Cytochemical Observations on the Interphase Nucleus of the Parasitic Ameba Hydramoeba hydroxena (Entz)

Abstract

The study was undertaken to determine the distribution of DNA, RNA, and histone in the nucleus, and thereby to clarify the nature of the peripheral layer. Whereas the endosome contains granules and a network which are definitely Feulgen-positive, the peripheral layer is only faintly so. Nevertheless, it contains histone and stains intensely red with methyl green-pyronin, and blue with toluidine blue. Ribonuclease removes nearly all its basophilic material, which is clearly RNA. Thus, the peripheral layer consists largely of chromatin. The original description of Hydramoeba hydroxena included an account of the general morphology of the nucleus (Entz, 1912). Further observations were made by Wermel (1925), who reported that the interphase nucleus is Feulgen-negative. A more thorough study was made by Reynolds and Threlkeld (1929), who interpreted the characteristic intranuclear mass as a compound karyosome. The peripheral karyosomal layer, in which discrete blocks are usually visible, was designated as ectokaryosomal chromatin and the central body as an endosome. The terminology is inconsistent in that it presents the paradoxical condition of an endosome within a karyosome, whereas these terms are generally used as synonyms (Mackinnon and Hawes, 1961, p. 12). For convenience, I shall retain the term endosome, but karyosome will not be used. Actually, the results of Reynolds and Threlkeld do not show conclusively that the peripheral layer contains chromatin. These authors did not use the Feulgen reaction, but based their conclusion on stainability in iron hematoxylin. Wermel's negative results are likewise inconclusive, since he counterstained all his specimens in light green. The counterstaining of Feulgen preparations in light green or fast green is not always advisable; if the reaction is weak, these dyes may obscure the characteristic reddish-purple color and lead to erroneous interpretations. It is evident that the distribution of chromatin in the nucleus of Hydramoeba hydroxena is not entirely decided. In the present study, several cytochemical methods, rather than conventional hematoxylin staining, were used in order to determine the distribution of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) in the nucleus, and thereby to clarify the nature of the peripheral layer. MATERIALS AND METHODS The host was the false brown hydra, Hydra pseudoligactis Hyman, which has relatively little resistance to Hydramoeba infections (Stiven, 1962). Heavily infected hydras were kindly supplied by my colleague, Dr. Alan E. Stiven. Since hydramebae attach firmly to hosts of low resistance, the infected hydras and their amebae were fixed simultaneously and subsequently sectioned in paraffin at 4 p. The ameba nuclei were studied in such sections. The following methods were used: Feulgen reaction without counterstaining; fixation, Carnoy (with chloroform) or Schaudinn (with acetic acid) (Pearse, 1960, p. 823). Methyl green-pyronin Y; fixation, Carnoy (Pearse, loc. cit., p. 826). Toluidine blue for basophilia; fixation, Carnoy; 5 to 10 min in a 0.1% aqueous solution of the dye, followed by dehydration in tertiary butyl alcohol. Ribonuclease extraction (Pearse, loc. cit., p. 916). Fast green for nuclear histone; fixation, 10% neutral formalin (Conn et al., 1960, p. 219). Received for publication 6 May 1964.