Interaction between flocculent and nonflocculent cells of Saccharomyces cerevisiae.

Interaction between nonflocculent and flocculent cells of Saccharomyces cerevisiae was studied. Adhesion experiments were done using three types of nonflocculent cells and a flocculent one. Two types of nonflocculent cells were obtained from the flocculent strain by changing environmental growth conditions. The integration of nonflocculent cells in the flocs was observed by two different methods: measurement of the sedimentation capacity of mixtures and microscopic observation of stained nonflocculent cells blended with flocculent cells. It was possible to verify that cell-cell interaction corresponds to a true stable binding and not to a simple entrapment inside the floc matrix.     

Fully hydrated yeast cells imaged with electron microscopy

We demonstrate electron microscopy of fully hydrated eukaryotic cells with nanometer resolution. Living Schizosaccaromyces pombe cells were loaded in a microfluidic chamber and imaged in liquid with scanning transmission electron microscopy (STEM). The native intracellular (ultra)structures of wild-type cells and three different mutants were studied without prior labeling, fixation, or staining. The STEM images revealed various intracellular components that were identified on the basis of their shape, size, location, and mass density. The maximal achieved spatial resolution in this initial study was 32 ± 8 nm, an order of magnitude better than achievable with light microscopy on pristine cells. Light-microscopy images of the same samples were correlated with the corresponding electron-microscopy images. Achieving synergy between the capabilities of light and electron microscopy, we anticipate that liquid STEM will be broadly applied to explore the ultrastructure of live cells.     

Use of iso-osmolality plots to characterize yeast fermentative performance

Yeast fermentation data (ethanol product, P, vs glucose substrate, S) can be plotted on a graph showing lines of equivalent osmolality or water activity. Comparison of such plots for different strains and for single strains under different environmental conditions shows the conditions for optimum performance. With these plots the product/substrate yield coefficient ( Y _p/s) can be shown to vary with time, contrary to its usually assumed constant value. Under conditions of optimum environment and nutrition, five of the yeast strains tested ceased growing at a characteristic value of solution osmolality, indicating that the inhibitory effects of ethanol and glucose, to a first approximation, are the result of their molar contribution to water activity rather than due to any specific cellular effect. In one yeast strain this characteristic behaviour was not affected by temperature variations between 30° and 36°C.     

Adhesion of cells to surfaces coated with polylysine. Applications to electron microscopy

Cells of many kinds adhere firmly to glass or plastic surfaces which have been pretreated with polylysine. The attachment takes place as soon as the cells make contact with the surfaces, and the flattening of the cells against the surfaces is quite rapid. Cells which do not normally adhere to solid surfaces, such as sea urchin eggs, attach as well as cells which normally do so, such as amebas or mammalian cells in culture. The adhesion is interpreted simply as the interaction between the polyanionic cell surfaces and the polycationic layer of adsorbed polylysine. The attachment of cells to the polylysine-treated surfaces can be exploited for a variety of experimental manipulations. In the preparation of samples for scanning or transmission electron microscopy, the living material may first be attached to a polylysine- coated plate or grid, subjected to some experimental treatment (fertilization of an egg, for example), then transferred rapidly to fixative and further passed through processing for observation; each step involves only the transfer of the plate or grid from one container to the next. The cells are not detached. The adhesion of the cell may be so firm that the body of the cell may be sheared away, leaving attached a patch of cell surface, face up, for observation of its inner aspect. For example, one may observe secretory vesicles on the inner face of the surface (3) or may study the association of filaments with the inner surface (Fig. 1). Subcellular structures may attach to the polylysine-coated surfaces. So far, we have found this to be the case for nuclei isolated from sea urchin embryos and for the microtubules of flagella, which are well displayed after the membrane has been disrupted by Triton X-100 (Fig. 2).     

