Part 18

"The investigations were performed after Koch's method in the following manner. The fluid (blood) or the parenchyma of the organs (bone-marrow, spleen, etc.) was spread on cover-slips in as thin a layer as possible, dried at room temperature, and after a convenient length of time stained. I had chosen this apparently coarse method for the special reason that for the histological recognition of new, possibly definite chemical combinations, corresponding to the granulations, all substances must be avoided that might act as solvents, e.g. water or alcohol, or as oxydising agents, such as osmic acid. In this instance only such procedures may be employed as will leave the simple drying of each single chemical substance as much as possible unchanged."

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.

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=.

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In his first publication Ehrlich described the =granules as products of the metabolism of the cells=, deposited within the protoplasm in a solid form, in part to serve as reserve material, in part to be cast off from the cell. On the ground of observations on the liver cells, described in detail in a paper of Frerichs (1883, page 43), Ehrlich gave up this position, though only temporarily. Ehrlich shewed that the liver cells of a rabbit's liver, rich in glycogen, appear in dry preparations as bulky polygonous elements, of a uniform homogeneous brown colour, surrounded by a thin, well-defined yellow membrane. In cells that were not too rich in glycogen, small roundish bodies, clearly of a protoplasmic nature, of a pure yellow, can be seen embedded in the homogeneous cells that are coloured brown with glycogen. "The hyaline cellular ground substance, carrying the glycogen, could not under any circumstances be stained, but the cell-granules above mentioned stained easily with all kinds of dyes. It was further possible to shew by staining that the membrane was chemically different from the granules, since with eosin-aurantia-indulin-glycerine, the membrane stained black, but the granules orange-red."

To these observations Ehrlich added the following conclusion, "that the cells of the liver after food really possess a thin =protoplasmic= membrane, and a homogeneous glycogen-bearing substance, in which the nucleus and =round granules= (? functionally active) of protoplasm are embedded.

"On comparing these results with those of more recent investigation of the cells, it is easy to determine the location of the glycogen very accurately. Kupffer has shewn, first for the liver cells--and this is now recognised as generally valid--that their contents do not represent a microscopically single substance. In the 'survival' preparation he found, in addition to the nucleus, two clearly distinct substances: a hyaline ground substance in preponderating amount, and a more scanty, finely granular, fibrillary substance embedded in it. Kupffer calls the first paraplasm, the latter protoplasm. On warming the preparation to about 22° C. manifest though feeble movements appeared in the network. It can hardly be doubted, that of these two substances the granular reticulated one--the protoplasm--is the more important; and it should not be erroneous to suppose that the granulations of the network form the =centre of the particular (specific) cell function=. In any case, it is desirable to give a special name, such as microsomes (Hanstein) to these forms, which in the liver cells are recognisable as distinct, round or oval granules, colouring yellow with iodine, and easily and deeply staining in other ways."

It was necessary to quote in full from this older paper, to shew that Ehrlich regarded the granules as the special carriers of the cell function so long ago as 1883, a view that Altmann advocated many years later, under the name "theory of bioblasts." Altmann's ever repeated assertion that no one before him had allotted so high an importance to the granules is consequently in disagreement with the facts we have above made sufficiently clear.

The importance Altmann ultimately gave to the granules, which he also calls by the name "=Ozonophores=" is shewn by his own words (Elementary Organisms, 1st edit., p. 39):

"Our conception of the ozonophores may therefore replace that of the living protoplasm, at least so far as vegetative function is concerned; and may serve us as an explanation of complicated organic processes. Once again, shortly summarising the properties of the ozonophores; as oxygen carriers they can perform reduction and oxydation, and can thus effect the decompositions and syntheses of the body, without losing their own individuality."

In the meantime Ehrlich had made various observations which could not be completely brought into line with his own earlier hypothesis or the far-reaching conclusions of Altmann. Studies in particular on the oxygen requirements of the organism, shewed that the "ozonophores" could certainly not be an important part of the cell. In addition it was found that normally cells occur in which no granules can be recognised by ordinary methods. Finally a pathological observation made untenable the view that the granules are the bearers of the cell function. In a case of pernicious anæmia (cp. Farbenanalytische untersuchungen) Ehrlich found the polynuclear cells of the blood and bone-marrow and their early forms free from all neutrophil granulation. On the grounds of this observation Ehrlich returned to his original assumption that the granules are secretory products of the cells, and defined his standpoint at that time as follows:

"Did the neutrophil granulations really represent the bodies which supply these cells with oxygen, as Altmann supposes, a condition such as we have here brought forward would be impossible, since with the disappearance of the granules death of the cells must follow. But from the point of view of the secretion theory the condition described is easily explainable. Just as under certain conditions fat-cells may completely lose their contents without dying, so the bone-marrow cell, if the blood fails to yield to it the necessary substances, may occasionally be unable to produce more neutrophil granules. And thus it becomes non-granular."

