Chapter 4

We will begin with the simplest example: the use of picro-carmine, a mixture of neutral ammonium carmine and ammonium picrate. In a tissue rich in protoplasm, carmine alone stains diffusely, though the nuclei are clearly brought out. But if we add an equally concentrated solution of ammonium picrate, the staining gains extraordinarily in distinctness, in as much as now certain parts are pure yellow, others pure red. The best known example is the staining of muscle with picro-carmine, by which the muscle substance appears pure yellow, the nuclei pure red. If, however, instead of ammonium picrate we add another nitro dye which contains more nitro-groups than picric acid, for example the ammonium salt of hexa-nitro-diphenylamine, the carmine stain is completely abolished, all parts stain in the pure aurantia colour. The explanation of this phenomenon is obvious. Myosin has a greater affinity for ammonium picrate than for the carmine salt, and therefore in a mixture of the two combines with the yellow dye. Owing to this combination it is not now in a condition to chemically fix even carmine. Further, the nuclei have a great affinity for the carmine, and therefore stain pure red in this process. If, however, nitro dyes be added to the carmine solution, which have an affinity for all tissues, and also for the nuclei, the sphere of action of the carmine becomes continually smaller, and finally by the addition of the most powerful nitro body, the hexa-nitro compound, is completely abolished. Connective tissue and bone substance, however, behave differently with the picro-carmine mixture, in as much as here the diffuse stain depends exclusively on the concentration of the carmine, and is quite uninfluenced by the addition of a chemical antidote. This staining can only be limited by dilution, but not by the addition of opposed dyes. We must look upon the latter kind of tissue stain not as a chemical combination, but as a mechanical attraction of the stain on the part of the tissue. We may also say: =chemical stains are to be recognised by the fact that they react to chemical antidotes; mechanical stains to physical influences=; of course always assuming, that purely neutral solutions are employed, and that all additions, which alter the chemical relation of the tissues such as alkalis and acids, or which raise or limit the affinity of the dye for the tissues, are avoided. A further consequence of this view is, that all successive double staining may be serviceably replaced by simultaneous multiple staining, if the chemical nature of the staining process is settled. In contradistinction, in all double stains, which can only be effected by successive staining, mechanical factors are concerned.

In the staining of the dry blood specimen, purely chemical staining processes are concerned, and therefore the polychromatic combination stain is possible in all cases.

The following combinations are possible for the blood:

1. Combined staining with acid dyes. The best known example is the eosin-aurantia-nigrosin mixture, in which the hæmoglobin takes on an orange, the nuclei a black, and the acidophil granulations a red hue.

2. Mixtures of basic dyes. It is possible straight away to make mixtures consisting of two basic dyes. As specially suitable we must mention fuchsin, methyl green, methyl violet, methylene blue. On the other hand, mixtures of three bases are fairly difficult to prepare, and the quantitative relations of the constituents must be exactly observed. For such mixtures, fuchsin, bismarck brown, chrome green, may be used.

3. Neutral mixtures. These have played an important part in general histology, from the time that they were first introduced by Ehrlich into the histology of the blood up to the present day; and deserve before all others a full consideration.

Neutral staining rests on the fact, that nearly all basic dyes (_i.e._ salts of the dye bases, for instance, rosanilin acetate) form combinations with acid dyes (_i.e._ salts of the dye acids, for instance, ammonium picrate) which are to be regarded as neutral dyes, such as rosanilin picrate. Their employment offers considerable difficulties as they are very imperfectly soluble in water. A practical application of them was first possible after Ehrlich had ascertained that certain series of the neutral dyes are easily soluble in excess of the acid dye, and so the preparation of solutions of the required strength, readily kept, was made possible. Among the basic dyes which are suitable for this purpose are those particularly which contain the ammonium group, especially methyl green, methylene blue, amethyst violet[5] (tetraethylsafraninchloride), and to a certain extent pyronin and rhodamin also. In contradistinction to these, the members of the triphenylmethan series, such as fuchsin, methyl violet, bismarck brown, phosphin, indazine, are in general less suited for the purpose, with the exception of methyl green already mentioned. The acid dyes specially suited for the production of soluble neutral stains are the easily soluble salts of the polysulpho-acids. The salts of the carbonyl acids and other acid phenol dyes are but little suitable: and least of all, the nitro dyes. Specially to be mentioned among the acid dye series are those which can be used for the preparation of the neutral mixtures: orange g., acid fuchsin, narcëin (an easy soluble yellow dye, the sodium salt of sulphanilic acid--hydrazo-[beta]-naphtholsulphonic acid).

