i. Crystal violet samples to be certified by the Commission must be of at least 

 80 per t!ent dye content as determined by reduction of TiCls in an atmosphere of 

 carbon dioxide. 



3. Gentian violet for general staining purposes, as defined by the Commission 

 on the Standardization of Biological Stains, must be either penta-methyl or hexa- 

 methyl pararosanilin, or else a mixture of methylated pararosanilins composed 

 primarily of the two compounds just named and having a shade at least as deep 

 as that recognized in the trade as methyl violet 2B. In other words the dye must 

 be Colour Index Xo. 680 or No. 681. 



4. Gentian violet samples to be certified by the Commission must be of at least 

 75 per cent dye content as determined by reduction of TiCl3 in an atmosphere of 

 carbon dioxide. The diluent used must be dextrin. 



5. The sample of crystal or gentian violet should prove satisfactory for staining 

 bacteria according to the Gram technic when made up in a 0.5 per cent aqueous 

 solution, without the use of anilin oil or any other mordant, and stained by the 

 following procedure: crystal or gentian violet 1 minute, Lugol's iodine solution 1 

 minute, 95 per cent alcohol 30 seconds, 0.05 per cent safranin solution 10 seconds. 

 In making the test, a weakly Gram-positive orgnism and a Gram-negative organism 

 should be tested. 



6. The sample should prove satisfactory in the Flemming triple stain when 

 used with safranin and orange G. For this purpose it must be tested by histologists 

 skilled in the technic in question. It should also prove a satisfactory nuclear 

 stain in general histology. 



7. Either gentian violet or crystal violet for certification must have sufficient 

 bacterostatic power so that, when added to nutrient agar in the proportion of one 

 part to a million, it will entirely prevent the growth of Bacillus suhtilis when this 

 organism is streaked over the surface of the hardened agar. It must be understood, 

 however, that this bacterostatic property of the dye is still under investigation 

 and has a relation not yet fully understood to the control of certain diseases. As 

 new investigations bring new light on the subject it may be necessary to draw up 

 new specifications to cover this point. 



SPECIFICATIONS FOR PYRONIN 



1. Pyronin designed for staining purposes must be either pyronin G (Colour 

 Index No. 739) or pyronin B (Colour Index No. 741). The container in which it is 

 sold should be marked plainly as to which of these two dyes is furnished. 



2. Samples of pyronin G must be characterized by an absorption curve showing 

 a maximum absorption at about oiSfifx Avhen determined in a 0.002 per cent solution 

 in a layer 1 milimeter thick. To be certified by the Commission these samples 

 should be of such a strength that the extinction coefficient at the point of maximum 

 absorption shall be not less than 1.20. 



3. Samples of pyronin B tested in the spectrophotometer under the above 

 mentioned conditions must have an absorption maximum at about 550;a/x, and must 

 be of such a strength that the extinction coefficient at the point of maximum ab- 

 sorption shall be not less than 1.00. 



4. The samples shall be satisfactory for use in the Pappenheim combination 

 with methyl green. In the case poor results are obtained with the formulae for 



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