STUDIES ON DEOXYRIBONUCLEOPROTEIN IN LEUKOCYTES AND RELATED CELLS OF MAMMALS VII. THE FAST GREEN HISTONE CONTENT OF RABBIT LEUKOCYTES AFTER HYPOTONIC TREATMENT

Abstract

Peripheral leukocytes of rabbit were subjected to the following treatments: (1) 3 parts blood to 2 parts water; (2) 0.95% sodium citrate; (3) Ohnuki's medium. Treated and control leukocytes were then smeared, fixed in 9:1 methanol-commercial formalin and stained for histones according to Alfert and Geschwind. Cytophotometry was carried out with the Barr and Stroud integrating microdensitometer. The cells treated with water increase their dye content by 50%; this increase is less marked (30%) in the cells incubated in Ohnuki's mixture, while there is a 15% decrease in case of citrate. The spread of values "between cells" is larger than the values found with the Feulgen reaction because of factors other than chromatin compaction. (1) There are fluctuations in staining intensity in different areas of the same slide. (2) Nonhistone substrates take the dye, such as hemoglobin of erythrocytes, cytoplasm and specific granules of heterophil granulocytes. (3) Apparently, a certain amount of histones is lost when nuclei swell in hypotonic citrate. It is concluded that, although histone dye binding behaves in a manner similar to Feulgen with respect to changes in the physical state of the deoxyribonucleoprotein complex, the quantitation of fast green histone is a less reliable method for the study of these changes.