If a solution of methyl green be allowed to fall drop by drop into a solution of an acid dye, for instance orange g., a coarse precipitate first results, which dissolves completely on the further addition of the orange. No more orange should be added than is necessary for complete solution. This is the type of a simple neutral staining fluid. Chemically the above-mentioned example may be thus explained; in this mixture all three basic groups of the methyl green are united with the acid dye, so that we have produced a triacid compound of methyl green.

Simple neutral mixtures, which have one constituent in common, may be combined together straight away. This is very important for triple staining, which can only be attained by mixing together two simple neutral mixtures, each consisting of two components. A chemical decomposition need not be feared. We thus get mixtures containing three and more colours. Theoretically there are two possibilities for such combinations:

1. Staining mixtures of 1 acid and 2 basic dyes,

_e.g._ orange--amethyst--methyl green; narcëin--pyronin--methyl green; narcëin--pyronin--methylene blue.

2. Staining mixtures of 2 acids and 1 base, in particular the mixture to be described later in detail of

orange g.--acid fuchsin--methyl green. Further narcëin--acid fuchsin--methyl green,

and the corresponding combinations with methylene blue, and amethyst violet may be mentioned.

The importance of these neutral staining solutions lies in the fact that they pick out definite substances, which would not be demonstrated by the individual components, and which we therefore call =neutrophil=.

Elements which have an affinity for basic dyes, such as nuclear substances, stain in these neutral mixtures purely in the colour of the basic dye; acidophil elements in that of one of the two acid dyes; whilst those portions of tissue which from their constitution have an equal affinity for acid and basic dyes, attract the neutral compound, as such, and therefore stain in the mixed colour.

* * * * *

The eosine-methylene blue mixtures are exceptional in so far, that it is possible with them, for a short time at least, to preserve active solutions, in which with an excess of basic methylene blue, enough eosin is dissolved for both to come into play. A drawback however of such mixtures is, that in them precipitates are very easily produced, which render the preparation quite useless. This danger is particularly great in freshly prepared solutions. In solutions, such as Chenzinsky's, which can be kept active for a longer time, it is less. Hence fresh solutions stain far more intensely and more variously than older ones, and are therefore used in special cases (see page 46). If the stain is successful the appearances are very instructive. Nuclei are blue, hæmoglobin red, neutrophil granulation violet, acidophil pure red, mast cell granulation deep blue, forming one of the most beautiful microscopic pictures.

For practical purposes, besides the iodine and iodine-eosine solution described below (see page 46) the following are especially used:

1. =Hæmatoxylin solution with eosin or orange g.=

Eosin (cryst.) 0.5 Hæmatoxylin 2.0 Alcohol abs. Aqu. dest. Glycerine _aa_ 100.0 Glacial acetic acid 10.0 Alum in excess

The fluid must stand for some weeks. The preparations, fixed in absolute alcohol, or by short heating, stain in from half-an-hour to two hours. The hæmoglobin and eosinophil granules are red, the nuclei stain in the colour of hæmatoxylin. The solution must be very carefully washed off.

2. In the practical application of the triacid fluid, particular care must be taken, as M. Heidenhain first shewed, that the dyes are =chemically pure=[6]. Formerly granules, apparently basophil, were frequently observed in the white blood corpuscles, particularly in the region of the nucleus. They were not recognised, even by practised observers (_e.g._ Neusser) as artificial, but were regarded as preformed, and were described as perinuclear forms. Since the employment of pure dyes these appearances, whose meaning for a long time puzzled us, are but seldom seen.

