246 Biological Stains 



products of methylene blue present in the polychromed solution 

 may combine individually with the eosin, thus yielding several 

 different eosinates in the precipitate. Unless the precipitate is 

 washed with two or three changes of water it usually contains a 

 considerable excess of one or the other of the uncombined dyes, and 

 this may further increase the complexity of the mixture. If 

 properly washed, the precipitate should be free, or almost free, of 

 uncombined basic or acid dye, because of their great solubility, 

 and possibly such is also the case in the methanolic solution of the 

 precipitate; but it is certain that when water is added in the process 

 of staining, dissociation must occur with the formation of the 

 acid and basic dyes from which the compound was derived. This 

 is another contribution to the complexity of the solution with 

 which the staining is accomplished. Complex as the mixture is, 

 Lillie (1942) has shown that it can be quite effectively analyzed 

 spectrophotometrica,lly ; and that from the position and width of 

 the absorption bands, one can get some idea as to the composition 

 of the polychrome methylene blue from which the eosinate was pre- 

 pared. By comparing such spectrophotometric results with the 

 behavior in staining, one can obtain practical information as to the 

 value of the different azures in a stain of this type. 



In addition to the chemistry of the constituent dyes, another 

 important factor in a blood stain of this type is the nature of the 

 solvent. Methyl alcohol, as explained above, is the usual solvent 

 employed; but there are many grades of this reagent available, 

 and not all are equally suitable for the purpose — a fact which at 

 one time was not fully appreciated. Apparently absolute purity of 

 the methanol is not needed; but one point is very important — the 

 solvent must be neutral in reaction. Methanol, as formerly pre- 

 pared by the destructive distillation of wood, often contained con- 

 siderable amounts of the other two major products of that decom- 

 position, acetone and acetic acid. The presence of acetone seems 

 to be unimportant (in spite of previous recommendations that 

 methanol for blood stains should be acetone-free); acetic acid, 

 however, is quite deleterious in that it tends to precipitate eosin 

 as its color acid, when the methanolic solution is diluted with 

 water. Special methyl alcohol for blood stains is on the market, 

 but the modern synthetic methanol of reagent grade proves quite 

 satisfactory; its further purification with Ag20, as sometimes 

 recommended to free it from aldehydes and ketones, seems to be 

 quite unnecessary. (See Lillie, 1944b). 



The two blood stains for which there is now most demand in 

 America are Wright stain and Giemsa stain, while the tetra- 

 chrome stain is used less frequently. Wright stain and the 

 tetrachrome stain both give almost identical results and are 

 handled very similarly. If one is to prepare the stain himself, 

 there are reasons for preferring the tetrachrome stain as it can be 



