ZOOLOGY AND BOTANY, MICROSCOPY, ETC. 715 



above method, working microscopists having long sought after a 

 double-staining process for blood-corpuscles in which Dr. Moore is 

 the first to succeed. 



Flemming's Modified Method for Staining Nuclei.* — A method 

 was published in 1875 by G. E. Hermann, f consisting essentially in 

 overstaining with anilin or azotized staining matters, and subsequently 

 extracting the colour, except from the nuclei, by means of absoluto 

 alcohol. 



On this W. Flemming suggests some improvements. He finds the 

 nitrogenized colouring-matters better than anilin colours for the 

 purpose. Chromic acid is also preferable to alcohol for hardening, as 

 it preserves the characters of the nuclei with more certainty. The 

 preparations are fixed in chromic acid of • 1 to '5 per cent, according 

 to their nature. Only sections or thin, flat, and readily penetrable 

 pieces should be used, and they must be thoroughly washed in dis- 

 tilled water. They are then placed for 12 to 24 hours in closed 

 vessels in about 1 ccm. of a solution of safranin (or one of the other 

 colouring matters mentioned below) absolute alcohol being used 

 diluted by about the same amount of distilled water. J The object is 

 now transferred to alcohol which frees it from part of the colour 

 by shaking for a short time, and then into absolute alcohol and 

 moved about for half a minute or more until no more colour is given 

 off, and the object appears transparent and of the desired tint. If 

 the object is to be permanently mounted it is placed in oil of cloves, 

 but only so long as will admit of the tissue becoming penetrated, as 

 it draws out the colour, and then mounted in cold dammar solution or 

 balsam. 



Out of a series of colours which were tested, manvein and fluores- 

 cent red, while staining the nuclei well, are yet somewhat unequal in 

 their action in that some nuclei will retain more colour than the rest. 

 Solid green has the property of being very readily extracted from the 

 intermediate substance of the nucleus, leaving the fibrillar network of 

 the latter well stained. If this is decolorized, the nucleoli long 

 retain the colour. Fuchsin gives excellent colours but somewhat 

 paler than safranin, magdala, dahlia, and mauvein. Bismarck broicn 

 is unsuitable for the above process with chromic acid. Safranin, 

 magdala-red (or naphthalin-rose), and dahlia (monophenylrosanilin) 

 are the most constant and satisfactory in their action. 



It must be noted, however, that practically the only application 

 of the method is for nuclei- staining in chromic acid preparations. 

 Where, however, it is desired to preserve and readily investigate the 

 true natural structure in cell-nuclei and divisions of nuclei (which 



* Arch. f. Mikr. Anat., xix. (1881) p. 317-30. 



t Flemming subsequently stated (torn, cit., pp. 742-3) that the credit of 

 priority so far as regards nuclei-staining with anilin colours, and decolorizing 

 with alcohol, is due to Professor Bottcher, whose method is not, however, to be 

 recommended for the same purposes as Hermann's, as he uses Midler's fluid and 

 alcohol, stains the sections with a solution of rosanilin-nitrate in dilute 

 glycerine, and after extracting the water with alcohol, clarifies with creosote. 



X Dahlia is best used in aqueous or acetic acid solutions. 



