Notes on the relationship between the permeability and viability of yeast cells

При инкубации с дeзоксихолатом клeтки дрожжeи станонятся проницаeмыми для соKeй янтарной кислоты и для фeррицианида. Чeрeз нeкотороe врeмя млeтки погибают и окрашиваютсн основными краситeлями. Так как коррeлпции мeжду проницаeмостью и окрашиваeмкстью клeток нe наблюдалось, высказываeтся врeдположeниe, что при частичном разрушeнии барьeра проницаeмости клeтки в тeчeниe нeкоторого врeмeни могут оставаться живыми.     

Effect of osmotic stress on the ultrastructure and viability of the yeast Saccharomyces cerevisiae

Exposure of the yeast Saccharomyces cerevisiae to hypertonic solutions of non-permeating compounds resulted in cell shrinkage, without plasmolysis. The relationship between cell volume and osmolality was non-linear; between 1 and 4 osM there was a plateau in cell volume, with apparently a resistance to further shrinkage; beyond 4 osM cell volume was reduced further. The loss of viability of S. cerevisiae after hypertonic stress was directly related to the reduction in cell volume in the shrunken state. The plasma membrane is often considered to be the primary site of osmotic injury, but on resuspension from a hypertonic stress, which would have resulted in a major loss of viability, all cells were osmotically responsive. The effects of osmotic stress on mitochondrial activity and structure were investigated using the fluorescent probe rhodamine 123. The patterns of rhodamine staining were altered only after extreme stress and are assumed to be a pathological feature rather than a primary cause of injury. Changes in the ultrastructure of the cell envelope were examined by freeze-fracture and scanning electron microscopy. In shrunken cells the wall increased in thickness, the outer surface remained unaltered, whilst the cytoplasmic side buckled with irregular projections into the cytoplasm. On return to isotonic solutions these structural alterations were reversible, suggesting a considerable degree of plasticity of the wall. However, the rate of enzyme digestion of the wall may have been modified, indicating that changes in wall structure persist.     

Mitotic viability and metabolic competence in UV-irradiated yeast cells

Colony formation is the classic method for measuring survival of yeast cells. This method measures mitotic viability and can underestimate the fraction of cells capable of carrying out other DNA processing events. Here, we report an alternative method, based on cell metabolism, to determine the fraction of surviving cells after ultraviolet (UV) irradiation. The reduction of 2,3,5-triphenyl tetrazolium chloride (or TTC) to formazan in mitochondria was compared with cell colony formation and DNA repair capacity in wt cells and two repair-deficient strains (rad1Delta and rad7Delta). Both TTC reduction and cell colony formation gave a linear response with different ratios of mitotically viable cells and heat-inactivated cells. However, monitoring the formation of formazan in non-dividing yeast cells that are partially (rad7Delta) or totally (wt) proficient at DNA repair is a more accurate measure of cell survival after UV irradiation. Before repair of UV photoproducts (cis-syn cyclobutane pyrimidine dimers or CPDs) is complete, these two assays give very different results, implying that many damaged cells are metabolically competent but cannot replicate. For example, only 25% of the rad7Delta cells are mitotically viable after a UV dose of 12 J/m(2)75% of these cells are metabolically competent and remove over 55% of the CPDs from their genomic DNA. Moreover, repair of CPDs in wt cells dramatically decreases after the first few hours of liquid holding (L.H.; incubation in water) and correlates with a substantial decrease in cell metabolism over the same time period. In contrast, cell colony formation may be the more accurate indicator of cell survival after UV irradiation of rad1Delta cells (i.e., cells with little DNA repair activity). These results indicate that the metabolic competence of UV-irradiated, non-dividing yeast cells is a much better indicator of cell survival than mitotic viability in partially (or totally) repair proficient yeast cultures.     

