Scanning electron microscopy of barley protoplasts

Summary Scanning electron micrographs of barley protoplasts were compared using various preparatory techniques. Numerous features were observed which turned out to be artifactual characteristics of the processing procedure used in collecting and dehydrating the samples. The most successful technique gave protoplasts which presumably maintained their natural structural integrity, as judged by retention of sphericity and absence of holes in the plasma membrane. The relative numbers of fragmented protoplasts and cellular organelles was also greatly reduced.     

Scanning electron microscopy of bone cells in culture

Summary Embryonic and young rat bone cells have been grown in culture and examined in the scanning electron microscope (SEM). Compared with cells fixed in situ and taken directly from the animal, the cultured osteoblastic cells were smoother, flatter and more extensive and showed tighter intercellular contacts. Some matrix is formed in culture and undergoes at least partial mineralization as judged by the accumulation of Ca and P measured by energy dispersive x-ray analysis. Findings concerning the morphology of the collagen arrangement were indecisive. Some superficial cells, free of surrounding matrix, resembled osteocytes in normal in vivo bone. This may indicate that a proportion of the extracellular matrix produced by the cultured cells failed to polymerise into recognizable bone matrix, and that osteocytic morphology is not dependent upon the physical characteristics of the bone matrix.     

Scanning electron microscopy of cultured cells of Aedes aegypti

Cultured cells of Aedes aegypti were fixed with glutaraldehyde and prepared for scanning electron microscopy by four procedures: air drying, lyophilization, ethanol dehydration and air drying, and ethanol dehydration and critical point drying. Comparison of the resulting electron micrographs with phase contrast photomicrographs of living cells revealed that although cultured insect cells dried by the critical point method are not completely without artifacts, this method of preservation is superior to other techniques currently used.     

Scanning electron microscopy analysis of aged Mycobacterium tuberculosis cells

Most electron microscopy studies of Mycobacterium tuberculosis ultrastructure were performed in the 1950s and 1960s and lack high resolution by modern standards. This study was performed to re-evaluate the fine structure of M. tuberculosis using modern scanning electron microscopy. Bacteria were grown in rich medium with a constant supply of oxygen for several weeks. Results show that surface bleb-like structures accumulate as cultures age. The most unusual feature of aging M. tuberculosis cultures is that they develop extracellular fibrils, which could play roles of adhering cells to surfaces and to one another.     

Scanning electron microscopy preparation protocol for differentiated stem cells

The lack of an established protocol for scanning electron microscopy (SEM) studies on stem cells differentiating into adipogenic lineage led us to develop a protocol for the preparation of differentiated adult bone marrow-derived mesenchymal stem cells (BMSC) for SEM. This protocol describes the procedure to maintain and preserve the structural organization of cellular components following differentiation, for morphological and physical characterization. The fixation of the differentiated cells was followed by dehydration using methanol, and vacuum desiccation before microscopy. The use of longer chain alcohols as dehydrating agents was avoided in our method to reduce the dissolution of lipid deposits in cells, thus allowing the maintenance of their structural integrity. The time period for the processing of samples was reduced by avoiding the osmium tetroxide postfixation and critical point drying. Thus, this protocol helps in determining the potential, fate, and degree of stem cell differentiation. This may be useful for SEM analysis of differentiated cells, especially those grown on various scaffolds.     

Scanning electron microscopy in nematode-induced giant transfer cells.

A study of giant cells induced by the root-knot nematode, Meloidogyne incognita, in roots of Impatiens balsamina was made by scanning electron microscopy. The cytoplasmic contents of giant cells were removed by a procedure based on KOH digestion, to reveal inner wall structure. Wall ingrowths typical of transfer cells are present in giant cells from six days onwards after induction. They develop on walls adjacent to vascular tissues, and their distribution and development was examined. Pit fields contianing plasmodesmata become elaborated in walls between giant cells, but pit fields are lost between giant cells and cells outside them. The distribution of plasmodesmata in pit fields suggests that de novo formation of plasmodesmata occurs in walls between giant cells. Various aspects of giant cell formation and function are discussed and wall ingrowth development is compared in giant cells and normal transfer cells.     

