STAINTNG WITH BRAZUJN. 469

Staining with Brazilin.

Sydney J. Hickson, F.R.S., Beyer Professor of Zoology in the Owens College, Manchester.

BEAZILIN is extracted from the wood of Caesalpinia echinata, growing in South America. Formerly, under the name Brazil-wood, it was used commercially as a dye; but of recent years it has been superseded by other colouring substances, and practically driven out of the market. Chemically it has considerable resemblance to hsematoxylin, which is also extracted from the wood of a tree belonging to the order Leguminosse, but brazilin (C1CHUO5) differs from haematoxylin (C10H14O,j) in having one hydroxyl less. Further information concerning the chemistry of brazilin may be found in the papers by Professor Perkin and Dr. Gilbody published in the 'Proceedings of the Chemical Society' during the past four or five years. Brazilin has been tried as a stain for animal tissues by Flechsig and Breglia, but apparently the results were not very satisfactory. Their methods, though mentioned in the third edition of Lee's ' Microtomist's Vade-Mecum/ are omitted from the fourth edition. Brazilin is mentioned as a chromatin stain in Rawitz's ' Leitfaden fin* histologische Untersuchungen,' p. 98, but it is not mentioned in the elaborate 'Tabellen zum Gebrauch bei mikroscopischen Arbeiten/ by W. Behrens, published in 1898, nor in Bbhm and Oppel's ' Taschenbuch der mikro- scopischen Technik/ published in 1896. My first experiment was to attempt to use brazilin instead of hEematoxylin in the method suggested by Heidenhain, 470 SYDNEY J. HI0KSON. and now widely used in England and elsewhere under the name of the " Iron-Hsematoxylin method." This experi- ment did not give results that were at all satisfactory. Another series of experiments which have been made in the Owens College laboratory by Mr. Wadsworth, under my direction, have shown that the most satisfactory results are obtained by the following method :—" The sections are placed in a solution of irou-alum (1 per cent, iron-alum in 70 per cent, alcohol) for one to three hours, and then placed, after slight washing in 70 per cent, alcohol, in a £ per cent, solution of pure brazilin in 70 per cent, spirit.'5 Brazilin stains much more slowly than hsematoxylin, and we have found that generally it takes several hours (three to sixteen) to give a good sharp definition. After staining, the sections are washed in pure 70 per cent, spirit, passed through the usual stages, and mounted. There is seldom any need to wash the sections in iron-alum after staining. It will thus be seen at the outset that this method possesses two advantages over iron-hasmatoxylin,—the sections are never taken down into water, and the number of washings is con- siderably reduced. The results are, to my mind, eminently satisfactory; for not only is brazilin a definite chromatin stain, but in nearly all tissues some parts of the cytoplasm are also stained, though of a different colour. It is in most cases a double stain, but with some tissues it is a treble stain. We have used as a test object a series of sections through the body of a larval newt. Each of these sections exhibits so many subjects of histological interest that it is impossible to describe them briefly. I think it would be admitted, however, by all observers that nearly, if not all the tissues are well stained. The chromosomes in the cells that are dividing in the skin and ovary are very sharply defined and of a deep purple colour, while the granules in the cytoplasm are'brown. On making a comparison of our preparations of newt's testis stained by the iron-brazilin method and by the iron-haema- toxylin method, it was found that the karyokinetic figures STAINING WITH BBAZILIN. 471 were equally well denned in the two preparations; but the iron-brazilin showed an advantage in that the spermatozoa were triple stained, the head, middle piece, and tail being clearly of different colours. A preparation of the sciatic nerve of a clog stained in brazilin exhibits the nenrokeratin remarkably clearly, but the axis-cylinder is not well stained. Our method has great advantages in showing the blood- supply of an organ, the red blood-corpuscles being stained very deeply. Preparations of ovary of a cat and kidney of a dog exhibit the capillary plexus excellently. It is, however, principally in cytological work I think that the new method will be .valuable, as we have found after many experiments, in our investigations on the very delicate nuclear structures of the Suctorian Dendroco metes, that it gives us by far the best general results. It is obvious that a good deal more must be done befoi'e an opinion can be expressed as to the general value of this method, but I have thought it right to bring it before the notice of workers in various branches of microscopic science at the present time, because I feel that it is a method well worthy of further trial. In conclusion I may say that the somewhat severe test of exposing slides to the action of direct sunlight for several mouths has not indicated any appreciable fading effect. I am indebted to Professor W. H. Perkin, jun., F.R.S., for much valuable advice and assistance in working out this method.