has been sectioned along the plane x —y of fig. 16, and is looked at from the direction m or n. Fig. 19 has been cut along the plane m—n of fig. 16, and is looked at from the direction x or y. (Protocol of experiment the same as for fig. 14.)

towards intercellular bile capillaries (C). The figures show that the dye accumulates as droplets (B) in relationship with the Golgi apparatus (G), preparatory to its excretion into the bile canaliculi.

7. Function of Golgi Apparatus and Mitochondria.The result of our observations are entirely confirmatory of the view of

Nassonov, that it is the function of the Golgi apparatus to bring about an elective concentration into droplets of the products of cellular activity. This



300 R. J. Ludford.

process is a physical one. What, then, is the function of mitochrondria ? This problem has been discussed recently by Horning and Petrie (1927). They refer to Cowdry’s (1926) surface-film theory of the function of mitochondria, and express the opinion that in the light of recent biochemical work the evidence is strongly in favour of enzymatic syntheses taking place at the mitochondria-cytoplasmic surface. Their own studies upon mitochrondria during the germination of cereals support this conception. In their opinion a starcli-splitting enzyme is located within, or at the surface of, the mitochondria of the cells of the scutellum-of the maize grain. A remarkable process is described by these authors. During germination, the mitochondria of the scutellum cells increase in numbers, and are “ secreted ” into the starch- containing endosperm cells, where they bring about the “ corrosion ” of the starch grains by liberation of the enzyme.

There is a great deal of evidence to show that increased cellular activity is expressed morphologically by an increase in the cytoplasmic-mitochondrial surface, and also in that of the surface of the Golgi apparatus. An explanation of this is afforded by a correlation of the new conception of the function of the Golgi apparatus with the idea of the enzymatic function of the mitochondria. At the mitochondrial-cytoplasmic surface synthesis by enzymes occur. The resulting products continually diffuse into the cytoplasm, preventing an accumulation at the surface of the mitochondria, which would inhibit further syntheses. At the surface of the Golgi apparatus the elaborated products are concentrated into droplets, as a preliminary to their elimination. Such an hypothesis is in accordance with our present knowledge of these matters, and affords a crude explanation of the functional inter-relationship of the cytoplasmic organs.

8. Summary.1. By means of the cytological technique described in this paper it is

possible to demonstrate in cells of the kidney and liver of animals stained intra-vitally with trypan blue—

(а) Dye droplets and mitochondria (figs. 3 and 15) ;(б) Dye droplets and Golgi apparatus (figs. 5-10 and 16-19).

2. No definite relationship can be established between dye droplets and mitochondria (figs. 3 and 15).

3. The dye droplets make their appearance in relationship with the Golgi apparatus, and when formed break away from it into the cytoplasm (figs. 5-10 and 16-19).



Vital Staining of Normal and Malignant Cells. 301

4. The formation of dye droplets in relationship with the Golgi apparatus resembles the process of formation of secretion granules in gland cells.

5. Regarded in the light of recent research these observations suggest that the following functional inter-relationship exists between the Golgi apparatus and mitochondria:—At the mitochondrial-cytoplasmic surface syntheses by enzymes occur. The resulting products continually diffuse into the cytoplasm, preventing an accumulation at the surface of the mitochondria, which would inhibit further syntheses. At the surface of the Golgi apparatus the elaborated products are concentrated into droplets preliminary to their elimination.

BIBLIOGRAPHY.

(1) Cowdry, E. V. (1926). “ Surface Film Theory of the Function of Mitochondria,”4 Amer. Nat.,’ vol. 60, p. 157.

(2) Cramer, W., and Ludford, R. J. (1926). “ On the Cellular Mechanism of Bile Secretionand its Relation to the Golgi Apparatus of the Liver Cell,” 4 Journ. Physiol.,’ vol. 62, p. 74.

(3) Glasunow, M. (1928). 44 Beobachtungen an den mit Trypanblau vitalgefarbtenMeerschweinchen. I. Morphologie der Trypanblauablagerungen in einigen Epithel- zellen,” 4 Zeit. f. Zellforsch. u. mikr. Anat.,’ vol. 6, p. 773.

(4) Horning, E. S., and Petrie, A. H. K. (1927). 44 The Enzymatic Function of Mitochondria in the Germination of Cereals,” 4 Roy. Soc. Proc.,’ B, vol. 102, p. 188.

(5) Jasswoin, G. (1925). 44 Zur Histophysiologie der Tubuli contorti der Amphibienniere,”4 Zeit. f. Zellforsch. u. mikr. Anat.,’ vol. 3, p. 741.

(6) Makarov, P. (1926). 44 Beobachtungen liber den Golgischen Apparat und die Ablager-ungen von Trypanblau in den Leberzellen verschiedener Wirbelthiere,” 4 Archives Russes d’Anat., d’Hist. et d’Embry.,’ vol. 5, p. 159.

(7) v. Mollendorff, W. (1920). 44 Vitale Farbungen an tierischen Zellen,” 4 Ergeb. d.Physiol.,’ vol. 18, p. 141.

(8) --------- (1921). 44 Methoden zu Studien iiber vitale Farbungen an Tier zellen,”4 Abderhalden’s Hdb. der biol. Arbeitsmeth.,’ Sect, v., Part 2, p. 97.

(9) ---------(1923). 44 Die Zelle in ihrer Umwelt, A. Farbenanalytische Untersuchung,”4 Hdb. der Bioch. d. Menschen u. d. Tiere,’ vol. 2, p. 273.

(10) --------- (1926). 44 Vitale Farbung,” 4 Enzykl. d. mikros. Technik.’ (edited byR. Krause), vol. 1, p. 697.

(11) Nassonov, D. (1926). 44 Die physiologische Bedeutung des Golgi-Apparats im Lichteder Vitalfarbungsmethode,” 4 Zeit. f. Zellforsch. u. mikr. Anat.,’ vol. 3, p. 472.

▼OL. era.—B. z



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