Histology of the Blood, Normal and Pathological

by Paul Ehrlich; Adolf Lazarus

A more detailed study of the process of staining, and of the relation between chemical constitution and staining power, enabled a further advance to be made. And the first result in this previously unworked direction, was the sharp distinction between acid, basic, and neutral dyes, and between the corresponding, oxy-, baso-, and neutrophil granulations. The triacid solution was only found after trial of many hundred combinations; and up to the present day this stain in its original form or in slight modifications has played a prominent part in various provinces of histology.

The classification of the cell-granules of the blood according to their various chemical affinities which was drawn up by this method is accepted to-day as the most valuable, and the only practical means of grouping the leucocytes. From the first Ehrlich has insisted, that different kinds of cells possess different granules, distinguished not only by their tinctorial properties, but also by their various reactions to solvents.

It is in this connection indeed, that Altmann's method, consisting of a complicated hardening process, and the use of a single, always similar stain, constitutes a retrograde step, in as much as it tends to obscure the principle of the specificity of each kind of granulation.

A further disadvantage of Altmann's hardening method lies in the circumstance, that the cell proteids are precipitated by it in a spherical form, and stain in the subsequent treatment. Hence it is extremely difficult to distinguish what is preformed, and what is artefact. Since A. Fischer's publication, where the formation of granule-like precipitates under the influence of various reagents is experimentally demonstrated, grave doubts as to the reality of Altmann's forms have been raised from various quarters. Ehrlich's dry process, on the contrary, is entirely free from error. Granules cannot be artificially produced, by desiccation, and the stained appearances correspond precisely to what is seen in fresh living blood. The greatest value of the dry method is that the chemical nature of the single granules remains unchanged, so that attempts at differentiation are made on a nearly unaltered object[22].

Another means of studying the nature of the granules depends on the principle of vital staining. The "vital methylene blue staining" (Ehrlich) that has since become so important, especially in neurology, led to the first attempts at staining the granules in living animals. One of the first publications on this subject is that of O. Schultze, who placed the larvæ of frogs in dilute methylene blue solution, and after a short period found the granules of the stomach, the red blood corpuscles and other cells stained blue. This method, however, cannot pass as entirely free from error, as Ehrlich frequently found that when the experiment lasts some time the methylene blue often forms granular precipitates that may be confused with the granules. Teichmann directs a detailed analysis to this point, and regards most of the granules described by Schultze as artificial products.

Neutral red is highly suitable for the study of vital granule-staining, a dye recommended by Ehrlich, and employed successfully since that time by Przesmycki, Prowazek, S. Mayer, Solger, Friedmann, Pappenheim and others. This dye was prepared by O. N. Witt from nitrosodimethylamin and metatoluylendiamin, and is the hydrochloric acid salt of a base which is soluble in pure water, yielding a fuchsin red colour, but which in weak alkaline solution—the alkalinity of mineral water suffices—is a yellow-orange hue.

Now neutral red is characterised by a really maximal affinity for the majority of the granules. Ehrlich was able by the aid of this dye to demonstrate granules, even in some vegetable cells. Moreover the method of using it is the simplest conceivable, as subcutaneous or intravenous injection, or even feeding, in the higher animals stains the granules; with frog's larvæ and invertebrates, to allow them to swim in a dilute solution of the dye is often sufficient. The staining also succeeds in "surviving" organs, and is best effected by allowing small pieces to float in physiological salt solution, to which a trace of neutral red is added, under plentiful access of air. When the object is macroscopically red it is ready for examination.

The finest results are naturally given by organs that are easily teased out, e.g. flies' eggs, or the Malpighian canals of insects. The staining solution is to be chosen so that the act of staining does not last too long, but on the other hand too high a concentration must not be used. About 1/50000 to 1/100000 is recommended, so that the protoplasm and nucleus remain quite uncoloured. Artificial products with this method cannot entirely be excluded, and, e.g. in plant-cells containing tannin, are to be explained by the production and precipitation of the salt of tannic acid. However it is not difficult for the experienced to recognise artificial products as such in individual cases. The kind of granulation, the typical distribution, a comparison with neighbouring cells, the combination of various methods, the comparison of the same object under vital and "survival" staining, facilitate judgment and obviate mistakes of this kind.

The majority of the granules of vertebrates are stained orange-red by neutral red, corresponding with the weakly alkaline reaction of these forms. Granules staining in pure fuchsin colour and which hence possess a weak acid reaction are much more rarely found.

Combination staining may be recommended as a valuable aid to the neutral red method. Ehrlich has used a double stain with neutral red and methylene blue. Frog's larvæ were allowed to remain in a solution of neutral red, to which a trace of methylene blue had been added. He then found red granulations almost exclusively, only the granules of the smooth musculature of the stomach were stained intensely blue. With the aid of a threefold combination Ehrlich obtained a still further differentiation of the living cell-granules. There is no doubt whatever that a thorough study of this neutral red method would lead to important conclusions as to the nature and function of the granules, and lead us to the most real problems of cell life. With our present information even we can get definite conceptions founded upon facts, as to the biological importance of the cell-granules.