Open-Face, Epoxy Embedding of Single Cells for Ultrathin Sections

Intact stamens of Tradescantia were fixed, dehydrated, and infiltrated with an epoxy resin. Each stamen was then put into a drop of resin on a microscope slide, which was transferred to the stage of a dissecting microscope so that individual hairs could be detached from the filament with fine tungsten needles. The detached hairs were transferred to drops of resin ca. 2 mm in diameter (6 or 7 in each of two rows) lying on a slide heavily coated with evaporated carbon. Polymerization was carried out in an oven until the resin attained a degree of viscosity that permitted orientation of the isolated hairs (by using a compound microscope) without their subsequent dislocation. When the small drops of resin had hardened after further polymerization, the positions of the hairs were marked by circumscribing the cells with India ink. The block was pried from the slide after rapid cooling with solid CO₂, and was then trimmed and sectioned. Cells suspended in culture medium were embedded in much the same way; they were centrifuged to obtain a pellet, which was fixed, dehydrated, and infiltrated. A small fragment of the pellet with a little resin was placed on a microscope slide, where the cells were dissociated under a dissecting microscope at ca. 100 × magnification. Individual cells were then picked up with tungsten needles and transferred to droplets of resin on a carbon-coated slide. The subsequent steps were similar to those described for the staminate hairs. Pieces of tissue in the 50-500 μ range were also handled by the foregoing technique. However, after infiltration they were put into large drops of resin on a slide coated with silicone mold-release rather than on a surface coated with carbon.     

Automatic search for model to simulate the differentiation of T lymphocytes within the thymus

The differentiation of T Lymphocytes within the thymus is an important biological phenomenon during wich these cell acquire their functions to further control the immune system. Numerous experiments under various conditions have been devised to understand the different mechanisms involved in this complex process. Nevertheless, interpretation of these experiments lead to still contradictory debatable hypotheses. Modelisation of this process through classical simulation methods cannot be envisaged because they are not adapted to modifications of the model structure, which is the point of interest. For these reasons, we proposed a new approach of automatic search for model. The program consists of four independent connected modules : The generator produces model, based on the rationale of formal grammars. Protocol and experimental data are stored in a set of experiments. The simulator using a protocol and a model provides simulated results. Finally, the supervisor by comparing simulated results and experimental data, adapts the model parameters to increase their fit and either chooses a new experiment to explore, or modifies the model structure. Change of the model structure is performed among still unexplored models according to their promise level, which is iteratively evaluated relatively to previously explored models through a proposed model distance. The generator is written in Prolog and the other modules in C++. The architecture of the program allows us to modify or complete a module without changing anything in the other modules. As a consequence, the proposed modeling approach conceived to study T lymphocyte differentiation within the thymus remains independent of this biological phenomenon and can be applied to other biological problems.     

Phenotypic variation during micropropagation of the chimeralRhododendron ‘President Roosevelt’

The putative periclinal chimeraRhododendron xlimbatum President Roosevelt was used to study the origin of shoots in vitro. Genotypic segregation readily occurred in vitro. Numerous phenotypes were observed, although most shoots were either entirely green or maintained the original variegation pattern. Derivatives of the third apical layer were rarely involved in shoot formation. A reversed chimeral form was isolated. Adventitious shoots were usually miniaturized and rapidly proliferating, but axillary shoots had thicker stems, larger leaves and proliferated more slowly. Corolla tissue produced stunted, leafy shoots; no variegated shoots were produced from floret explants. In shoot tip cultures the addition of 40M 2iP without IBA resulted in the greatest number of shoots. Explant choice was the most critical factor for maintenance of foliar variegation.     

MPphiWR-2, a cryptic phage associated withMicromonospora purpurea ATCC 15835

Summary A previously undescribed cryptic phage was found associated withMicromonospora purpurea ATCC 15835.     

Growth of food-borne pathogenic bacteria in oil-in-water emulsions: I—Methods for investigating the form of growth

Methods are presented for investigating the site and form of growth of bacteria in model oil-in-water emulsions and in dairy cream. Following growth of the bacteria, the continuous aqueous phase is gelled using agarose and the oil phase removed using a mixture of chloroform and methanol. Using this method, the authors have found that Listeria monocytogenes, Salmonella typhimurium and Yersinia enterocolitica grow in the form of colonies in concentrated oil-in-water emulsions. Colonies of L. monocytogenes and Y. enterocolitica also form in artificially-inoculated fresh and tinned dairy cream. If information about the precise site of growth is not required, the authors have discovered that intact colonies can be liberated from the model emulsions by dissolving away the oil phase with chloroform: methanol.