270 



METHODS AND FORMULAS 



DSOO 



be to the great advantage of students pre- 

 paring microscope slides if they were de- 

 prived of access to coloring reagents until 

 they had made at least a hundred shdes 

 without the aid of the reagents. This is 

 particularly true in the preparation of 

 wholemounts, in many of which more de- 

 tails of structure are brought out by the 

 varying refractive index of the organs, 

 than they do by soaking them in red and 

 blue solutions which tend in many in- 

 stances only to obscure the finer struc- 

 tures. These remarks do not apply with 

 nearly so much force to the staining of 

 sections, though it is probable that such 

 structures as ciliated epithelium can be 

 better distinguished in an unstained than 

 in a stained preparation. The true value 

 of staining is realized when it provides 

 either a specific coloration of an organ or 

 cellular structure which is to be studied, 

 or alternatively when it provides a con- 

 trast between two such structures. A 

 lesser, and a less necessary, purpose is to 

 render apparent through the introduction 

 of color those very few structures, the re- 

 fractive index of which so closely approxi- 

 mates either that of the mounting medium 

 or of their neighboring structures as to 

 render them indistinguishable as un- 

 colored objects. 



Contemporary opinion on the theoretical 

 composition of materials used for staining 

 has altered very httle since the original 

 account of Witt 1876 (2627, 9:522) who 

 first advanced the now widely held theory 

 that the presence of color in a chemical is 

 conditioned by the presence of certain 

 groups or radicals known as chromophores, 

 and that materials known to contain 

 these groups should be called chromogene. 

 The power of imparting this color to other 

 substances is given to a chromogen by 

 the presence of an auxochrome. The ma- 

 jority of auxochromes are either alkali or 

 acid radicals which impart the property 

 of solubility to the materials under dis- 

 cussion. It is unfortunate that the partial 

 absorption of these theories by biologists 

 should have left them with the almost 

 universal habit of classifying dyes either 

 as basic or acidic, according to the nature 

 of the auxochrome, and left them also en- 

 deavoring theoretically to forecast the 



performance of such a dye on the basis of 

 its alleged physical reaction. 



There are in point of fact four main 

 ways in which a color may be caused to 

 remain diffused throughout or adherent 

 to, a particular structure. The first of 

 these, surface adsorption, is a physical re- 

 action and is dependent both on the 

 charge upon the ionized dye and upon the 

 materials on which this dj-e is precipitated. 

 It might be imagined, and has indeed been 

 widely stated, that it is thus only neces- 

 sary to know the isoelectric point of the 

 protein involved and the pH of the dye 

 solution to be able to secure a perfect 

 differential absorption at all times. This 

 is to a considerable extent true in the case 

 of vital staining or of proteins whose 

 nature has not been altered. In the latter 

 instance, for example, the dried smears, 

 used either in the staining of blood or the 

 staining of bacteria, fulfill the require- 

 ments. In these circumstances the control 

 of the pH of the staining solution is all 

 that is required to secure reproducible and 

 perfect results. As soon, however, as a 

 fixative is employed which denatures the 

 proteins, the problem becomes more com- 

 plex; and there is no method save that of 

 trial and error by which the desirable pH 

 of a dye solution may be determined. This 

 does not mean that pH should not be more 

 widely controlled, for there is little doubt 

 that many of the troubles of the early 

 microtomists, who were forced to specify 

 a particular source for a particular dye, 

 were due to the influence on the pH of the 

 final solution of various impurities left in 

 the commercial product by different 

 manufacturers. 



Another physical reaction involved in 

 the staining of tissues is the saturation of 

 a material with a dye and the subsequent 

 precipitation of the dye in place by the use 

 of solvents for dehydrating in which the 

 dye is not soluble. These methods are 

 little used and difficult to control. Lastly, 

 in a few cases, a definite chemical com- 

 bination is entered into between the dye- 

 stuff and the tissue which is being differ- 

 entially stained. A common and obvious 

 case of this is the use of alizarin red S to 

 stain boney structures or the calcareous 

 plates of invertebrates. In this case a 



