elo-splenic) or lymphatic, in corpuscles may so increase red, while the latter are w poikilocytosis.

., Fig. 3) is characterised by and e), and particularly by und mononuclear leucocytes. rrow of bones (myelocytes, a),

The dange des af normal blood, with which

3. Melanæmia and Lipæmia.-For Melanamia, see pp. 59 and 168. Lipamia occurs physiologically during digestion, and otherwise is present in chronic alcoholism, chronic nephritis, and diabetes, as well as after fractures, where fat enters the blood in consequence of injury to the marrow. Numerous small shining globules, some of them enclosed in white corpuscles, are found under the microscope.

4. Animal and Vegetable Parasites in the Blood.-The only vegetable parasites met with are Bacteria, of which the following are found:(1) Anthrax bacilli in anthrax, when a general infection has taken place (p. 128).

(2) Spirilla Obermeieri in relapsing fever (p. 156).

(3) The following also occur inconstantly, and in isolated cases:— Streptococcus and Staphylococcus pyogenes in pyæmia, and in the cases of ulcerative endocarditis due to these micro-organisms; tubercle bacilli in general acute tuberculosis; glanders bacilli in acute glanders; and, with still less constancy, Diplococcus pneumonia in pneumonia and the form of endocarditis produced by this species of bacterium; and lastly, Streptococcus erysipelatis in severe cases of erysipelas. In many of these cases, however, even when the attempt to find the respective bacteria with the microscope has failed, it will still be possible to demonstrate their presence by the process of cultivation.

Of the animal parasites, the Plasmodium malaria must first be mentioned, then also Filaria sanguinis hominis and Distoma hamatobium. The two latter, however, only occur in the tropics.

Examination of the Blood.—A. HISTOLOGICAL EXAMINATION. 1. General.-Blood is obtained from the living by puncturing the skin (previously purified) of the finger-tip or lobule of the ear with a sterilised needle, bringing a cover-glass down upon the blood as it oozes out, and spreading a very small quantity of the latter in a very thin film upon a slide If it is desired to prevent shrivelling of the red corpuscles, the examination should be carried out in 0.75 per cent. salt solution, a drop of which may be placed upon the skin and the puncture made through it; but otherwise it is better to examine the blood undiluted.

A simple examination of this kind is not usually sufficient, however, for the recognition of the different diseases of the blood, accurate counting, and perhaps also measurement and special staining, of the blood-cells being necessary in addition, as well as a determination of the contained hæmoglobin.

2. Counting and measurement of the corpuscles. For accurate determination of the number of the red and white corpuscles the Thoma-Zeiss apparatus should be used. This consists of a chamber 01 mm. in depth, upon the bottom of which are ruled 400 squares, each having a superficial area of of a square millimetre, so that the cubic contents of the column of fluid over each square amount to 1000 of a cubic millimetre. The blood must be previously diluted, which is most simply done by means of the bulbed pipette (mélangeur) supplied with the apparatus, using a 3 per cent. solution of common salt coloured with a little methyl or gentian violet (0·1 grm. dye to 150 grm. of the 3 per

4

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cent. salt solution) in order to stain the white corpuscles. The blood obtained by pricking the skin is first sucked up as far as the mark at the commencement of the bulb, and the filtered diluting fluid drawn in immediately afterwards until the mixture reaches the mark above the bulb. Since the latter holds one hundred times as much as the portion of pipette below it, a dilution of the blood of one hundred times is thus obtained. An even distribution of the blood-cells having been secured by shaking the fluid so as to mix it, the Thoma-Zeiss apparatus is filled from the pipette, a cover-glass laid over it, and the blood corpuscles then counted under a medium power in as many squares as possible, including not only the corpuscles within the squares but also those on the boundary lines. If now the sum-total thus obtained is multiplied by 4000 (ie., the cubic contents of the counting chamber), and by the number expressing the degree of dilution (ie., usually 100), and divided by the number of squares counted, we obtain the total number of corpuscles in a cubic millimetre of undiluted blood.

To measure the size of the corpuscles, a cover-glass is slightly warmed and brought over the drop of blood as it oozes out, and the latter is then examined in the dry state, the actual measurement being made by means of an eyepiece micrometer (see p. 23, note).

