On Double Staining Nucleated Blood-Corpuscles with Anilin Dyes.

292 DE. VINCENT HAEBIS. On Double Staining Nucleated Blood-Corpuscles with Anilin Dyes. Vincent Harris, M.D., Demonstrator of Physiology at St. Bartholomew's Hospital. THE usefulness of the process of staining tissues with several dyes has been abundantly proved. The general effect aimed at is the staining of each separate part in a* different colour, so that for the purposes of histological demonstration each shall be distinct and clear. In the use of certain of the most commonly employed and easily manipulated dyes, e. g. hematoxylin and picrocarmine, it is believed that a definite effect may be always calculated upon when they are used in combination. With anilin stains, however, the results arrived at appear to differ very materially if the methods of employment are made to vary in even a very slight degree, and this has been one of the causes of the restricted use of very beautiful staining colours. It has been shown by several experimenters that with combinations of anilin colours, there is a tendency at any rate for certain dyes to pick out and stain different parts of a tissue; but I think I am right in believing that no certain result has hitherto been expected, except in the case of a very few combinations. There is no doubt that unless more definite results be obtained with combinations of these dyes, the hope that with them it will be possible to pick out each element of a tissue in a different colour, and each kind of morbid growth in a similar manner, will be long deferred. The following notes were made during a series of experiments, the object of which was to find out the best combination of anilin

k totjble STAINING NUCLEATED BLOOD-COEPTJSOLES. 29$ ^. ' dyes for double staining, as some of the methods recommended? had, in my hands, turned out very unsatisfactorily. During the year 1881 I had, at the request of Dr. Vandyke Carter, devoted a considerable time to the preparation and investigation of the organs of patients who had died of "spirillum fever," and also of those of animals which had been experimentally inoculated Avith the fever virus. We wished to demonstrate in these tissues and organs the presence of spirilla \ as had been done with ease in the blood. We used, according \ to the advice of Professor Koch, several anilin dyes in turn with anything but certain results. The indefiniteness, therefore, of the staining struck me much, and induced me to think that something might be done in the way of obtaining more satisfactory combinations. The demonstration of the bacillus in tubercle has brought into prominence the manipulation of combinations of these dye3, and the fact that since the first process of Koch was introduced, large numbers of modifications of it have been brought forward, notably by Ehrlich, Baumgarten, Ermengen, Gibbes, and others, point to the conclusion that a considerable amount of work remains to be done before the subject is fully develoded. These experiments in staining the nucleated corpuscles were undertaken, as I have said, with a view of finding out whether, with such definite elements of tissue, certain results could be obtained by staining with certain anilin dyes in solutions of definite strength in regular sequence. Method of experiment. 1 After trying several methods, the one I adopted was as follows : Blood from various animals frogs, newts, &c. was spread in thin layers upon -inch cover-glasses and allowed to dry in the air. Certain of the anilin colours were then chosen as the first or primary colours; these were fuchsin, eosin, rosein, and rosanilin, in aqueous or dilute spirit solution, as red dyes; iodine or malachite green, as green dyes; and methyl violet, Hoffman's violet, or gentian violet, as violet dyes. All of these were in aqueous or dilute spirit solution. With each primary colour was included a separate series of experiments. Several drops VOL. XXUI. NEW SEE. V

294 DR. VINCENT HARRIS. of the first solution were allowed to remain upon the dried blood for about five minutes, and were then washed off with a gentle stream of water from a wash-bottle. The cover-glass was then dried in the flame of a spirit-lamp and allowed to cool. When ready for the second dye, a small quantity was dropped upon the cover-glass and allowed to remain the same length of time. A second washing with a stream of distilled water followed until the washings were all but colourless; dry- j ing and mounting in Canada balsam concluded the process. / Treatment with alcohol and clove oil previous to mounting in j several instances quite vitiated my result, and so I gave up *. that method of dehydration and clearing. In cases where I thought it possible to employ three dyes, the third was used in a manner almost exactly similar to the one described above. The method of fixing the corpuscles with osmic acid did not in the least improve the staining of the corpuscles. The chief precaution which appeared to be necessary was not to allow the blood to coagulate, and to place it under conditions of rapid, but natural, drying, e. g. in the sun's rays. ARRANGEMENT OF THE COMBINATIONS OF COLOURS : Series A. Primary colour, red; tried with orange, yellow, green, blue, violet, and brown. 1ST DM. Substances used. Red Fucksin Lake. Rosein Crimson. Eosin Pink. 2ND BYES. Substances used. Yellow Anilin Primrose. Orange Tropaeolin, or Aurin. Green Iodine Green. Blue Metbylen Blue. Violet Hoffman's. Brown Bismarck. SERIES A. Red and orange. (Eosin and aurin.) This combination was unsuccessful, as the solution of aurin had to be made with absolute alcohol, it being such a very in-

