342 



METHODS AND FORMULAS 



DS 13.1 



and 13.2) employ the thiazins and their 

 related compounds either in combination 

 with eosin, or in the next class, with such 

 other dyes as have been employed. The 

 next class (DS 13.3) takes up the methyl 

 green combinations and is followed by the 

 group (DS 13.4) of complex formulas in 

 which the Mallory reaction is employed. 

 This is followed by a small group (DS 13.5), 

 largely of French origin, in which safranin 

 is employed as the nuclear stain, and 

 another small group (DS 13.6) in which 

 hematoxylin is employed. This still leaves 

 for the last class (DS 13.7) a considerable 

 miscellaneous group. 



Most of these complex techniques are 

 better employed for class demonstration 

 than for research purposes. This does not 

 apply with any force to the meth3dene 

 blue-eosinates, so widely employed in 

 blood staining, in which the material to 

 be stained is customarily an unfixed, heat- 

 dried smear, the staining reactions of the 

 constituents of which can be controlled 

 by the accurate adjustment of the pH of 

 the staining solution. When, however, as 

 in the other classes, the staining method 

 is intended to operate on materials which 

 have been fixed and sectioned, there is the 

 greatest difficulty in securing a reproduci- 

 bihty of results, and the utmost attention 

 should be paid to the recommendations of 

 the original author with regard to the 

 fixative to be employed. When, as in the 

 case of the methylene blue-eosinates, the 

 entire staining materials are usually ap- 

 plied from a single solution, there is 

 nothing save the isoelectric point of the 

 proteins on which they act which controls 

 the differential staining. This factor of 

 lack of reproducibility of results tends to 

 render dangerous a publication of research 

 observations based only on these staining 

 methods for the slightest variation in the 

 material, source of material, or fixative, 

 and would cause anyone endeavoring to 

 check the results to come to conclude that 

 the method described is not applicable to 

 his case. There is no finer group of stains 

 which may be employed for class teaching 

 purposes, particularly in sections of em- 

 bryos and the like where a great range of 

 tissues are available. Even in this instance, 

 however, care should be taken that the 



entire batch of sections required is pre- 

 pared at the same time, or students may 

 become confused by the apparent abnor- 

 malities presented by successive batches 

 of material. 



13.1 Techniques Employing 



THE "EOSINATES" OF THE ThIAZINS 



WITHOUT Other Admixture 



The eosiimtes of methylene blue and its 

 oxidation products are compound dyes 

 which are usually utilized in solution in 

 methanol. They may, however, be pre- 

 pared either directly in solution or in the 

 form of the dry powder, and in the latter 

 state are at present widely found in com- 

 merce. For the purposes of the present 

 work these techniques are divided into 

 three classes. First (DS 13.11) are the 

 straight methylene blue-eosinates, among 

 which the original formulas of Jenner 1899 

 and May-Griinwald 1902 are the best 

 known. These two techniques are not very 

 widely used today, save as a preliminary 

 to staining with other mixed polychrome 

 methylene blue-eosinates. An interesting 

 variation of these is the method of 

 Sabrazes 1911, in which the stain is pre- 

 pared in actual contact with the object 

 to be colored. 



The next class of these stains (DS 13.12) 

 is usually incorrectly identified as Giemsa 

 stains, though to the parasitologist the 

 formulas of Leishman 1901 and Roma- 

 nowski are possibly better known. These 

 formulas are irrational, for they contain 

 the eosinates of polychrome methylene 

 blue, which is itself a mixture of varying 

 composition, and which often widely 

 varies in nature even though the method 

 of preparation is specified. Giemsa 1902 

 prepared his mixture from ingredients of 

 known composition. His formula is there- 

 fore given in the next section. 



The most logical stains to use are those 

 of the third class (DS 13.13), which are 

 the eosinates of methylene blue and its 

 varying oxidation products. They are pre- 

 pared, however, from reagents of known 

 composition. The formula of Giemsa 1902 

 is the best known of these, but the name 

 has unfortunately been used to describe 

 almost any azur-eosinate without specific 

 reference to the exact azur intended. The 