Saturated watery solutions of the three dyes are first prepared, and cleared by standing for some considerable time. The following mixture is now made:

13-14 c.c. Orange-g. solution 6-7 c.c. Acid fuchsin solution 15 c.c. Aqu. dest. 15 c.c. Alcohol 12.5 c.c. Methyl green 10 c.c. Alcohol 10 c.c. Glycerine

These fluids are measured in the above-mentioned order, with the same measuring glass; and from the addition of methyl green onwards the fluid is thoroughly shaken. The solution can be used at once, and keeps indefinitely. The staining of the blood specimen in triacid requires only a little fixation, cp. page 35. The stain is completed in five minutes at most.

The nuclei are greenish, the red blood corpuscles orange, the acidophil granulation copper red, the neutrophil violet. The mast cells stand out by "negative staining" as peculiar bright, almost white cells, with nuclei of a pale green colour.

The triacid stain is very convenient. It is much to be recommended for good general preparations; =it is indispensable in all cases where the study of the neutrophil granulations is concerned=.

3. =Basic double staining.= Saturated, watery methyl-green solution is mixed with alcoholic fuchsin.

The stain, which only requires a small fixation, is completed in a few minutes, and colours the nuclei green, the red blood corpuscles red, the protoplasm of the leucocytes fuchsin colour. It is therefore specially suited for demonstration preparations of lymphatic leukæmia.

4. Eosin-methylene blue mixtures, for example Chenzinsky's fluid:

Concentrated watery methylene blue solution 40 c.c. 1/2% eosin solution in 70% alcohol 20 c.c. Aqua dest. 40 c.c.

This fluid is fairly stable, but must always be filtered before use. It only requires a fixation of the specimen for five minutes in absolute alcohol. The staining takes 6-24 hours (in air-tight watch-glasses) at blood temperature. The nuclei and the mast cell granulations stain deep blue, malaria plasmodia light sky blue, red corpuscles and eosinophil granules a fine red.

This solution is particularly suited for the study of the nuclei, the baso and eosinophil granulations, and it is used by preference for anæmic blood, and also for lymphatic leukæmia.

5. 10 c.c. of a 1 per cent. watery eosin solution, with 8 c.c. methylal, and 10 c.c. of a saturated watery solution of methylene blue are mixed, and used at once, see page 41. Time of staining 1, at most 2 minutes. The staining is characteristic only in preparations very carefully fixed by heat. The mast cell granulations are stained pure blue, the eosinophil red, the neutrophil in mixed colour.

6. Jenner's stain consists of a solution in methyl alcohol of the precipitate formed by adding eosine to methylene blue.

Grubler's water soluble eosine, yellow 1.25% } a.a. watery " medicinal, methylene blue 1% } solutions.

Precipitate allowed to stand 24 hours, and then dried at 55°. It is then made up to 1/2% in methyl alcohol (Merck). The stain may be obtained from R. Kanthack, 18, Berners Street, London, ready for use. It is exceedingly sensitive to acids and alkalis. Fixation is effected by heat. Time of staining 1-4 minutes.

Before we pass to the histology of the blood, two important methods may be described, for which the dried blood preparation is employed directly, without previous fixation: 1. the recognition of glycogen in the blood; 2. the microscopic test of the distribution of the alkali of the blood.

1. _Recognition of glycogen in blood._

This may be effected in two ways. The original procedure consisted in putting the preparation into a drop of thick cleared iodine-indiarubber solution under the microscope, as had been already recommended by Ehrlich for the recognition of glycogen.

The following method is still better. The preparation is placed in a closed vessel containing iodine crystals. Within a few minutes it takes on a dark brown colour, and is then mounted in a saturated lævulose solution, whose index of refraction is very high. To preserve these specimens they must be surrounded with some kind of cover-glass cement.

