For the fixation of material used in these studies, it has been found that the osmic acid mixtures of Flemming and Hermann are the most generally applicable and are productive of the best results. In connection with these, however, Gilson’s acetonitric-sublimate mixture has been tried, and frequently affords an excellent fixation. Extensive shrinkage in the melted paraffin is sure to follow the use of sublimate mixtures unless celloidin is used to support the soft tissue. This double infiltration of celloidin, followed by paraffin, has been found the best method of securing clear and accurate figures, for, because of the lessened shrinkage, the elements are not crowded together and rendered indistinct. This circumstance is particularly fortunate in the case of the Locustid cells, where the nuclear elements are so numerous and crowded.
The stains employed are the iron-hæmatoxylin of Heidenhain and the safranin-gentian violet-orange combination of Flemming. For general purposes, nothing excels the hæmatoxylin stain, but it is frequently advantageous to trace the chemical changes undergone by the different cell elements in the process of mitosis, and the aniline stain above mentioned serves excellently for this. Kernschwarz has also been found a valuable stain for some purposes.
III. NOMENCLATURE.
The terminology as outlined in a former paper (17) will be followed in the present one.
IV. OBSERVATIONS.
(a) General Form and Structure of the Testes.
The testes of the Locustidæ are paired structures lying in the anterior dorsal portion of the abdomen. Each organ is made up of numerous short follicles, which are bound together by a connective tissue investment. In adult animals the testes are a bright yellow color, while in nymphs the color varies from white in the youngest to yellow in the oldest. The pigment is lodged in the connective tissue sheath about the testis, and is seen in sections as irregularly rounded masses in the cytoplasm.
(b) The Spermatogonia.
No further discussion of the spermatogonia will be given here than is necessary for an understanding of the derivation of the first spermatocytes. As appears to be universally the case, the second spermatogonia, in their last generation at least, are much reduced in size as compared with the primary spermatogonia that preceded them and with the first spermatocytes that arise from them. The entire cell stains dark with almost all stains and, as the nucleus occupies nearly the whole cell body, the chromatin appears relatively large in amount. A cyst of spermatogonia, therefore, looks as if composed almost entirely of chromatin aggregated into rounded masses—the nuclei.
The chromosomes are of the rod type, and divide longitudinally in each mitosis. The number of chromosomes is large and could not be determined with absolute certainty, but a number of careful enumerations makes it evident that there are most probably thirty-three. In most species of Locustids, one chromosome is easily distinguished from the others by its larger size and tardy division in the act of metakinesis. This is the element as described for Xiphidium, which passes into the first spermatocyte as a formed chromosome, while its fellows break up into the spireme.