3. Staining of the corpuscles.-For the purpose of staining the different forms of blood-cells (in leucæmia, anæmia, etc.), the methods introduced by Ehrlich are resorted to. The blood is smeared upon cover-glasses, previously well cleaned1 with alcohol, in a very thin and equal layer, by laying two cover-glasses one upon the other and drawing them quickly and evenly apart. When dry, they are heated for half-an-hour to two hours at 120°-130° C. (in a small dry oven or in the hot-air sterilising apparatus, p. 34) in order to fix the hæmoglobin, and then stained with one of the two following mixtures:

(1) Thoroughly saturated aqueous solutions are first prepared of orange G, acid fuchsin, and methyl green-some undissolved pigment must still remain on the bottom of each bottle after standing for several days in a warm place-50 c.cm. of each solution are then, without shaking the bottles, poured together into a fresh flask, and a further addition of 50 c.cm. absolute alcohol, and 100 c.cm. water, is made to the mixture. The latter must be left standing for several weeks until the precipitate formed has completely subsided to the bottom, and should then be of a rusty-brown colour. The fluid must not be filtered, so that it is always used by conveying to the cover-glass to be stained a few drops taken from the central part by means of a dry pipette or glass rod. By rocking the cover glass continually to and fro the stain is kept from drying up anywhere for some minutes, after which the glass is rinsed in water and examined in thin Canada balsam. The nuclei of the cells now appear from greenish-blue or lightblue to bluish-violet (Plate I., Fig. 3), the neutrophil granules greyish- to reddishviolet (Plate J., Fig. 3, c), the eosinophil granulations yellowish-red to dark-red (Plate I., Fig. 3, d), and the haemoglobin orange-yellow (Plate I., Fig. 3, ƒ and g). Unfortunately the mixture just given is not constant in its action, in many

For the mode of cleaning, see also above, p. 26, note.

"It is impossible to give the exact time, as the behaviour of the hæmoglobin in this respect is very different in different cases. Instead of heating, the cover-glasses may also be deposited for two hours or longer in a mixture of equal parts alcohol and ether,

cases not staining well until it has become rather old, whilst at other times the reverse is the case. It may also happen that one or other of the pigments in the mixture fails to make itself sufficiently prominent in the staining of preparations, in which case more must be added.

(2) The second staining-fluid is prepared as follows:-10 grm. crystallised eosin, 13 to 15 grm. crystallised nigrosin, 8 grm. orange, and 70 to 100 grm. glycerin are well rubbed up together and allowed to stand for some days at 60° C. This mixture also is not filtered. In use, a drop having been placed upon a slide, the cover-glass after being smeared with the blood and heated is laid upon it, and the dye allowed to act for a considerable time, six hours at least. The cover-glass is then lastly rinsed in water and examined in Canada balsam. By this method the nuclei are stained black, the hæmoglobin yellow, and the eosinophil granules red, while the neutrophil granules remain unstained.

Besides these two staining solutions the double staining with methyl blue and eosin mentioned amongst the methods of examining Protozoa (p. 170) may be resorted to with advantage, as the different formed elements of the blood are characteristically stained in this way likewise.

Sections may also be prepared from blood. For this purpose some drops are let fall into a glass of 20 c.cm. capacity containing 5 c.cm. of a 2 per cent. osmic acid solution, the blood is distributed through it by shaking to and fro, and the vessel is then allowed to stand at rest. (As the red corpuscles sink first, then the white corpuscles, and lastly the blood-plates, the elements can also be obtained isolated in this way.) In twenty-four hours the mixture is shaken up afresh, and four or five drops transferred from it by means of a pipette to about 5 c.cm. of agar, kept fluid at a temperature of 37° C. This is poured into a little paper box or a capsule, and is hardened in 83 per cent. alcohol after it has set. It can then be cut into sections.

