STUDIES ON CELL MORPHOLOGY AND FUNCTIONS: METHODS AND RESULTS

To a large extent, our present knowledge of cell morphology has derived from observations of cells and tissues under the microscope, but inquiries by the microscopical method alone have repeatedly failed to give conclusive information on the chemical constitution of the various cell structures and the topography of biochemical functions. The chief difficulties inherent in histochemical methods are : (1) the chemical tests which otherwise may be specific must be carried out on cell structures altered by fixation and dehydration to an extent which cannot be ascertained; (2) the chemical reactions studied must take place on structures, and in surroundings, of unknown and uncontrollable complexity; and ( 3 ) a t the microscopical level, one must deal with exceedingly small amounts of substances, which often places the tests beyond the sensitivity of the reaction to be employed. Since Miescher, attempts have been made to apply analytical methods to the study of cell structures and to isolate known cell components mechanically or through mild chemical manipulations. Separation of nuclei led to the discovery of nucleic acids and the recognition of certain basic proteins.' In 1913, Warburg segregated cytoplasmic granules by centrifugation and was able to show that these were responsible for most of the oxygen uptake of cell-free extracts of guinea-pig liver? In 1934, Bensley and Hoerr succeeded in separating, also by centrifugation, large cellular elements which they identified as mit~chondria.~ In 1938, submicroscopic components of the ground substance were isolated by means of centrifugation at high speed.4 These results indicated that, given the proper equipment and a systematic application of the method of differential centrifugation, it would be possible to separate the various cell components by mechanical means.&* In this paper, I shall attempt to review and discuss briefly the usefulness and the difficulties of the method. The Meihod of Dijerential Centrifugatioit. The method of fractionation by differential centrifugation, as applied to cell studies, precisely overcomes the most serious limitations of the microscopical technique. By means of centrifugation, the whole range of particle and molecular sizes can be investigated, since centrifuges can be made to provide centrifugal fields of practically any given strength. In practice, however, the use of very high centrifugal force is not necessary. Under ordinary conditions, i.e., when the viscosity and density of the medium is approximately that of water or saline, the cellular components with a diameter equal to, or greater than, 50 p can be separated completely by centrifugation of one hour at 20,000 xg. In this manner, particulate components of cytoplasm (microsomes) too small to be detected by light microscopy could be demonstrated and isolated in