the two nuclei thus formed are of equal number, and now come to lie together. After a longitudinal division of the chromosomes, the daughter chromosomes glide along the filaments of the achromatic spindle, developed from the centrosome of the male pronucleus, toward its two poles, as in ordinary mitosis. This they do in such a manner that an equal distribution of the male and female daughter chromosomes results. Then follow. the stages of the anaphase.

From the above description of the process of fertilization it is seen that it consists, in the end, of a union of the nuclei of both sexual cells.

If paternal qualities are inherited by the offspring, this can only take place through the nucleus, or through the centrosome of the male sexual cell. In other words, it can be safely said that these structures, or the nucleus alone, are the principal means of transmitting inherited qualities. The same may also be said of the female pronucleus. There is no doubt that the first two segmentation cells of the ovum are equally provided with male and female nuclear elements. Since all future cells are derivatives of these two, it is possible that the nucleus of every somatic cell (body-cell) is hermaphroditic.

E. CHROMATOLYSIS.

In the living organism many cells are destroyed during the various physiologic processes and replaced by new ones. On the death of a cell, changes take place in its nucleus which result in its gradual disappearance. These processes, which seem to follow certain definite but as yet unfamiliar laws, have been known since their study by Flemming (85, I) by the name of chromatolysis (karyolysis). The nuclei during the course of these changes show many varied pictures.

TECHNIC.

113. In a fresh condition, cells do not show much of their internal structure. Epithelial cells of the oral cavity, which can easily be obtained and examined in the saliva, show really nothing except the cell outlines and the nuclei. More, however, can be seen in young ova isolated from the Graafian follicles of mammalia; or the examination may be facilitated by using the ovary of a young frog. Tissues that are especially adapted for the observation of cells in a fresh condition are small ova, blood-corpuscles, and epithelia of certain invertebrate animals (shellfish,

etc.).

Unicellular organisms such as amebæ, infusoria, and many low forms of vegetable life make also good material for this purpose.

Protoplasmic currents are best seen in the tactile hairs of the nettle. Should fresh animal cells be desired, amebæ can occasionally be found in muddy or marshy water. The same phenomena may be observed in the leucocytes of the frog or, better still, in the blood of the crab.

114. In order to make a detailed study of the minute relationship of the different cellular structures, it is necessary to fix the cells; the same is true of nuclear division and cell proliferation. Although this process has been observed in living cells, it was not until it had been thoroughly worked out in preserved preparations. The best results in the study of the cell are obtained by methods that will be subsequently described. Fresh tissues are absolutely essential.

According to Hammer, mitosis in man does not cease immediately after death. The nuclei suffer chromatolytic destruction, and the achromatic spindle is the last element to disappear.

115. Flemming's solution (vid. T. 17) here deserves first mention as a fixative. The tissues are imbedded, sectioned, and stained with safranin (vid. T. 66). An equally good fixative is Hermann's solution, which may be combined with a subsequent treatment with pyroligneous acid (vid. T. 18). Rabl fixes with a 0.1-0.12% solution of chlorid of platinum, washes with water, passes into gradually stronger alcohols, then stains with Delafield's hematoxylin (vid. T. 62), and finally examines the preparation in methyl alcohol.

116. Mitoses can also be seen by fixing in corrosive sublimate, picric acid, chromic acid, etc., and staining in bulk with hematoxylin or carmin, although perhaps not so well as by the preceding method. The objects to be examined are best when obtained from young and growing animals, especially those possessing large cells. Above all are to be recommended the larvæ of amphibia, like the frog, triton, and salamander. If examination by means of sections be undesirable, thin structures should be procured, such as the mesentery, alveoli of the lungs, epithelium of the pharynx, urinary bladder, etc. These have the advantage of enabling one to observe the whole cell instead of parts or fragments of cellular structures. In sections of a larva that has been fixed in toto, mitotic figures can be seen in almost all the organs, and are particularly numerous in the epithelium of the epidermis, gills, central canal of the brain and spinal cord, etc. Other organs, such as the blood, liver,

and muscle, also show mitoses.

