Demonstration of yeast scars by fluorescence microscopy

Abstract

D URING the multiplication of yeast cells scars develop at cell-wall sites after separation of the daughter cell, appearing in the optical microscope as an unevenness of the surface. Barton [4] was the first to study systematically the types of scars in living, plasmolysed and stained cells of Saccharomyces cerevisiae. Later the structure of the cell wall in whole cells [3], in isolated membranes [l, 6, 10) and in ultrathin sections [I, 2, 91 was studied with the aid of the electron microscope. Since one can determine the relative age of cells from the number of scars, methods designed to demonstrate yeast bud scars should be of interest to cytologists [3], but hitherto there has been no method for studying scars on intact cells [5]. In the course of cytological studies of yeasts in this laboratory with aid of the fluorescence technique, the fluorochrome primulin was used among other agents. This non-toxic vital dye was used for the differentiation of viable and non-viable yeast cells [7, 8, 121. It was found, however, that at higher concentrations primulin causes an intense greenish fluorescence of the scars on the cell wall. The species studied were grown in modified medium of Olson and Johnson [ll] for 24-48 hr at 28°C on a shaker. The centrifuged and washed cells were suspended in the primulin dye solution in concentrations ranging from 1: 1000 to 1: 5000. Specimens were prepared from the suspension and the coverglass embedded in Vaseline. They were stained for 5-30 min. The specimens were observed in the Nf microscope (Zeiss) with an illuminator 01-17 (USSR) fixed on the microscope tubus. The source of illumination was a highpressure mercury vapour lamp DRSH-250. The following filters were used: FS-12 mm, exciter filter with 85 per cent transmission at 380 my, and the S3S-7, S3S-14, BS-8 filters and the ZHS-18 suppression filters. The photographs were made with the Miflex (Zeiss) camera, Ilford HPS film was used. Fluorescence microscopy was used for studying the yeast cell surface with the scars developing during budding, fission and reproduction by the intermediate process. For this purpose 28 species of various genera of yeasts were used. Budding yeasts.-In the majority of the strains investigated two kinds of scars were found, birth scars and bud scars, which have been previously described [l, 4, 61. The birth scar (Fig. la) indicates the site of the separation of the new cell from the mother cell. No fluorescent substance was found to accumulate at its margin. The bud scars (Fig. 1 b and 5) are formed after the separation of the daughter cells. On their margin a circular fluorescent thickening was observed so that these scars resemble a crater. In Saccharomyces cerevisiae the maximum of 25 bud scars were counted at the end of the logarithmic phase of growth.