• DOURINE OR MALADIE DU COIT 19 



For the differential staining of the white blood cells we have used stains pre- 

 pared by following the directions of Jenner, Leishman, Nocht-Jenner, Hastings- 

 Nocht-Jenner, Wright, «S:c. The essential dyes in each of the foregoing, while being 

 the same — eosin and methylene blue — possess certain advantages in particular com- 

 binations, although unavoidable difficulties are encountered with the above-mentioned 

 preparations, and these difficulties are familiar to those who have \ised. these dyes in 

 the enumeration of the various blood cells. 



As uniformity is an essential feature in connection with a staining process 

 where examinations are to continue for weeks, months, or even years, it is essential 

 that a stain possessing the same characteristics shoi;ld be available at all times. To 

 this end, Watson and I, during the winter of 1906, endeavoured to obtain a more 

 uniform staining material. Our experiments were very encouraging and we were 

 able to obtain a staining material which we could modify almost at will to suit our 

 special requirements. This staining material was made in the following manner — 

 .-. polychrome methylene blue solution is prepared, using the formula of Eomanowsky: 

 Methylene blue (med. pur.), 10 G. 

 riodium carbonate C. P., 5. 

 Distilled water, 1000. 



This mixture is steamed over a water bath with occasional stirring and sufficient 

 water should be added from time to time to cover the loss by evaporation. When a 

 rich purple coloration is noted, which is usually after two on three hours, the solu- 

 tion is evaporated to dryness. After cooling the residue is dissolved in pure methylic 

 alcohol, making a saturated solution. To this alcoholic solution of the polychromed 

 methylene blue, is added a solution of eosin in methylic alcohol of the following 

 strengtii : — 



Grubler's yellow eosin, 1 gramme. 



Methylic alcohol, C.P., 1,000 grammes. 

 After the preparation of these two solutions the stain is made by mixing them 

 in the following proportions, which, after much experimenting, has been found to 

 give the best results : — 



Polychrome methylene blue solution, 70 parts. 



Eosin solution, .30 parts. 

 After this has been allowed to stand for an hour the staining of a blood film 

 will reveal any error in the stain, which can be remedied according to its, cause, 

 provided, however, that the methylene blue has been prepared in a proper manner. 

 If it is found that the solution is too alkaline, the addition of a few drops of acetic 

 acid (0 '5 — 1 -0 per cent of acetic acid in methylic alcohol) will remedy the difficulty. 

 In the addition of this acid great care should be exercised for the reason that if the 

 solution is rendered too acid it is very difficult and may be impossible to fully restore 

 its characteristic properties. If it is only very slightly acid it may be brought 

 back to the proper degree by the addition of an alkaline solution such as 10 per 

 cent caustic potash in methylic alcohol. 



By preparing the stock solutions in large quantities and working out the details 

 for mixing in small quantities only (100 cc.) as required, the stain gives excellent 

 results. Many of the tiresome details connected with the preparation of blood stains 

 are overcome by following the above technique and it is not necessary to have such 

 a large amount of the staining fluid to manipulate during the process of preparation, 

 a serious consideration with many of the other differential stains. 



In staining the blood film, the slide or coverslip preparation of blood is completely 

 flooded with the undiluted stain for about one minute, when two or three times tho 

 volume of water is added and the film remaining immersed in this for from three to 

 five minutes for purposes of differentiation as indicated by Leishman or until a green- 

 ish yellow scum appears on the surface. If this does not appear some error in prepa- 

 ration has been made. After this differential staining, rinse in water for about thirty 



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