Cell Constitution 



41 



FRACTIONATION OF CELL 

 PARTICULATES 



The development of the technique origi- 

 nally employed by that pioneer of analytical 

 cytology, Robert R. Bensley, has led to 

 great advance in our knowledge of the con- 

 stitution and function of subcellular systems. 

 Cells are fragmented in a medium usually 

 containing an indifferent non-electrolyte. By 

 differential centrifugation, particles of vary- 

 ing size, density and composition may be 

 isolated. Particulates so obtained exist in an 

 environment very different from that obtain- 

 ing in the cell. Biochemical tests show that 

 certain of the properties of the particulates 

 may be retained after isolation. However, 

 much further progress may be expected from 

 further investigation of the effect of the 

 fragmentation medium on the system. The 

 work of Kopac ('50a,b) strongly indicates 

 that mere cytolysis, to say nothing of com- 

 plete cell fragmentation, produces surface 

 denaturation of cytoplasmic proteins, as in- 

 dicated by the spontaneous Devaux effect. A 

 truly indifferent "Ringer's solution" for the 

 suspension of cellular constituents has yet to 

 be devised. 



The term "particulate" has been used to 

 connote subcellular aggregates of dimensions 

 near or below the resolution of the light 

 microscope. Fractionation and characteriza- 

 tion of much smaller particles, such as pro- 

 tein macromolecules and complexes, is a 

 field which is destined to play a very sig- 

 nificant role in the next few decades. This 

 will be facilitated by adaptation of ultra- 

 centrifuge, electrophoresis and other physical 

 chemical methods to deal with very small 

 amounts of sample. References: Click ('49), 

 Schneider and Hogeboom ('51). 



THE PROBLEM OF FIXATION 



The purpose of fixation is to treat cells in 

 a manner such that their structure may be 

 examined in the greatest detail with minimal 

 alteration of the normal state, and also to 

 gain information concerning the chemical 

 properties of cell constitvients by interpreta- 

 tion of fixation reactions. The advent of 

 electron microscopy has required a reinvesti- 

 gation of the mechanism of fixation (see 

 Palade, '52a). The present problem is essen- 

 tially similar to that which faced cytologists 

 of old. Aside from the fundamental indeter- 

 minacy arising from the fact that any treat- 

 ment must alter the cell, one is faced with 

 the difficulty that, until one knows the struc- 

 ture of the normal living cell, one can only 



guess whether a given method of preparation 

 preserves that structure unaltered. Except 

 for gross alterations (shrinkage, swelling, 

 etc.), judgment as to the value of a fixative 

 depends upon what the investigator chooses 

 to regard as normal structure. When dealing 

 with a system such as the cytoplasmic 

 "ground substance" where the components 

 are of colloidal dimensions, the electron mi- 

 croscopist's problem is at least as great as 

 that of the histologist working at light micro- 

 scope resolution. No doubt we may expect in 

 the next few decades description of new 

 "fundamental" protoplasmic structures ob- 

 served with the electron microscope (EM) in 

 tissues fixed in various ways. The extent to 

 which soimd interpretation of such fixation 

 "artifacts" can be made will probably depend 

 upon concurrent advances in the physical 

 and analytical chemistry of protoplasmic 

 systems. 



The problem of fixation is amenable to 

 systematic investigation and some prelimi- 

 nary investigations have already been made. 

 It has long been realized that the fixative 

 should be isotonic with the tissue (especially 

 true in the case of solutions of osmium tetrox- 

 ide, which is un-ionized). The importance 

 of appropriate carbon dioxide tension has not 

 yet been sufficiently realized. The effect of 

 pH, ionic strength, redox potential and the 

 like have been studied (Zeiger, '49), as well 

 as the chemical properties of particular fixa- 

 tives, such as osmium tetroxide (Hirschler, 

 '43; Schmidt, '47) and Formalin (French and 

 Edsall, '45; Crawford and Barer, '51). Since 

 many structures can be seen in the living 

 cells by phase contrast microscopy, useful 

 comparisons have been made before and after 

 fixation (Buchsbaum, '48; Zollinger, '48a,b). 

 Most cellular objects exist as organized sys- 

 tems of components. It is therefore necessary 

 to preserve not only the individual entities 

 but the organization of the various systems 

 with respect to each other as well. It seems 

 improbable that any procedure will be 

 found which will fully meet these require- 

 ments. The feeling is growing that it may 

 be better not to try to "fix" the various com- 

 ponents chemically but rather merely to 

 remove water by freeze-drying (see Sylven, 

 '51). This has proven useful to many who 

 work with the EM. 



THE COLLOIDAL ORGANIZATION OF 

 PROTOPLASM 



The cell consists of a highly aqueous col- 

 loidal system, enclosed in a complex limiting 