By the use of better methods the red blood corpuscles which have taken on the iodine stain stand out, without having undergone any morphological change. The white blood corpuscles are only slightly stained. All parts containing glycogen on the contrary, whether the glycogen be in the white blood corpuscles, or extracellular, are characterised by a beautiful mahogany brown colour. The second modification of this method is specially to be recommended on account of the strong clearing action of the lævulose syrup. In using the iodine-indiarubber solution a small quantity of glycogen in the cells may escape observation owing to the opaqueness of the indiarubber, and occasionally too by the separate staining of the same. The second more delicate method is for this reason recommended, in the investigation of cases of diabetes and other diseases[7].

2. The microscopic test of the distribution of alkali in the blood.

These methods rest on a procedure of Mylius for the estimation of the amount of alkali in glass. Iodine-eosine is a red compound easily soluble in water, which is not soluble in ether, chloroform, or toluol. But the free coloured acid, which is precipitated by acidifying solutions of the salt, is very sparingly soluble in water. It is, on the contrary, very easily soluble in organic solvents, so that by shaking, it completely passes over into an etherial solution, which becomes yellow. If this solution be allowed to fall on glass, on which deposits of alkali have been formed by decomposition, they stand out in a fine red colour as the result of the production of the deeply coloured salt.

In its application to the blood, of course the vessels used for staining as well as the cover-glasses must be cleaned from all adhering traces of alkali by means of acids. The dry specimen is thrown directly after its preparation into a glass vessel containing a chloroform or chloroform-toluol solution of free iodine-eosine. In a short time it becomes dark red. It is then quickly transferred to another vessel containing pure chloroform, which is once more changed, and the preparation still wet from the chloroform is then mounted in canada balsam. In such preparations the morphological elements have preserved their shape completely. The plasma shews a distinct red colour, whilst the red corpuscles have taken up no colour. The protoplasm of the white corpuscles is red, the nuclei appear as spaces, because unstained (=negative nuclear staining=). The disintegrated corpuscles and the fibrin which is produced, shew an intense red stain. These stains are peculiarly instructive, and shew many details which are not visible in other methods. The study of these preparations is really of the highest value, since they allow the products of manipulation of the dry preparation and every error of production to stand out in the most reliable manner, and so render possible a kind of automatic control. The scientific value of this method lies in the fact that it throws light on the distribution of the alkali in the individual elements of the blood. It appears that free alkali reacting on iodine-eosine is not present in the nuclei; these must therefore have a neutral or an acid reaction. On the contrary the protoplasm of the leucocytes is always alkaline, and the largest amount of alkali is held by the protoplasm of the lymphocytes. We call particular attention, in this connection, to the strong alkalinity of the blood platelets.

FOOTNOTES:

[4] Klönne and Müller, Berlin, supply these after Ehrlich's directions.

[5] Baden Anilin and Soda manufactory, Kalle and Co.

[6] At M. Heidenhain's instigation, the Anilin-dye Company of Berlin have prepared the three dyes in the crystalline form.

[7] It may also be used for the recognition of glycogen in secretions. For instance, gonorrhoeal pus always shews a considerable glycogen reaction of the pus cells. It is found, moreover, in cells which originate from tumours, whether these be present in exudations, or obtained by scraping.

B. NORMAL AND PATHOLOGICAL HISTOLOGY OF THE BLOOD.

In satisfactorily prepared dry specimens =the red blood corpuscles= keep their natural size and shape, and their biconcavity is plainly seen. They present a distinct round homogeneous form, of about 7.5 µ in diameter. They are most intensely coloured in a broad peripheral layer, and most faintly in the centre corresponding to their depression. With all stains mentioned above the stroma is quite uncoloured, and the hæmoglobin exclusively attracts the stain, so that for a practised observer the depth of stain gives a certain indication of the hæmoglobin equivalent of each cell, and a better one than the natural colour of the hæmoglobin in the fresh specimen. Corpuscles poor in hæmoglobin are easily recognised by their fainter staining, especially by the still greater brightness of the central zone. When somewhat more marked, they present appearances which from the isolated staining of the periphery Litten has happily named "pessary" forms. The faint staining of a red corpuscle cannot be explained, as E. Grawitz assumes, by a diminished affinity of the hæmoglobin for the dye. Qualitative changes of this kind of the hæmoglobin, expressing themselves in an altered relationship towards dyes, do not occur, even in anæmic blood. If in the latter, the blood discs stain less intensely, this is due exclusively to the smaller amount of hæmoglobin.

