GALLOCYANIN-CHROMALUM 137 



STAINING 



comes weaker. Thns, for safety the 

 solution should not be used for more 

 than 3 weeks. Lake-sulphate and lake- 

 hydroxide are the main constituents of 

 the precipitate filtered off the solution 

 before use. Although such inner-com- 

 plex salts all have very low solubilities 

 in water, the lake-sulphate, however, 

 reamins dissolved in a sufficient amount 

 to become dissociated into the mono- 

 valent lake-cation and an anion ac- 

 cording to the equation: 



(gallocyanin Cr(H20)4)2 S04^ 2 galloc 

 ^ Cr(H20)4+ + SO4 



In the lake-cation, Cr(H20)6+++ is 

 attached to one gallocyanin molecule 

 by two valency bonds to its OH and 

 COOH, and by two coordinating bonds 

 to its =0 and/0\ through substitu- 

 tion of two HoO-molecules from the 

 internal sphere of the Cr-atom. The 

 designations =0 and /0\ are used 

 for the sake of brevity. =0 means 

 the 2-carbonvl-O, and /0\ means the 

 oxazin-0 of the chromophor-group). 



The reddish lake-cations, the actual 

 staining compound, unite progressively 

 and selectively with the nucleoproteins 

 of the fixed cell structures to form a blue 

 salt of the lake-cation and the tissue. 

 This proceeds until maximum occupa- 

 tion has taken place; then no further 

 attachment of the stain to the baso- 

 philic cell structures is possible. When 

 the staining has attained a certain in- 

 tensity it will not be further increased, 

 even if the tissue be left in the staining 

 solution for a considerable length of 

 time; in this respect the staining is 

 independent of the staining time {ex- 

 quisite progressivity) ; no other histo- 

 logical staining known to me possesses 

 this quality in the same degree. Owing 

 to the great stability of the staining it 

 is also completely unaffected by alcohol 

 and xylol during the procedure of de- 

 hydration and mounting. In consid- 

 eration of these qualities the staining 

 intensity depends directly on the in- 

 herent capacity to bind the stain, pos- 

 sessed by the living cell at the moment 

 of fixation. 



Briefly the staining consists of : 



1. A specific binding of the lake- 

 cation to the nucleic acids, in which it 

 reacts with the phosphoric acid groups 

 to form a lake-salt of nucleic acid; 

 within the range from pH 0.83 to 4.35 

 this specific binding always takes place 

 by means of the free -f-valency of the 

 lake-cation, regardless of the type of 

 nucleic acid. 



2. An adsorption of lake-sulphate 

 to the tissue, i.e., an unspecific co- 

 staining of non-basophilic structures, 



G ALLOC Y AN IN -CHROMA LUM 

 STAINING 



which increases directly with the pH 

 of the dye-solution; it mainly takes 

 place by the basic 7-(CHj)2N-group of 

 the dye molecule; by staining at a suffi- 

 ciently low pH the co-staining is re- 

 duced to a minimum. 



3. A binding of the lake-cation to the 

 proteins of the basophilic structures, 

 which, however, only takes place in a 

 definite range of pH on the alkaline 

 side of the iso-electric point of the pro- 

 teins and in fact is unimportant; on the 

 acid side of the iso-electric point the 

 lake-cation combines with the nucleic 

 acids alone. 



As the lake compounds stabilize 

 (buffer) the staining solution towards 

 acids and alkalies its pH can be varied 

 simply bv adding 1 to 10 cc of N/1 

 HCl and' N/1 NaOH respectively to 

 each 40 cc. of stock solution. In the 

 range from pH 1.50 to 1.80 the specific 

 staining reaches its maximum intensity 

 at the same time as the co-staining re- 

 mains completely negligible; pH 1 .64 

 represents the optimum acidity for the 

 specific attachment of the lake-cations to 

 the nucleic acids, the relative quantity 

 of which can be estimated by photometric 

 or densitometric measurements. The in- 

 crease of the co-staining first sets in at 

 pH 1.80 to reach its maximum between 

 pH 3.4 and 3.5 whereupon every stain- 

 ing fades away; at pH 4.27 it is barely 

 visible. The co-staining at pH 2.90- 

 3.42 may be used for demonstrating 

 axons and neuroglia (Einarson & 

 Ringsted 1938, p. 43). After the addi- 

 tion of alkali the solution should not be 

 used longer than 4-8 days. 



The usual basic blues preferably stain 

 proteins and other amphoteric electro- 

 lytes which display ''facultative baso- 

 phily",\.e., under another set of condi- 

 tions they are less basophilic or even 

 acidophilic; they are amphophilic sub- 

 stances, and the staining is a mere ad- 

 sorption of the dye. 



Gallocyanin-chromalum indicates the 

 degree of "genuine basophily" which 

 depends directly on the quantity of 

 nucleic acids present; genuine baso- 

 phily is not in the same way dependent 

 on the external conditions as the 

 facultative one, and its changes reflect 

 the decomposition during neuronal 

 function or regeneration and the sub- 

 sequent repair of cytoplasmic nucleic 

 acid from the nucleus (Einarson, L., 

 Am. J. Anat., 1933, 53, 141-176). 



Gallocyanin (CI. 883)-alizarin blue 

 RBN, chrom blue GCB and fast violet. 

 It is a basic oxazin dye. 



Gamma = 



1000 



mg. or 0.001 mg. 