DOUBLE STAINING NUCLEATED BLOOD-COBPUSOLBS. 295 soluble substance, and entirely replaced the eosin, which was a saturated watery solution. The whole of the corpuscles were stained a deep orange. So far I have been able to do little with aurin as a dye, its great insolubility causes every solution speedily to deposit crystals. Red and yellow. (Fuchsin and anilin primrose.) Fuchsin, a salt of rosanilin, is a fine lake dye, partly soluble in water, freely soluble in dilute Anilin primrose, a penetrating yellow dye of the colour of picric acid, almost insoluble in water, and only partly in methylated spirit, from which it quickly deposits crystals. After some difficulty in obtaining a good specimen the corpuscles were found to have stained thus. The nucleus, a yellowish red, not unmixed crimson, the remainder of the coloured corpuscles a light yellow, and the colourless corpuscles a light red. The combination was not a good one, as the yellow proved a very difficult dye to turn out, but, judging by results, it had the greater affinity for the protoplasm of the corpuscles, and less for the nucleus. Red and green. (Rosein and iodine green.) Rosein, similar to fuchsin, but of a more deep crimson, is partly soluble in water, very soluble in dilute Iodine green is freely soluble in water; an excellent combination. The coloured corpuscles were stained a bright red, with bluishgreen nuclei. The colourless corpuscles were easily made out to be of three varieties in the blood (of newt): 1, entirely stained in green; 2, partly stained in green and partly yellowish red, the nucleus green, and the surrounding protoplasm of the Other colour; 3, the large masses of nuclei-like bodies, said to be developing colourless corpuscles, were deeply stained in green. The relation in size of the nuclei of the colourless corpuscles to the stroma is a very variable one. Sometimes the nucleus appears to occupy nearly the whole cell, at others, perhaps, not more than a fourth or fifth of it. It would be possible to describe a larger number of different kinds of colourless corpuscles than the above if their reaction to the dyes were alone

266 DR. VINCENT HARBlg..,. considered, but no doubt the effects of the staining fluids were / not constant. V Red and blue. (Fuchsin and methylen blue.) Certainly one of the most successful combinations. The methylin blue was used as a saturated solution in absolute alcohol. In this case, as in most of the others, the blue was used second to the fuchsin, and vice vers&, with similar results. In these specimens the nuclei of the coloured corpuscles were deeply stained blue. Of the remainder of the corpuscle, a light greenish hue with the edge a bright pink, or where the staining had been less deep the whole of the corpuscle, except the nucleus, was stained pink with an edge of a deeper but similar colour. The staining of the colourless corpuscles was peculiar j some were a light bluish green. These were irregular and branched; others had a deeper purple colour with unstained spots (vacuoles?); and a third variety appeared to be stained in two colours; in these a large central mass looked like an immense nucleus. Other varieties might be mentioned, as in the last combination, but as it is possible that the size, amount of granules, and the staining are, except in the three varieties, mere differences in amount not in kind, it is scarcely necessary to mention them. For purposes of demonstrating the divisions, irregularities in shape and in varieties of colourless corpuscles, I am strongly inclined to recommend staining with methylen blue and fuchsin as the best combination possible. Red and blue, 2nd combination. (Fuchsin and soluble anilin blue.) Nuclei stained red, as well as the colourless corpuscles; stroma a light blue. It is as good a combination as the last. Red and violet. (Eosin and methyl violet.) With these colours there appeared to be a mixture of the dyes in the corpuscle, and the nuclei were not distinct. Red and brown. (Fuchsin and Bismarck Brown.) Bismarck brown is an anilin dye of considerable utility; it is partially soluble in water, more so in water with a few drops of glycerin added to it, and easily soluble in dilute