A diminution in the hæmoglobin content can in this way be shewn in all anæmic conditions, especially in posthæmorrhagic, secondary and chlorotic cases. On the contrary, as Laache first observed, in the pernicious anæmias, the hæmoglobin equivalent of the individual discs is raised.

To appreciate correctly pathological conditions, it must always be borne in mind, that in normal blood the individual red blood corpuscles are by no means of the same value. Step by step some of the cells are used up and replaced by new. Every drop of blood contains, side by side, the most various stages of life of fully formed erythrocytes. For this reason influences which affect the blood--provided their intensity does not exceed a certain degree--cannot equally influence all red corpuscles. The least resistant elements, that is, the oldest, will succumb to the effect of influences, to which other and more vigorous cells adapt themselves.

To influences, of this moderate degree, belongs without doubt the anæmic constitution of the blood as such, the effect of which in this direction one can best investigate in cases of posthæmorrhagic anæmia.

In all anæmic conditions we observe characteristic changes in the blood discs.

A. =Anæmic or polychromatophil degeneration.=

This change in the red blood corpuscles, first described by Ehrlich, to which the second name was given later by Gabritschewski, is =only recognisable in stained preparations=. The red blood discs, which under normal circumstances stain in pure hæmoglobin colour, now take on a mixed colour. For instance, the red corpuscles are pure red in preparations of normal blood, stained with hæmatoxylin-eosine mixture. But in preparations of blood of a chromic anæmia stained with the same solution, in which possibly all stages of the degeneration in question are present, one sees red discs with a faint inclination to violet; others which are bluish red; and at the end of the series, forms stained a fairly intense blue, in which scarcely a trace of red can be seen, and which by their peculiar notched periphery are evidently to be regarded as dying elements.

Ehrlich put forward the theory, that this remarkable behaviour towards dyes indicates a gradual death of the red blood corpuscles, that is of the old forms, leading to a coagulation necrosis of the discoplasm. The latter takes up, as is the case in coagulation necrosis, the proteids of the blood, and acquires thereby the power of combining with nuclear stains. At the same time the discoplasm loses its power of retaining the hæmoglobin, and gives it up to the blood plasma in ever increasing quantity as the change proceeds. Hence the disc continues to lose the capacity for the specific hæmoglobin stain.

Objection has been raised to these views from many quarters, especially from Gabritschewski, and afterwards from Askanazy, Dunin and others. The polychromatophil discs are not, they say, dying forms, but on the contrary represent young individuals. The circumstance, that in certain anæmias the early stages of the nucleated red corpuscles are variously polychromatic, was evidence for this opinion.

In view of the great theoretical importance which attaches to this subject, the grounds for regarding this change as degenerative, may be here shortly brought forward.

1. The appearance of the erythrocytes which shew polychromatophilia in the highest degree. By the notching of their margins they appear to eyes practised in the judgment of morphological conditions, in a stage almost of dissolution, and as well-pronounced degeneration forms.

2. The fact that by animal experiment, for instance, in inanition, their appearance in large numbers in the blood can be produced. That is, precisely in conditions, where there can be least question of a fresh production of red blood corpuscles.

3. The clinical experience, that in acute losses of blood in man, these staining anomalies, can be observed in numerous cells, within so short a time as the first 24 hours. Whilst in our observations, which are very numerous upon this point, embracing several hundred cases, and carried out with particular care, no nucleated red blood corpuscles in this space of time can be found in man[8].