117. Certain vegetable cells are peculiarly adapted to the study of mitosis, as, for instance, those in the ends of young roots of the onion. The onion should be placed in a hyacinth glass filled with water and kept in a warm place. After two or three days numbers of small roots will be found to have developed. Beginning at the points, pieces 5 millimeters in length are cut, which are treated in the same manner as animal tissues. These are then cut, either transversely or longitudinally, into very thin sections (not over 5 μ in thickness). In one plane, polar views of the mitoses are obtained; in the other, lateral views.

118. The methods used for demonstrating the remaining parts of the cell and its nucleus (except the chromatin) are, as a rule, more complicated, and consequently less reliable. In order to see the centrosome,

the spindle fibrils, the linin threads, and the polar rays, one of the methods already described may be used; viz., the treatment with pyroligneous acid of objects previously fixed in osmic acid mixtures.

119. According to Hermann (93, II), sections from such preparations can be double-stained as well as those that have not been treated with pyroligneous acid. They are accordingly stained with safranin in the usual manner, and afterward treated from three to five minutes with

the following solution of gentian violet: 5 c.c. of a saturated alcoholic solution of the stain is dissolved in 100 c.c. of anilin water. The latter is composed of 4 c.c. of anilin oil in 100 c.c. of distilled water. This is shaken in a test-tube and then filtered through a wet filter. The sections are then placed in a solution of iodin and iodid of potassium (iodin 1 gm., iodid of potassium 2 gm., water 300 c.c.) until they have become entirely black, after which they are immersed in alcohol until they receive a violet tinge with a slight dash of brown. By this means the chromatin network, the resting nuclei, and the chromosomes in both of the spirem stages appear bluish-violet, while the true nucleoli are pink. The chromosomes of the aster and diaster are colored red.

120. Flemming (91, III) recommends the following method: Fixation. by his mixture (T. 17); the specimens or thin sections are then placed in safranin from two to six days (T. 66), washed for a short time in distilled water, and then immersed in absolute alcohol weakly acidulated with hydrochloric acid (1 : 1000), until no more color is given off. They are then washed again with distilled water and placed in a concentrated solution of anilin-water-gentian-violet from one to three hours. After a third rinsing in distilled water, they come into a concentrated aqueous solution of orange G, until they begin to assume a violet color. Then wash with absolute alcohol, clear in clove or bergamot oil, and mount in Canada balsam.

121. A comparatively simple method showing the different structures of the cell and its nucleus with great clearness consists in staining with Heidenhain's hematoxylin (vid. T. 65).

122. Solger (89, I and 91) has discovered that both chromosomes and polar rays are shown in an exquisite manner in the pigment cells of the skin (corium) of the frontal and ethmoidal regions of the common pike (vid. Fig. 35). The preliminary treatment is optional, Flemming's solution or corrosive sublimate being the best. These cells illustrate the stability of the radiate structures of protoplasm, the polar rays showing as parallel rows of pigment granules.

123. The various structures of resting and dividing nuclei and cells are of such a complicated nature that they can be observed only with great difficulty in ordinary objects, because of the crowding of so many elements into a comparatively small space. For example, salamandra maculosa, which has become a classic histologic object through the researches of Flemming, possesses somatic cells whose nuclei have no less than twenty-four chromosomes. (It may here be remarked that, curiously enough, salamandra atra has only half this number.) Consequently, van Beneden's discovery (83), that the somatic cells of ascaris megalocephala have only four primary chromosomes, is a fact of considerable importance. Boveri (87, II and 88) has even found an ascaris showing only two chromosomes. As these animals also show distinct achromatic figures in the protoplasm of their ova and sperm cells, they are certainly worthy of being regarded as typic specimens for laboratory purposes. The processes of cell-proliferation are almost diagrammatic in their dis

tinctness.

After opening the abdominal wall of the animal, the ovisacs are removed, their numerous convolutions separated as much as possible, and then fixed for twenty-four hours in a picric-acetic acid solution

(a concentrated aqueous solution of picric acid diluted with 2 vols. of water to which per cent. glacial acetic acid is added). Then follows washing for twenty-four hours with water, after which the specimen is transferred to increasing strengths of alcohol (Boveri, ibid.). Different regions of the ovisacs contain ova in various stages of development, those nearest the head containing cells ripe and ready for fecundation, while in the more posterior regions are ova in varying stages of segmentation showing mitoses. Specimens fixed in the manner above described can be stained with a borax-carmin solution. After staining, the ova are gently pressed out with needles upon a slide, separated, covered with a cover-glass, and cleared by gradual irrigation with glycerin. The ova, especially the segmentation spheres, are very small, and can be examined only under high magnification. In spite of the minuteness of the object and the fact that the yolk does not take the stain, and, on account of

its high refractive index, distorts the picture to a considerable extent, the mitotic figures are beautifully distinct.

