The structure of the nucleus in saccharomyces cerevisiar

Abstract

The Living Cell as the Standard. The investigations embodied in this thesis were planned to elucidate the structure of the nucleus in Saccharomyces cerevisiae using the living cell as the standard. Subsequent to the publication of Parts III, IV and V (Royan and Subramaniam, April 1956; Royan, July 1956; September 1956) the author came across Müller’s (September, 1956) observations on the nucleus of living yeast. According to his details of the structure of the nucleus, conclusions can be clarified only by a comparison of fixed and stained material with living cells. Bright and dark field illumination were found unsuitable for observations on living yeast. Even the phase contrast did not improve the situation when yeast cells were examined in normal media. By suitable adjustment of the refractive index of the medium, Müller obtained the photographs illustrating his contribution. The strain of Saccharomyces carlsbergensis employed as material for the observations reported in this thesis: the nucleus could be photographed in 66–120 hr old cultures not only under phase contrast but also under bright as well as dark-field illumination (Parts III, IV and V). Müller’s photographs are of cells of a strain of Saccharomyces carlsbergensis. Very recently Thyagarajan and Subramaniam (1956) have been able to observe and photograph under phase contrast the nucleus in 160–190 hr wort cultures of a strain of S. carlsbergensis without any special adjustment of the refractive index of the medium.

The Behaviour of the Nucleus during Fixation. Müller (1956) was unsuccessful in his attempts to follow, under the microscope, the behaviour of the nucleus during fixation and remarks that no fixing fluid gives a faithful reproduction of the structure of the living nucleus. The photomicrographs presented in Part IV would indicate that the nucleus could be followed up during the cytological procedures like fixation, hydrolysis and staining.

The Reduction in Cell Size on Fixation. It would be obvious from Parts IV, VII and VIII that among the fixatives tried, only iodine–formaldehyde–acetic acid gives life-like preservation of nuclear details. As there is an overall reduction in the size of the cells on fixation, since the orientation and morphology of the structures visible in the living cell are preserved in the fixed, it could be assumed that the contraction or reduction is uniform throughout the cell. It is only when cell organelles like the vacuole are not fixed properly that distortion in the orientation or even the morphology of the nucleus is to be expected.

The Necessity for Hydrolysis. The high RNA content of the yeast cell and the resultant basophilia necessitate its removal by acid hydrolysis as an essential step preceding staining. Hydrolysis of fixed material obviates the difficulties in staining referred to by Müller (1956).

Heidenhain’s Haematoxylin and its Importance. Though the hydrolysed cells could be stained by different stains like leuco-basic fuchsin, Giemsa’s solution, and iron alum–haematoxylin, the pictures seen in haematoxylin alone reveal accurately the structural details observed in the living nucleus.

The Feulgen Technique and its Importance. The Feulgen technique enables the confirmation of identifications. Even in hydrolysed cells there are granules scattered in the cytoplasm which are stained by haematoxylin. They are Feulgen-negative and hence can be presumed to be non-cytoplasmic organelles. The vacuole is usually bereft of any structure stainable by haematoxylin. Since the vacuole and its contents are uniformly Feulgen-negative, the vacuole turns out to be a cytoplasmic inclusion unrelated to the nucleus. It was indicated in Part III that if we have to concede the possibility that the vacuole is also “nuclear” in character, the probability of the yeast cell being binucleate may have to be considered. The Feulgen-negative character of the vacuole emphasises that yeast is uninucleate and that the nucleus occupies an extra-vacuolar position.

The Structure of the Yeast Nucleus and its Significance. On the basis of their structure in living cells, Müller (1956) classifies the nuclei into two types. While it is an optically homogeneous vesicle in Saccharomyces cerevisiae, in Saccharomycodes ludwigii the nucleus is said to be more differentiated in that it contains a centrally located, optically dense body. It was shown in Part IV that the nucleus of S. cerevisiae exhibits a range of variation in its structure excepting when it is homogenously refractive (Photo 15, Part IV). The nucleus is seen to possess a distinct nuclear membrane enclosing formed structures having varying orientation inside it. The occurrence of a deeply distinct, Feulgen-positive granule inside the nucleus is suggestive of the presence of a heterochromatic–chromocentre inside the nucleus (Lindsey, 1953; Müller, 1956). The nucleus of yeast resembles those of higher organisms. There have been periodic reports on the presence of a nuclear membrane in yeast (Hannaguy, 1896; Guilliermond, 1920; Subramaniam, 1946; Linde, 1951; Windisch, 1952). Proof for its existence has been offered in this thesis (Part VI) from observations on living cells under phase contrast and dark-ground illumination as also from stained preparations (Part III). The presence of a nuclear membrane enables a study of the orientation of the nucleus in relation to the vacuole and to distinguish structures lying inside and outside the nucleus. It is hoped that a clear idea of the structure of the nucleus and its reactions to fixatives and stains would render it possible to elucidate its behaviour during cell division.