A colorimetric method is proposed for in situ cytophotometric estimation of ribonucleic acid (RNA) in biologic material, using a gelatin-RNA model system as a reference. Both specificity and stoichiometry of azure B dye binding to RNA in this model system were demonstrated. As the amount of dye binding is stoichiometric with the concentration of RNA, the regression equation generated from cytophotometric data of relative concentration ( E/d) of azure B-RNA complex as a function of concentration of RNA (picograms per cubic micron) can be used to calculate the absolute concentration (picograms per cubic micron) of RNA in biologic material. Comparison of such an estimate for nucleolar RNA in Purkinje cell nuclei of the mouse with an estimate derived from available experimental data in the literature is made to assess the validity of the reference. These estimates are in agreement, supporting the applicability of the proposed method.

The nucleoli of cells of the adult mouse were examined by staining with toluidine blue after removal of deoxyribonucleic acid from tissue sections by deoxyribonuclease treatment.The nuclei of each cell type examined contained one or more nucleoli. This was observed even in lymphocytes and neuroglia, although these cells have occasionally been described as anucleolated. In mature spermatids and spermatozoa, however, it was not possible to detect a nucleolus.The distribution of the number of nucleoli in many diploid cells exhibited a m o d e of two or three nucleoli per nucleus, and a range from 1 to 6 nucleoli.In presumedly diploid hepatic nuclei, the maximum number of nucleoli was six; but in presumedly tetraploid hepatic nuclei, it was 11. Thus, nearly twice as many nucleoli are present when the chromosome number is doubled. In view of this observation, it is suggested that six nucleolar organizers are present in the diploid chromosomal complement of the mouse. However, through failure of some nucleolar organizers or more probably through fusion of nucleoli, the number of these organelles in most nuclei is less than six.Since the time of the discovery of the nucleolus by Fontana in 1781 (Montgomery, 1898), the morphology and cytochemistry of this organelle have been the subject of numerous investigations.' Nevertheless, there is little precise data on the number of nucleoli present in each nucleus. Text-books state merely that there is one or more nucleoli per nucleus. It was therefore decided to count the number of nucleoli in a variety of cells of the mouse. In doing so, an opportunity was afforded to re-examine the belief of some hematologists that small lymphocytes and granulocytes have no nucleoli (Osgood and Ashworth, '37) and the similar opinion of some neuroanatomists about neuroglia (cf. Cajal, '13; Del Rio-Hortega, '32).A basophilic component exists in nucleoli and in chromatin; as a result, the staining of the two structures may be the same in routinely processed sections, making it difficult to distinguish and count all the nucleoli of a cell. An attempt was therefore made to establish a method by which nucleoli would be the only nuclear component stained.It is known that chromatin contains DNA with a small amount of RNA (de Robertis et al., '65). In contrast, the nucleolus contains RNA (Vincent, '52; Baltus, '54) as well as a small quantity of DNA (Lettr6 and Siebs, '54; Mulnard, '56; Gran-J. MOEPH., 119: 425-434.boulan and Granboulan, '64). Since both DNA and RNA are basophilic, the selective removal of DNA by enzymatic treatment will leave only RNA in the sections. After staining with a basic dye, the RNA-rich nucleoli are then demonstrated without ambiguity. If care is taken to use sections thick enough to include entire nuclei, the total number of nucleoli per nucleus may be readily counted. MATERIALS AND METHODSTissues and organs were excised from an adult male, albino mouse, weighing 30 gm. Under ether anesthesia, the animal was exsanguinated from the abdominal aorta by means of a hep...