154 Biological Stains 



order to account for the fact that although only two of the sulfonic 

 groups are neutralized with sodium, the compound acts as though 

 it has no free acid. In other words, it is a case of intramolecular 

 salt formation. Now w^hen the tri-sodium salt is formed, this bond 

 is broken down, whereupon the quinoid ring disappears and the fol- 

 lowing compound is produced : 



SOsNa 



/ 



/~\_NH2 



\ / 



H,N_/— \_C— OH SOaNa 



\_/ \ / 



/ \/~\_NH2 



NaSO 



3 



This compound, it will be seen, is a carbinol in structure, and as it 

 lacks the quinoid ring it is colorless; but it is very readily converted 

 into the di-sodium salt by the addition of acid, whereupon the color 

 again appears. This property makes acid fuchsin of use as an 

 indicator. The decolorized solution of acid fuchsin neutralized 

 with sodium hydroxide is called the Andrade indicator. It was 

 once used extensively in bacteriological work, because of the strik- 

 ing reaction when its color is restored by acid-forming bacteria. 

 (See Andrade-Penny, 1895.) As an indicator to show hydrogen- 

 ion concentration at all accurately, however, it is found to have 

 much less value than the phthalein and sulphonphthalein dyes (see 

 pp. 197 to 206). 



Acid fuchsin is a widely used plasma stain, which has also been 

 recommended for a number of special uses. Among the best 

 known are: the Van Gieson (1889) connective tissue stain, in which 

 it is used with picric acid after hematoxylin to differentiate smooth 

 muscle from connective tissue; the Ehrlich-Biondi tri-acid stain 

 for blood, which is a "neutral" combination with orange G and 

 methyl green (see Krause, 1926-7; pp. 457, 1707). In plant his- 

 tology it is used to stain the cortex, pith and cellulose walls: 

 while the Pianese (1896) stain (with malachite green and martins 

 yellow), which was originally applied to cancer tissue, is now used 

 by plant pathologists in studying infected vascular plants. It is 

 used with methyl green, by Altmann, Bensley and Cowdry as a 

 stain for mitochondria (see Lee, 1937, pp. 167-8). To the pathol- 

 ogist it is quite valuable as a constituent (with anilin blue and 

 orange G) of the Mallory (1900) connective tissue stain. 



Scanlan, French and Holmes, (1927) showed that many lots of 

 acid fuchsin are unsatisfactory because of the rapidity with which 

 they fade in Van Gieson preparations. These writers found that 

 this fault arises from lack of control of the process of sulfonation. 

 Their results indicated that a satisfactory product could be ob- 

 tained if this process were carefully controlled by the use of a low 



