APPENDIX II 

 COMMISSION SPECIFICATIONS OF CERTAIN STAINS 



As announced in several notes published by the Commission in Science, it is 

 planned to draw up specifications for all the stains put on a certification basis. 

 So far as these specifications have been prepared they are given on the following 

 pages. Similar specifications for other stains will be prepared later. 



It must be understood that these specifications are not intended to furnish 

 definite statements as to the chemistry of satisfactory stains. The object with 

 which they were drawn up was to allow the manufacturers as much latitude in the 

 matter of chemical composition as has been found consistent with good results in 

 practice, and to lay the greatest stress upon the performance of the stains in actual 

 laboratory use. The requirements listed are those which must be met by dyes 

 submitted to the Commission for certification. 



These specifications are published, moreover, with the understanding that they 

 are subject to revision at any time. Further investigations are in progress con- 

 cerning the adaptability of various dye products for different purposes, and also 

 as to the relation of optical characters to performance in staining. The Commission 

 reserves the right to modify the specifications for any stain in regard to either of 

 these two points as data accumulate to show the need of modification. 



SPECIFICATIOXS FOR METHYLENE BLUE 



1. Samples of methylene blue to be considered must be of the so-called medicinal 

 grade. It is expected that they will meet the U. S. P. requirements, but less weight 

 will be attached to this consideration than to those following. In other words, a 

 sample giving satisfactory performance will not be excluded because of failure in 

 some particular to meet these chemical requirements. 



2. Methylene blue for the purpose above specified must contain at least 75 

 per cent total color, this to be determined by reduction with titanous chloride. 

 When reduced by titanous chloride in an atmosphere of carbon dioxide, 1 gram of 

 the dye must consume at least 4.69 cc. normal titanous chloride solution. 



3. The methylene blue must have no solvent action on casein. This is to be 

 determined as follows: Prepare two 1 per cent solutions of this stain, one in distilled 

 water, the other in tap water. Place single drops of skimmed milk on each of two 

 clean glass slides and smear each drop over a surface of about one square centimeter 

 so as to form a very thin film of milk; allow this film to dry without heat or at a 

 temperature not over 60°C., immerse for about a minute in xyol to dissolve the fat, 

 then for the same length of time in alcohol to coagulate the casein. After this 

 immerse one slide in the distilled water solution of methylene blue and the other 

 slide in the tap water solution, allowing them to stand for three minutes; at the 

 end of this period there should be no action of the stain on the casein. 



4. The methylene blue should stain the diphtheria organism in any of the types 

 of solution^ ordinarily employed. It should be tested as follows: Prepare three 

 solutions of the stain, one a 1 per cent solution in distilled water, the second a 

 mixture of three parts saturated alcoholic solution to 10 parts of distilled water, 



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