\ \ DOUBLE STAINING NUCLEATED BLOOD-COBPUSOLES. 297 i methylated The solution used was 2 per cent, in 1 dilute The corpuscles stained easily "and in a fairly regular manner, the nucleus a deep red, the stroma a fine brown. The colourless corpuscles pinkish red. In a few cases I noticed a mixture of the colours in the corpuscles. In using Bismarck brown, a dye much employed on the Continent, I find it best to immerse the specimens in it for twenty \ t. to thirty hours, and then they will retain their colour even \ if passed slowly through the dehydrating and clearing fluids. ' Red and brown, 2nd combination. (Eosin and ve- : suvin.) Vesuvin and Bismarck Brown are said to have the same chemical formula, and are probably identical. The vesuvin was used in a strong aqueous solution. The corpuscles easily stained with eosin, nuclei and colourless corpuscles a deep pinkish colour, and the stroma of the coloured corpuscles.a light pink. After double staining with vesuvin the stroma stained a light yellowish brown, leaving the nuclei and the colourless corpuscles stained as before. A very successful combination. Series B. Primary colour green ; tried with brown, red, orange, yellow, blue, and violet. 1ST DYE. 2ND DYES. Substances used.. Substances used. Green Iodine or Malachite Brown Bismarck. Green. Red flamingo or Ponceau. Orange Aurinand Anilin Orange. Yellow Anilin Primrose. Blue Bleu de Ljon. Violet Methyl Violet. SERIES B. Green and brown. (Iodine green and Bismarck brown.) The colourless corpuscles and the nuclei of the coloured corpuscles distinctly green; the stroma brown. Green and red. (Iodine green and flamingo.) Flamingo, a deep brownish red, soluble in water partly, hut freely in dilute The latter solution was the one employed. The nuclei of the coloured and the whole of the

298 DR. VINCENT HARRIS. colourless corpuscle showed a deep bluish green, and the stroma was coloured pink. Green and red, 2nd combination. (Malachite green and ponceau.) Malachite green is freely soluble in water, and was used in aqueous solution. The nuclei of the coloured corpuscles stained green and the stroma a light pink. The colourless corpuscles of the same shade as the nuclei. On keeping the /' specimens it was found that the green dye almost entirely / disappeared. Green and orange. (Malachite green and fluorescin.) The nuclei of the coloured corpuscles stained a light yellowish green, as did also the colourless corpuscles. The stroma stained a yellow colour. The green stain was very temporary. Green and orange. (Malachite green and aurin.) Entirely unsuccessful. Green and yellow. (Iodine green and anilin primrose.) The colourless corpuscles and the nuclei of coloured corpuscles stained green ; the stroma of the latter yellow. Green and blue. (Iodine green and Bleu de Lyon.) Double staining quite unsuccessful. The Bleu de Lyon was employed in a dilute spirit solution. Green and violet. (Malachite green and methyl violet.) Combination not good. The nuclei stained a very light purple, as did also the colourless corpuscles, whilst the stroma was a pinkish yellow. The green and violet apparently mingled in staining the nuclei. Series C. Primary colour, violet; tried with brown, red, orange, yellow, green, 'and blue. 1ST DYE. 2ND DYES. Substances used. Substances used. Violet Brown Bismarck. Methyl Violet. Red Mamingo, Eosin, Anilin Scarlet. Gentian Violet. Hoffman's Violet. Orange Tropseolin. Yellow Anilin Primrose. Green Iodine Green. Blue Metbylen Blue.

