38 Biological Stains 



dyers can obtain the desired shade without having to measure out 

 dyes in very small quantities. Inasmuch as the early biological 

 stains were textile dyes without much, if any, modification, it is 

 natural that some of them should also have been of low dye con- 

 tent, and also that different batches should have been of various 

 degrees of purity. In general the present dyes are much more 

 pure than those available before the war of 1914-18. This makes 

 it difficult to prepare stain solutions identical in strength with those 

 prepared before that time. 



There are two general types of stain formulae: in one a definite 

 weight of dry dye is specified; in the other a certain volume of a 

 saturated (generally alcoholic) solution of the dye. Each type of 

 formula has its own possibilities of error; and to appreciate the 

 problem it is necessary to understand certain facts in regard to the 

 solubilities of dyes. 



The error inherent in the first type of formula is plain at a 

 glance. If two different staining solutions are made up containing 

 1 g. per 100 ml. of dry methylene blue, and in one case the actual 

 dye content of the dry stain is 90 percent, while in the other only 

 55 percent (a difference actually observed in samples at one time 

 on the market), it is plain that the two solutions must differ greatly 

 in their strength. For this reason an early recommendation of the 

 (commission (1928b) was that formulae of the second type be pre- 

 ferred, on the assumption that a saturated solution of a dye would 

 be more likely to be of constant dve content than different lots of 

 dry stain bought in the market. 



This recommendation, however, was made without complete 

 understanding of the actual facts of the case. The amount of a 

 dye that will go into solution in either water or alcohol depends 

 upon the amount of mineral salts present. If a dye contains a 

 large percentage of sodium chloride, for instance, a saturated solu- 

 tion will be of considerably lower actual dye content than if the 

 dye were free or nearly free from salt; the sodium chloride prevents 

 the solvent from taking up as much of the dye as it would nor- 

 mally. For this reason two staining solutions each containing 10 

 percent by volume of a saturated solution of the two methylene 

 blues above mentioned would be quite different from each other 

 in actual dye content, although possibly more nearly alike than if 

 they had been prepared with identical weights of the dry stain. 



As soon as the facts were fully understood the Commission 

 (1933e) modified its recommendation. It is plain that the only 

 way two stain solutions can be made identical, if different batches 

 of stain are used, is to make them up on the basis of the weight of 

 actual dye present in the stain samples employed. For some time 

 now, all staining formulae recommended by the Commission have 

 been put in such a form as to call for a definite weight of dye of a 

 specified dye content — thus enabling the user to recognize any 