Use of reflectance interference contrast microscopy to characterize the endothelial glycocalyx stiffness

Reflectance interference contrast microscopy (RICM) was used to study the mechanics of the endothelial glycocalyx. This technique tracks the vertical position of a glass microsphere probe that applies very light fluctuating loads to the outermost layer of the bovine lung microvascular endothelial cell (BLMVEC) glycocalyx. Fluctuations in probe vertical position are used to estimate the effective stiffness of the underlying layer. Stiffness was measured before and after removal of specific glycocalyx components. The mean stiffness of BLMVEC glycocalyx was found to be ~7.5 kT/nm(2) (or ~31 pN/nm). Enzymatic digestion of the glycocalyx with pronase or hyaluronan with hyaluronidase increased the mean effective stiffness of the glycocalyx; however, the increase of the mean stiffness on digestion of heparan sulfate with heparinase III was not significant. The results imply that hyaluronan chains act as a cushioning layer to distribute applied forces to the glycocalyx structure. Effective stiffness was also measured for the glycocalyx exposed to 0.1%, 1.0%, and 4.0% BSA; glycocalyx compliance increased at two extreme BSA concentrations. The RICM images indicated that glycocalyx thickness increases with BSA concentrations. Results demonstrate that RICM is sensitive to detect the subtle changes of glycocalyx compliance at the fluid-fiber interface.     

Staining yeast cells for electron microscopy by growth in copper-containing nutrient broth

Copper attaches to the nucleoli and the chromosomes of yeast cells. Lindegren and Zink (1969) showed that lipolated mitochondria contain fat and inferred that fat was an energy source. The present study shows that fat is present in the nucleolus and fat is inferred to be an energy source for nucleolar metabolism. The extrusion of nucleoprotein from the nucleolus into the cytoplasm is demonstrated for the first time by electron microscopy. The copper-stained chromosomes are about 90 å in diameter and tightly packed in the nuclear vacuole.     

Scanning electron microscopy of exposed surfaces of the porcine placenta

The three-dimensional development of the separated parts of the porcine placenta from 9 Danish Landrace sows at gestational stages from 20 to 100 days was studied. After cautious separation of the allantochorion and the endometrium in a 1 mM buffered ethylenediaminetetraacetic acid solution, the separated parts were processed for scanning electron microscopy by routine methods. The macroscopic enlargement resulted from primary and secondary circular folds or plicae, which were permanent on the maternal side, whereas they were mainly non-permanent on the fetal side. The areolar placenta and interareolar placenta with formation of permanent microscopic folding on both sides were described. The observations of the separated parts yielded new information on the development of surface enlargement during gestation and revealed a hitherto unknown regular architecture of the endometrium by the formation of parallel primary ridges or rugae with secondary ridges or rugae at their sides subdividing the maternal troughs or fossae. This configuration on the maternal side explains the transformation of the regular chorionic ridges from the 63-day stage into bulbous protrusions at the 100-day stage. Based on these observations the precise terminology used above was proposed.     

Enhancement of metabolic rates of yeast flocculent cells through the use of polymeric additives

The influence of several polymeric additives on specific glucose uptake rate of flocs of a S. cerevisiae strain — S. cerevisiae NRRLY 265 was studied. A special continuous membrane microreactor was used to measure glucose uptake on the presence of calcium and of the tested additives — two cationic polymers — bis(polyoxyethylene-bis(amine)) 20,000 and BPA 1,000 and one anionic polymer — Magna Floc LT25.An increase on glucose uptake rate was always observed when comparing with calcium bound flocs. For bis(polyoxyethylene-bis(amine)) 20,000 the increase was only 19% but for BPA 1,000 a value of more than 50% was observed. For Magna Floc LT25 a two fold increase was measured.The determination of floc size and porosity in the presence of the additives indicated that, on the basis of these parameters, it was not possible to explain the observed glucose uptake rates. The floc porosites in additive bound flocs were similar and 10% larger than for calcium bound flocs and glucose uptake rate was larger for the largest flocs — Magna Floc LT25 bound flocs were the largest followed by BPA 1,000, bis(polyoxyethylene-bis(amine)) 20,000 and calcium bound flocs. These values disagree with what should be expected in diffusion controlled processes.The calculation of intercellular floc distance indicated that polymeric additives act on the reduction of diffusional limitations by increasing the available flux area for glucose inside the flocs. By analysing different kinds of packings, it was also observed that the packing arrangement for yeast cells in flocs is close to the cubic packing. The simulation of this arrangement for the obtained floc sizes confirmed that the 10% increase in floc porosity is sufficient to explain the increase in the available flux area.     