Effect of low doses of ionizing irradiation on viability of cortical thymocytes

The cortical thymocytes of rats in whole organism, isolated lobes of thymus and cells suspension were exposed to ionizing radiation in a wide range of doses (0.1-200 cGy). In contrast to relatively high dose radiation (50-200 cGy), exposure to doses of 10 cGy resulted in cell death without DNA degradation. The level of doses lower than 10 cGy (0.5-5 cGy) induced thymocyte death which is independent of DNA degradation, RNA and protein synthesis. With decrease in radiation dose, the increase of latent period preceding cell death took place.     

Effect of low-power red light laser irradiation on the viability of human skin fibroblast

Human skin fibroblast monolayers (S-126 cell line) were exposed to laser radiation (wavelength 670 nm, power density 40 mW/cm²). The energy densities were 2 J/cm² and 12 J/cm², respectively, and the irradiation was carried out at a temperature of 22°C. For fibroblast viability evaluation, the colorimetric assay (conversion of thiazolyl blue to formazan) was used. The experiments were carried out at 37°C, in the presence of 5% CO₂, and at different time periods of incubation after irradiation (2, 4, 8 h and 1, 2, 3, 4, 5 days). The results indicated that there was a certain stimulating effect on the long-term proliferation of skin fibroblasts and that the stimulation proceeded in two stages, the first one 2 h and the second one 3 days post-irradiation. Received 7 January 1998 / Accepted in revised form: 11 June 1998     

Effect of centrifugation on the viability of Burkitt lymphoma cells

This study reports some findings on the effects of centrifugation on the viability of mammalian cells. The authors used Burkitt lymphoma cells cultivated in a synthetic medium containing 10% fetal calf serum for all experiments. Batch centrifugations were conducted in a RC2-B centrifuge (Ivan Sorvall, Incorporated, Norwalk, Connecticut USA) operated at 0 and 25°C. During centrifugation we exposed the cells to gravitational fields ranging from 24,800 to 42.200g. The results showed that at, 0°C and 25,800 or 42,000g no loss in cell viability occurred for up to 90 min exposures in the centrifugal field. However, at 25°C and for gravitational fields of 24,800 and 42,000g, there were appreciable losses in cell viability. Continuous centrifugation studies in the Sharples supercentrifuge (Division of Penn Salt Corporation, Warminister, Pennsylvania USA) were also conducted with bowl speeds up to 28,000 rpm (19,000g) and flow rates ranging from 1.4 to 20 1, hr. Slight, losses in cell viability were noted and postulated as caused by the shear stresses encountered by the cells. Some pumping studies using the lymphoma cells substantiate this conclusion.     

Effect of autologous marrow transplantation on bone marrow recovery following large-field fractionated irradiation of dogs

After large-field fractionated irradiation of dogs at a cumulative dose of 54 Gy, a stable bone marrow depletion occurs persisting for a year following irradiation. The automyelotransplantation after the end of the exposure elicits a transient recovery of the exposed bone marrow, 1.5-2 months after the beginning of irradiation, followed by a secondary depletion of the exposed haemopoietic sites. The control and the automyelotransplanted animals exhibited bone marrow recovery one year and six months after irradiation, respectively, the cellularity being maintained at a high level for 3 years of observation.     

Effect of radiosensitivity gene irradiation and genotype on yeast cytoduction]

Effects of gamma- and UV-irradiation of one parent on progenies of yeast crosses are studied. In the crosses of the type: p+ a alc4 X p-alpha ade2 (alc4--nuclear respiration deficiency) zygotic colonies (white) and haploid p+ cytoductant colonies (red) were scored. The irradiation of p+ parent increases the cytoductant frequency up to 17%. When both parents are radiation-sensitive (xrs1, allelic to rad54), the frequency of cytoduction reaches 90%. Crosses p+xrs1 X p-xrs+ or p+xrs+ X p-xrs1 are similar to crosses of wild types. The same results were observed in the case of xrs4 mutants as well as for UV-irradiation. In the progenies of crosses irradiated p+ a alc4 (xrs+ or xrs1) X non-irradiated p-alpha ade2 ura (xrs+ or xrs1) the genotype of red colonies was analysed: in most cases (82.3%-99.8%) they were p+ alpha ade2 ura, that was true haploid cytoductants. It is concluded that irradiation induces particular damages of yeast nuclei leading to a block of normal karyogamy and thus to a cytoductant formation. The highest frequency of cytoduction was observed in crosses of radiosensitive mutants.     

