Analytic applications of diazo reactions in histology

Abstract

As consequence of previous papers of the author, an es- sential difference is stablished between the histochemical dia- zoreactions, leading to the formation of triazenes, and the coupling processes, by means of which the surface of histological cuts is trajisformed in the corresponding azoproteinic dyes.

These reactions are utilized fot the study of histological structures, and for the investigation of their more important constituents and components.

The influence of the surface phenomena on these processes is studied too, as a particular case of the general heterogeneous acid-base calalysis, of biochemical interest.

As diazonium salt, the puré crystallized anthraquinoncdia- zonium chloride, is utilized, obtained of a very high qualityby means of a method of the author.

A buffered coid solution of 20 mgr/25 mi of anthraquino- nediazonium chloride is prepared and the histological cuts, ob­ tained by means of a freezing microtome, are introduced in this solution, maintained at O’-IO’C, for instance in an ice box.

The rate of the azo-coupling speed is dcpending of the />H of the médium, which enables the easy regulation of the azo

proteinic dye layer speed formation, in the histological cuts surface. Generally the author works at /?H=6,5 or 7,5 at 0°C and in these conditions the corresponding coloring processes are accomplished in no more time than one hour, depending chiefly of the tissue structure and of the fixation technique utilized.

Several figures of the corresponding microphotographs, are included in this paper, showing very clear differences in nor­ mal and leucosic hen liver and too in blood preparations, these last dyed by means of the common histological hematoxylin- eosin method and, in an other way, utilizing the azo-dye pro­ teinic coupling method. It enables the clear observation of the nucleus structure of hen erytrocytes, which is not possible by means of hematoxylin-eosin method.

The intensity of the azo-proteinic dye coloration formed de- pends in all cases of the capacity for coupling of the amino groups of termináis proteinic peptid chains aminoacides. In normal tissues, being the proteinic structure more compact than in sarcomatosic ones, the concentration of these amino groups is greater in normal than in sarcomatosic tissues. The inten­ sity of the coloration formed is then much smaller in the pa- thological zones of the tissue studied.

The method is also applied to the study of the more com- plexes phytochemical structures and several data are reported showing too very marked differences, according to the nature of their Chemical constituents and components.