DOUBLE STAINING NUCLEATED BLOOD-CORPUSOLES. 299 Violet and brown. (Hoffman's violet and Bismarck brown.) An excellent combination. An aqueous solution of the violet was used, and a dilute spirit solution of the Bismarck, brown. The result showed excellent double staining. The nuclei of the coloured corpuscles and the colourless corpuscles were stained a reddish brown, and the stroma a light brown. The brown evidently stained the whole of the corpuscle stroma, and nucleus, but met the violet in the nucleus, and together stained it the colour it presented. Violet and red. (Hoffman's violet and flamingo.) Flamingo is a mixture of rosanilin and Bismarck brown. The nuclei and stroma stained two shades of the same colour, probably a mixture of the two used, mauve. Violet and red, 2nd combination. (Gentian violet and anilin scarlet.) Unsuccessful. Violet and red, 3rd combination. (Gentian violet and eosin.) Nuclei of coloured corpuscles, deeply stained red, with the colourless corpuscles a similar colour with their nuclei. Stroma a light pink. Violet and orange. (Hoffman's violet and tropseolin.) Tropseolin used in 1 % watery solution. Double staining entirely failed. Violet and yellow. (Gentian violet and anilin primrose). Strangely enough the corpuscles were stained two shades of green. Violet and blue. (Methyl violet and methylen blue.) Certainly one of the best combinations tried. The methyl violet is a very pink dye, and the blue a very deep blue. The latter stained the nuclei, the former the stroma. Results. From my experiments I draw the following conclusions as to double anilin staining of the nucleated corpuscles. It seems reasonable to look upon such corpuscles as made up of only a few varieties of tissue, and as such I have spoken of them.

300 DB. VINCENT HARRIS. 1. The only entirely successful combinations were the following:,' Rosein and anilin green. Fuchsin and methylen blue. Fuchsin and Bismarck brown. Eosin and vesuvin. Iodine green and Bismarck brown. Hoffman's violet and Bismarck brown. Anilin violet and methylen blue. I 2. The green dyes were not at all permanent. I have proved / this with both malachite and iodine greens. 3. Even with the above successful combinations the results varied in a most extraordinary manner, whilst the circumstances of the staining operation and the solutions appeared to be unvaried, the very greatest care being required to produce a constant result. One thing necessary for success was certainly that the solutions should be quite fresh. This is likely to prove a great objection to the general introduction of anilin dyes into use. The simple method of dehydration employed, of course, could not be employed in the preparation of tissues, although it does for blood, sputa, &c. 4. The result was materially affected by the time each dye was allowed to remain in contact with the blood. It is worthy of note that according to the evidence of competent authorities, various chemically diffused anilin dyes have been sold under the same commercial name ; and so, both in the preceding notes and also in the annexed table, it should be said that the anilin dyes used were obtained from Messrs. Hopkins and Williams, Hatton Garden, W.C. The following table (drawn up August, 1882) includes the dyes used in above experiments : I am much indebted to Mr. Meldola, of Messrs. Brooks, Spiller & Co., for valuable information as to the chemical composition and relations of many of the above anilines, and of the commercial names, &c, of others. This information he kindly furnished at the request of Dr. Russell,

CLASSIFIED LIST or THE CHIEF ANILIN DYES, WITH THEIR SOLUBILITIES IN WATEK AND IN SPIRIT. BROWN. RED. ORANGE. YELLOW. GBEEN. BLTJE. VIOLET. Bismarck partially sol. in water; sol. in dilute Vesuvin sol. in water. Chrysoidin sol. in water. Eosin, Pink freely sol. in water. Anilin Scarlet insol. in water; freely so in methylated Flamingo, deep brownish red partly sol. in water; freely so in meth. Ponceau, 1 deep red crimson partly sol. in water; freely in dilute Rosanilin partly sol. in water; freely sol. in dilute F u c h s i n partly sol in water; sol. in dilute Aurin insol. in water; partly sol. in strong spirit; more so in absolute alcohol. Anilin Orange ditto, ditto. Tropasolin, in Deep Yellow Glistening Scales partly sol. in water; more so in meth. Phosphin, Yellowish Orange partially sol. in water; more so, but not freely, in Saffranin sol. in water and in Fluorescin, Greenish Yellow insol. in water; sol. in spirit, the solution being beautifully fluorescent. Anilin Primrose only partly sol. in meth. Iodine Green, Blue Green freely sol. in water or Malachite Green, a less Blue Green freely sol. in water and in Soluble Anilin Blue freely sol. in water. Bleu de Lyon insol. in water; freely so in strong Methylen. Blue, a very Deep Blue freely sol. in water and in China Blue freely sol. in water. Serge Blue ditto. Blue Blackfreely sol. in water. Hoffman's Violet freely sol. in water and in dilute Methyl Violet, the Red predominating sol. in water partially; freely sol. in Gentian Violet, the Blue predominating freely sol. in water. Tyrian Blue, near to Violet sol. in water. Spiller'a Purple solublein Ponceau is a mixture of rosanilin and phosphin.