Use of the cytocentrifuge for electron microscopy investigations

A simple method of cytocentrifugally processing cell suspensions for conventional and histochemical investigations at the ultrastructural level is described. Fixed sediments from cell-poor suspensions are resuspended in an albumin-buffered solution. A few drops of the albumin cell solution are cytocentrifuged, leaving a cell disc on a plastic support. A brief dipping in a paraformaldehyde-glutaraldehyde mixture transforms the cell disc into a compact thin fragment attached to the plastic support. Cytocentrifugation of cell-rich suspensions, on the other hand, produces a thicker cell disc, which can be easily detached from the plastic slide. In both cases, the postfixation, dehydration and infiltration are directly carried out on the cell disc. The present method is particularly useful for the ultrastructural study of cell-poor suspensions and can also be performed on cell suspensions previously stained with several histochemical procedures.     

Scanning electron microscopy of dissociated pancreatic acinar cell surfaces

Summary The pancreatic acinar cell surfaces have been studied by SEM with a dissection technique and correlated with results obtained by TEM. The SEM results demonstrate characteristic arrangement of microplicae which in some areas are densely packed.In many areas, the microplicae are distributed in such a manner that they create zones with typical geometrical shapes and show a relatively smooth surface.These smooth areas may coincide, as indicated by correlated TEM results, with the limits of intimate contact between adjacent acinar cells which, in turn, represent part of the junctional complex. Another aspect revealed by these SEM preparations concerns the presence of groups of densely packed microplicae, arranged in regular rows and distributed along some grooves and/or infoldings of the cellular surface. On the basis of SEM and TEM information, it is likely that these structures correspond to intercellular (and possibly, in some cases, intracellular) canaliculi which topographically form a kind of extensive microlabyrinthine arrangement running along all the cell sides.One final point revealed by fractured samples concerns the finding of spherical zymogen droplets within the vesicles of the Golgi complex. Because in many scanning images these vesicles appear connected by small openings, it is suggested that they may represent a system of intercommunicating chambers (vacuoles) through which the zymogen droplets can be continuously accumulated and discharged into the acinar lumen.     

Scanning electron microscopy of the integumental surfaces of Schistosoma haematobium.

The integumental surfaces of critical point dried S. haematobium were studied by scanning electron microscopy at 34 to 8,000 magnifications. There are marked differences between the surface structures of male and female as well as from one part of the same parasite to another. The surface of the male schistosome is moderately rough while that of the female is relatively smooth. SEM reveals certain basic features such as spines in the oral sucker and the acetabulum of both sexes which may facilitate rasping and/or attachment of the parasite for residence in the bloodstream of the definitive host. The lining of the gynecophoral canal is roughened by minute spines. The presence of a gynecophoral fold may enhance anchorage of the female in the grasp of the male. The significance of visualization of surface features by SEM as a means for differentiating species is not yet known.     

A glass bead treatment facilitating the fixation and infiltration of yeast and other refractory cells for electron microscopy

Summary Physical removal of the cell wall of yeast and other fungal cells, by rapid mixing of the cells with glass beads after preliminary fixation in glutaraldehyde or formalin, removes the cell wall barrier to fixation and/or infiltration of the cells for electron microscopy. The technique has been used on a variety of fungal cells which have been difficult to fix for electron microscopy, and appears to have wide applicability.