In case it is desired to stain the blood-cells in such sections, or in sections of hardened tissues and organs, by Ehrlich's method, Heidenhain recommends that thoroughly saturated (but filtered) solutions of orange, acid fuchsin, and methyl green, should also be first prepared, and 100 c.cm. of orange solution then mixed with 20 c.cm. of acid fuchsin, to which 50 c.cm. of methyl green are added with gentle agitation. For actual staining this mixture is diluted with water

in the proportion of 1 to 60-100. The fluid when diluted to this extent should become a deeper red on the addition of acetic acid, and should make a spot upon filter-paper which appears bluish-green in the centre and orange towards the margin. Should any one of the colours come out too strongly, or not strongly enough, in this test, the proportions of the mixture must be altered accordingly. The sections remain in the solution from six to twenty-four hours, and are decolorised with alcohol. The karyokinetic figures and the fragmented nuclei of the leucocytes are stained green, the resting nuclei blue, and the red corpuscles red.

4. Determination of the hæmoglobin.-This does not, strictly speaking, come within the bounds of Pathological Histology, but as it is indispensable for the differential diagnosis of certain diseases of the blood, it is proposed to describe the process briefly in this place. For this estimation Fleischl's hæmometer may be employed with advantage. In this instrument the colour of the blood to be examined, which is dissolved in water, is compared with that of a wedge of red-tinted glass, which is pushed beneath a little table, arranged after the manner of the stage of a microscope, and having a circular aperture in its

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191

centre. Over this aperture fits a short metal tube closed below by a little plate of glass, and divided by a vertical partition into two halves, one of which is adjusted so as to stand above the coloured wedge, the other merely over the empty aperture. Some water is first delivered into both halves, and then the blood, having been collected by means of a capillary tube which is supplied with the apparatus, is transferred to the half which does not lie above the coloured wedge. Both halves are now completely filled with water, and the glass wedge is then pushed along until the fluid in the two appears to be of the same depth of red, when the scale is lastly read off. The apparatus can only be used by artificial light, the illumination coming from a plate of gypsum occupying the position of the mirror in a microscope, and reflecting the light from a lamp or gas-flame.

The number read off on the scale, 70 for instance, signifies that the blood contains 70 per cent. of the normal quantity of haemoglobin; and if we now compare the contained hæmoglobin thus estimated with the number of the red corpuscles as previously ascertained, we learn whether the amount of hæmoglobin, i.e., the colour index, of the individual red corpuscle remains normal or has become larger or smaller than it should be. This determination is of importance from the point of view of diagnosis, inasmuch as in chlorosis the number of the red corpuscles is frequently not diminished, although the colour index is; whereas in pernicious anæmia this is reversed, and in other forms of anæmia both numbers may be reduced.

5. Recognition of blood and fibrin.-Should proof be needed in the case of dried blood that it really is blood which has to be dealt with, the required evidence may be supplied by the recognition of red corpuscles or the demonstration of hæmin crystals. The former result can be gained by softening in a 0·8 per cent. solution of common salt or a 33 per cent. solution of caustic potash; but no conclusion must be drawn from the relative sizes of the softened red corpuscles as to the source of the blood, i.e., whether from men or animals.

To show Teichmann's hæmin crystals, some drops of glacial acetic acid and a minute grain of salt are added to small dry particles of the substance under examination which have been placed upon a slide, and the whole is carefully heated until bubbles are given off. The crystals are dark-brown rhombic plates. For the special staining of fibrin, see p. 75.

B. BACTERIOLOGICAL EXAMINATION.-As in the case of other fluids, this is done as a rule by making stained cover-glass preparations (p. 26). If it is desired to stain the red corpuscles as well as the bacteria, the preparations after treatment by Gram's method should be transferred to eosin as a counter-stain (p. 29); but where simple decolorisation of the red corpuscles is required, the coverglasses, after being passed through the flame, are first laid for about ten seconds in 1 to 5 per cent. acetic acid (which decolorises the red corpuscles), then thoroughly washed with water and dried, and lastly stained. With regard to the choice of stain and the nature of the process required for each individual bacterium and for the Plasmodium malaria, see Part II., Chapters V. and VI. desired to make cultivation experiments with the blood of living skin must be disinfected in the manner described on p. 35. carefully done the blood may sometimes be transferred directly to oblique agar or to bouillon,- that is, when but few bacteria are suspected to be present in it.

Should it be individuals, the If this is very

1 When the bacteria in question admit of staining by this method.