Part 8

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.

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:

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

Baden Anilin and Soda manufactory, Kalle and Co.

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

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.