THE OSMIUM TETROXIDE-P-PHENYLENEDIAMINE PROCEDURE REVEALS THE CHROMATID CORES AND KINETOCHORES OF MEIOTIC CHROMOSOMES BY LIGHT AND ELECTRON-MICROSCOPY

We analyzed first-metaphase meiotic chromosomes of the grasshopper Cho rthippus jucundus by two different methods, i.e., a silver impregnatio n technique and the osmium tetroxide-p-phenylenediamine (Os-PPD) proce dure, The former was applied on squashed testes previously fixed in et hanol-acetic acid, whereas for Os-PPD the material was not subjected t o any previous extraction treatment but was fixed in OsO4, treated wit h PPD, and embedded in Epon 812. Both techniques revealed chromatid co res and kinetochores regardless of the processing of the material (squ ashed or sectioned), Unstained Os-PPD sections were analyzed by light microscopy and transmission electron microscopy (TEM), The Os-PPD tech nique provided a high contrast of chromatid cores and kinetochores in relation to the chromatin, which revealed a low electron density, To d etermine the Os-PPD reaction mechanism, the PAS procedure, as well as scanning electron microscopy (SEM) backscattering and SEM X-ray microa nalysis, was performed on sections, By use of the Os-PPD-PAS procedure , glycol groups formed by oxidation of osmium bound to aromatic substr ates were detected in chromatid cores and kinetochores by brightfield and fluorescence microscopy. A high Z contrast was detected in these s tructures by backscattered electron imaging. SEM X-ray microanalysis s howed osmium and phosphorus to be the main elements present on the chr omatid cores, Taking into account the known reactivity of OsO4 and the present results, the possible participation of nucleic acids as well as proteins in the Os-PPD reaction mechanism and in the composition of chromatid cores and kinetochores is discussed.