The effect of 5-fluorouracil on cells and regenerating protoplasts ofSaccharomyces cerevisiae

The regeneration of the yeast cell-wall was studied using 5-fluorouracil and yeast protoplasts. Protein synthesis in yeast cells (Saccharomyces cerevisiae) was kept reduced in the presence of this inhibitor at a rate corresponding to that before inhibition and was independent on the concentration of the inhibitor (10 or 100 μg/ml). The inhibition of the RNA synthesis was incomplete and dependent on the concentration of the inhibitor. Synthesis of thymidine and DNA was not inhibited as indicated by the growth tests. On the basis of the obtained data it may be concluded that fluorouracil inhibits only thede novo and the induced protein synthesis while permitting protein synthesis that has already been started before inhibition. Fluorouracil was then applied during the regeneration of yeast protoplasts. The results obtained have shown that fluorouracil does not inhibit the synthesis of the yeast cell wall but that the normal course of cell division is impaired by fluorouracil. The low efficiency of the fluorouracil inhibition of the cell wall synthesis indicates that processes leading to the regeneration of the cell wall are in fact only a continuation of those taking place under normal growth conditions.     

Scanning electron microscopy of rat neurointermediate lobe cells in culture

Enzymatically dispersed cells of the rat pars nervosa -- pars intermedia (NI) were observed by scanning electron microscopy after 1, 2 and 4 days in culture. The cells attached to the plate slowly, requiring about 3--4 days for the majority to adhere. The epithelial cells became attached singly and in clumps and branched chains, often over a bed of fibroblast-like cells. After the first day in culture, few surface features were evident on the NI cells, but by day 2, the surfaces showed a small number of blebs. In 4-day cultures, the cells revealed extensive areas with blebs and microvilli, and a few structures resembling cilia were observed. The adrenocorticotrophic hormone content of the cells after 4 days in culture was similar to that found in fresh tissue.     

Scanning electron microscopy of aggregating embryonic neural retina cells.

Scanning electron microscopy of in vitro reaggregation of trypsin-dissociated neural retina cells from 10-day chick embryos revealed that filopodial projections participate in the assembly of the dispersed cells into clusters. Freshly dissociated cells displayed numerous elongated, randomly projecting filopodia. With the onset of cell reaggregation these filopodia bridged and connected distant cells becoming shorter as the cells came together and formed aggregates. In 24-h cell aggregates only short microvilli were seen, mostly on cell surfaces facing the periphery of the aggregate. Cells dissociated from retina tissue pre-treated with inhibitors of protein synthesis, or cells exposed to these inhibitors immediately after dissociation were mostly devoid of filopodial projections; such cells failed to re-aggregate histotypically. Thus, metabolic and biosynthetic processes are required for the changes in the cell periphery which result in formation or maintenance of filopodia, and which enable trypsin-dissociated cells to reform histotypic associations. Possible relationships between the formation of filopodia and histotypic reaggregation of cells is discussed.     

Scanning electron microscopy of Penicillium conidia

The morphology of conidia in 211 species and 12 varieties belonging to the genus Penicillium Link ex Gray have been studied and compared.According to surface ornamentation, conidia have been classified into six groups: A, smooth-walled (7% of the species); B, delicately roughened (13%); C, warty (28%); D, echinate (10%); E, striate with low irregular ridges (36%); and F, striate with scarce high ridges or bars (6%). Whereas the first two groups are closely related in both shape and average size, a gradual reduction was observed in size and in the length/width (l/w) ratio in the remaining groups. Echinate conidia were globose, having the largest average size. Only four species produced conidia not surpassing 2 m in diameter. Maximum length observed was 8 m, and most elongated conidia had a l/w ratio of 3.5. Forty per cent of the species studied had globose conidia.Conidia of the monoverticillate species were generally smaller, more globose and frequently with ridges. In the Asymmetrica, the conidia were generally larger, and showed ridges in comparatively few species. Conidia of the Symmetrica, which were frequently striate with ridges, presented the most elongated forms. The largest average size was found in the conidia of the Polyverticillata which were generally warty. Finally, we have considered the variations in surface ornamentation of conidia during the evolution of the genus Penicillium and drawn attention to their possible relationship with certain habitats and ways of conidial dispersion.