Electron microscopy; proceedings of the Electron microscopy; proceedings of the Stockholm Conference, September, 1956 . electronmicrosco00euro Year: 1957 126 M. S. C. BIRBECK AND E. H. MERCER mentation in fresh medium. We have found that an improved separation may be obtained more quickly by using layering or gradient methods in a swing-out head centrifuge. When the medium contains high molecular weight components, which are required to improve the morphological appearance of the fractions, density gradients may be obtained without increasing the osmotic strength of the medium. Fixation of ce
Electron microscopy; proceedings of the Electron microscopy; proceedings of the Stockholm Conference, September, 1956 . electronmicrosco00euro Year: 1957 126 M. S. C. BIRBECK AND E. H. MERCER mentation in fresh medium. We have found that an improved separation may be obtained more quickly by using layering or gradient methods in a swing-out head centrifuge. When the medium contains high molecular weight components, which are required to improve the morphological appearance of the fractions, density gradients may be obtained without increasing the osmotic strength of the medium. Fixation of cell fractions by the standard buffered osmium tetroxide solution (7) is satisfactory; no improvement has been found either by the addition of sucrose or high molecular weight components to the fixative, or by using a solution of osmium tetr- oxide in the suspending medium as a fixative. A small volume of a resuspended fraction is added to a larger volume of fixative; the fixed suspension is then spun to produce a pellet. The pellet, which should be less than mm thick, is treated as a lump of tissue. It is dehydrated and embedded in methacrylate as in the conventional method. The pellet is oriented on the microtome so that a single section contains all the strata produced by sedimen- tation; in this way a proper estimate of the distribu- tion of material in the pellet may be obtained. A promising variant of these methods has been devised by us in which no attempt is made to isolate separate fractions. The tissue is homogenised in a medium, and the suspension is 'layered' over a medium of higher density in a centrifuge tube. Using a swing-out head, the suspension issedimented to form a pellet about mm thick. This will con- tain 'strata' of all components of the suspension separated according to their sedimentation rate. The whole pellet may then be fixed, sectioned and the composition of the various layers determined electron microscopically. As an example of its use, cons
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