Effect of ultraviolet light irradiation on viability and aflatoxin production by Aspergillus parasiticus

The effect of ultraviolet light irradiation (254 nm) on both the viability and the aflatoxin-producing ability of the fungus Aspergillus parasiticus NRRL 2999, a good aflatoxin-producing strain, was studied. This strain showed noticeable resistance and irradiation for more than 10 min was necessary to reduce survival to under 10%, while the white mutants were more susceptible (5 min of irradiation reduced survival to under 1%). Induction of mutants with complete loss of aflatoxigenicity was rare and only 3 of the 1463 survivors tested were aflatoxinless.     

Quantitative studies on the viability of yeast protoplasts following dielectrophoresis

Abstract Protoplasts from Saccharomyces cerevisiae and Saccharomyces diastaticus were collected in a non-homogeneous alternating electric field. The dependence of the viability of the protoplasts on different conditions of collection was tested by determining the regeneration rates in each case. The parameters varied in collection were the field strength (0.33 kV/cm–6.67 kV/cm), the frequency of the alternating field (1–2 MHz) and the collection time (2–10 min). The introduction of a new type of fusion chamber (meander chamber) permitted, for the first time, quantitative exposure of protoplasts to the electric field as well as their complete transference into the regeneration medium. The regeneration rates of yeast protoplasts collected under those conditions employed for electrofusion did not differ from those of protoplasts which had been maintained under the same experimental conditions but were not subject to the influence of an alternating electric field. The two yeast strains were fused together (collection 1 kV/cm; pulse 15 kV/cm; duration of pulse 40 μs) and the fusion products were introduced into a selection medium for regeneration. The fusion rate was about 4.8 × 10⁻⁴; on average 272 colonies grew on the selection medium for each chamber filling.     

Correlation between the cellular content of mobile water and the viability of lyophilized yeast cells

Spin-echo NMR studies showed that lyophilized yeast cells contain isolated mobile water (IMW), whose content varied from 0.25% (of the dry weight of cells) in the lyophilized exponential-phase yeast cells to 3.8% in the lyophilized lag-phase and stationary-phase yeast cells. The viability rate of yeast cells varied from 20% in the lyophilized preparation of exponential-phase cells to 86% in the lyophilized preparation of early-stationary-phase cells. In the lyophilized preparation of yeast cells grown in a chemostat mode at a constant specific rate, the content of IMW depended on the growth-limiting factor, being minimal in the case of growth limitation by carbon source. In the latter case, the viability rate of cells was also minimal. The data obtained show that there is a correlation between the IMW content and the viability rate of yeast cells in lyophilized preparations.     

Studies on plating efficiency and estimation of viability of suspensions of Paracoccidioides brasiliensis yeast cells

Mild sonication was used to obtain single cell suspensions of Paracoccidioides brasiliensis. These cells were intact by microscopic criteria. Direct cell counts in a given inoculum and colony formation on various media were used to determine plating efficiency. Sonicated and nonsonicated cell suspensions were used to study plating efficiency and to estimate viability by means of vital dyes. Methylene blue, Erythrosin B, and Janus green were unreliable when used with P. brasiliensis, but vital dyes were accurate when tested with Candida albicans.Acridine orange gave more meaningful results of viability. Estimates of viability, however, changed significantly as a result of relatively minor alterations in the composition of the suspending medium.In initial experiments, the plating efficiency of P. brasiliensis was dismally low. It descended abruptly with increasing dilution of inoculum. Efficiency was much improved if horse serum was added to brain heart infusion plates or if glucose glycine yeast extract (GGY) plates were incubated at room temperature and mycelial colonies were counted. With the technique we report, current plating efficiency of sonicated suspensions is of the order of 25 %. Our results and procedures have an important bearing upon those studies concerned with in vitro killing of P. brasiliensis in suspensions or with isolating this fungus from clinical or environmental specimens.