124. Certain methods of treatment bring out in both cells and nuclei the presence of peculiar granules. The latter have been especially studied and described by v. Altmann (94, 2d ed.). The methods that he applies are as follows: The specimens of organs of recently killed animals are fixed in a mixture consisting of equal volumes of a 5% aqueous solution of potassium bichromate and a 2% solution of osmic acid, remaining in the mixture for twenty-four hours. They are then washed for several hours in water and treated with ascending strengths of alcohol; viz., 70, 90, and 100%. The specimens are now placed in a solution of 3 parts of xylol and I part of absolute alcohol, then in pure xylol, and finally in paraffin. The tissues imbedded in paraffin must not be cut thicker than 1 to 2 p. Altmann mounts according to the following method: A rather thick

solution of caoutchouc in chloroform (the so-called traumaticin of the Pharmacopeia-1 vol. guttapercha dissolved in 6 vols. chloroform) is diluted before use with 25 vols. of chloroform and the resulting mixture poured upon a slide. The latter is tilted, and after evaporation of the chloroform, heated over a gas flame. The paraffin sections are mounted upon the slides so prepared and then painted with a solution of guncotton in aceton and alcohol (2 gm. guncotton dissolved in 50 c.c. of aceton, 5 c.c. of which is diluted with 20 c.c. of absolute alcohol). After painting with this solution, the sections are firmly pressed upon the slide with tissue paper, and after drying are made to adhere more closely by slight warming. Fixation to the slide with water is equally good. The sections can now be treated with various staining solutions without becoming detached from the slides. The paraffin is gotten rid of by immersing in xylol, after which the specimens are placed in absolute alcohol. Fuchsin S. can be used as a stain (20 gm. fuchsin S. dissolved in 100 c.c. anilin water). A small quantity of this solution is placed upon the section, and the slide warmed over a flame until its lower surface becomes quite perceptibly warm and the staining solution begins to evaporate. The slide is then allowed to cool, washed with picric acid (concentrated alcoholic solution of picric acid diluted with 2 vols. of water), after which it is covered with a fresh quantity of picric acid, and again, but this time vigorously, heated (one-half to one minute). Occasionally the same results can be obtained by covering the section for five minutes with a cold solution of picric acid of the above strength. This last procedure has a decided influence upon the granula, and gives rise to a distinct differentiation between them and the remaining portions of the cell, the latter appearing grayish-yellow, while the granula themselves appear bright red. In some cases where the granula can not be sharply differentiated from the remaining structures, it may be necessary to repeat the staining process. Xylol-Canada balsam should not be used for mounting, as it has a bleaching effect upon the osmic acid in the specimen. Mount either in liquid paraffin (Altmann) or in undiluted. Canada balsam, which is easily reduced to a fluid state, whenever needed, by heating.

There is another method used by Altmann which deserves mention, but practical application of which must be improved upon in the future; this consists in freezing the specimens and drying them for a few days in the frozen condition in a vacuum over sulphuric acid at a temperature of about -30° C.

According to Fischer, dilute solutions of pepton when treated with various reagents (especially with a potassium bichromate-osmium mixture) form precipitates and granules which are remarkable in that they react to stains exactly as do Altmann's granula. It is, therefore, doubtful whether Altmann's granules should be regarded as vital structures.

125. Altmann (92) has also devised a simpler negative method for demonstrating the granula. Fresh specimens are placed for twenty-four hours in a solution consisting of molybdate of ammonium 2.5 gm., chromic acid 0.35 gm., and water 100 c.c.; then treated for several days with absolute alcohol, sectioned in paraffin, and colored with a nuclear stain such as hematoxylin or gentian. The intergranular network is colored, while the granula remain colorless. The amount of chromic acid used (0.25 to 1%) varies according to the object treated; if molybdate of ammonium alone be used, the nuclei will appear homogeneous,