308 



METHODS AND FORMULAS 



DS 11.3-DS 11.4 



11.3 Bkazilin and Other Natural Stains 



11.3 Bensley 1916 brazilin 590,29:37 



foemula: water 10, brazilin 0.05, water 90, phosphotungstic acid 1 

 preparation : Add the acid dissolved in 90 water to the dye dissolved in 10. 

 method: water -^ stain, 12 to 3 hrs. -^ water, wash -^ balsam, via usual reagents 



11.3 Guinard 1890 alkand 11071,6:447 



preparation: Infuse 20 alkanet in 60 abs. ale. I'ilter. Evaporate to dryness and dissolve 

 residue in 10 acetic acid. Add 100 50% ale. 



11.3 Hickson 1901 brazilin 17510, 44:470 



REAGENTS REQUIRED: A. 1% ferric alum in 70% ale; B. 10% ale. 100, brazilin 0.5 

 method: 70% ale. -» A, 3 hrs. -^ 70% ale, quick rinses B, overnight -> 70% ale. 

 thorough wash — > balsam, via usual reagents 



11.3 O'Leary see DS 21.213 O'Leary 



11.4 Synthetic Nuclear Stains 



The term synthetic nuclear stains is here 

 used in contrast to those stains, such as 

 hematoxylin and carmine, which are ex- 

 tracted from natural sources. These syn- 

 thetic dyes are widely called aniline dyes 

 without very much justification, since by 

 no means are all of them directly derived 

 from aniline. The now obsolete term coal- 

 tar dyes was far more accurate. Almost any 

 stain can be used to color nuclei differ- 

 entially by the adjustment either of the 

 pH or tlie chemical composition of the 

 solution in which it is employed. Those 

 here fisted are those most widelj^ employed 

 for the purpose either alone or in combina- 

 tions in complex contrast formulas. 



The most interesting of these reagents, 

 grouped in the first class, are the oxazine 

 dyes, which are turned into their metallic 

 lakes in the course of the preparation of 

 the staining solutions. These were intro- 

 duced by Becher 1921, in a book which 

 never received a very wide circulation. In 

 their staining reactions they strongly re- 

 semble hematoxylin, giving eitlier black 

 or blue nuclei, but they do so without the 

 necessity for differentiating, and are far 

 less liable either to fading or to altei'ation 

 in color. They have been rediscovered at 

 various intervals since 1921, and it is a 

 matter of some astonishment to those 

 who have employed them that they have 

 not received wider acceptance as a substi- 

 tute for hematoxylin. The author's choice 

 among the formulas is the anonymous 

 one, given first. Probably the one most 

 widely employed is the formula of Proe- 



scher and Arkush, 1928 (20540b, 3:36). 

 The publication of this paper in the 

 United States started a brief vogue for 

 these materials. The original publication 

 of Becher quoted not only the utilization 

 of these oxazine dyes for nuclear staining, 

 but also for the staining of the central 

 nervous system and for some general- 

 purpose polychrome stains. These will be 

 found in their appropriate divisions later. 



Safranin, in the Engfish-speaking coun- 

 tries, is more widely employed in botanical 

 tlian in zoological technique. In Europe, 

 however, particularly in France, it re- 

 mained the standard nuclear stain for 

 many years, and has not yet been regu- 

 larly supplanted by hematoxyfin. Thus a 

 standard French technique for a histo- 

 logical preparation will be safranin-light 

 green, in which the corresponding British 

 or American usage calls for hematoxyfin- 

 eosin. This safranin-light green combina- 

 tion has found occasional acceptance in 

 the literature as Benda's stain. There is 

 little to choose between any of the for- 

 mulas, it being a matter of convention 

 that the techniques of Chamberlain 1915, 

 and Johansen 1940, are customarily em- 

 ployed for plant material, while Babes, 

 1887 and Bohm and Oppel 1907 are com- 

 monly employed for animal materials. 



Magenta is less widely employed as a 

 single nuclear stain than it is in combina- 

 tion with plasma stains in complex tech- 

 niques. The formula of Ziehl 1882, though 

 customarily confined to bacterial staining, 

 is here included for the reason that it is 

 just as good a nuclear stain as it is a stain 