The view, that the granules are special metabolic products of the specific cellular activity, is strongly supported by the great chemical differences between them. Ehrlich made these peculiarities clear for the blood-cells, and found that their granulations differ from one another, not only in their colour reactions, but also in their =shape= and =solubility=; so that they must be sharply distinguished.

Whilst for instance the majority of the granules are more or less rounded forms, in some classes of animals, e.g. in birds, the analogues of the granules of mammalian blood are characterised by a decided crystalline form, and a strong oxyphilia. The substance of the mast cell granulations is also crystalline in some species of animals.

The size of the individual granules is constant in any animal species for every kind of granule--excepting only the mast cells. The eosinophil granulation reaches its greatest size in the horse, where really gigantic examples are found.

The presence of granulated colourless blood-cells has been demonstrated in the most various classes of animals, and even in the blood of many invertebrates, particularly, as Knoll has shewn, in the Lamellibranchiates, Polychætes, Pedates, Tunicates and Cephalopods. Concerning vertebrates, especially the higher classes, accurate and ample researches are to hand. In birds we recognise two oxyphil granulations, of which one is embedded in the cells in the crystalline, the other in the usual granular form. Amongst the vertebrates most investigated classes possess granulated polynuclear cells. To this circumstance Hirschfeld has recently devoted a thorough paper containing many details worthy of note. In the majority of the animals observed, he found too that the polynuclear cells contained neutrophil granules; in only one animal, the white mouse, did he find them, or granulations analogous to them, completely wanting.

According to the investigations carried out some years back in Ehrlich's laboratory by Dr Franz Müller, these results of Hirschfeld's must be described as inaccurate. After many vain endeavours, Dr Müller was able to find a method by which numerous though very minute granules could be found in the polynuclear cells of the mouse. The case shews that it is not permissible to assume the absence of granules, when the ordinary staining methods are not at once successful. There is no universal method for the staining of granules, any more than for the staining of various kinds of bacteria. Indeed all granules, that are easily soluble, vanish when the triacid method is used, and so a homogeneous cell protoplasm is simulated.

But naturally, the occurrence of non-granulated polynuclear cells in certain classes of animals is not to be denied from these considerations. Hirschfeld asserts that such cells occur side by side with granulated cells, for instance in the dog; and draws from them far-reaching conclusions as to the meaning of the granules. From Kurloff's work (see p. 85) we must insist, on the contrary, that there is no evidence that the non-granulated polynuclears are identical with the granulated cells. Kurloff has shewn, at least for guinea-pig's blood, that these two heterogeneous elements are to be sharply separated one from the other, and that they have an entirely different origin.

Specially important for a theory of the nature of the granules is the circumstance, that generally speaking in all species of animals =they are present in those cells of the blood only which are adapted to and capable of emigration=. That a certain nutritive function is to be ascribed to the emigration of the granulated cells is a very obvious supposition, scarcely to be denied; and naturally cells with a plentiful store of reserve material are eminently suited for this purpose. The lymphocytes on the contrary, incapable of emigration, are almost totally devoid of specific granulations.

A further indication that =the granulations really are connected with a specific cell activity lies in the fact, that one cell bears but one specific granulation=. The contrary assertions that neutrophil and eosinophil, or eosinophil and mast cell granulations occur in the same cell Ehrlich regards as unfounded, from extensive researches specially directed to this point. Nor has Ehrlich seen a pseudoeosinophil cell of the rabbit change to a true eosinophil. That such a transition does not occur is most distinctly shewn by the fact that the various granulations behave entirely differently towards solvents. With the aid of acids, for example, the pseudoeosinophil granules can be completely extracted from the cells, whilst the eosinophil granules remain whole under this process, and can now be stained by themselves.