TECHNIQUES OF ANATOMY — ROOFE AND LESHER 335 



Centrif ugation. — The possibility that various enzymes may be bound 

 to or absorbed on mitochondria motivated the development of the 

 technique of the separation of cellular components by centrifugation. 

 The earliest important separation was achieved by Bensley and Hoerr 

 (1934). In their paper they describe the process of isolating large 

 granules which proved to be mitochondria. Subsequent investiga- 

 tors (Claude, Dounce, Hogeboom, Lazarow, Mirsky, Moog, Palade, 

 Pollister, and others) perfected the differential centrifugation tech- 

 nique and isolated in addition to mitochondria the much smaller mi- 

 crosomes, glycogen particles, whole nuclei, and chromosomes. Tests 

 for enzyme activity confirmed the original postulation that mitochon- 

 dria are the most important site of intracellular enzymes. If one 

 recognizes the obvious limitation of this technique which destroys the 

 intracellular relationships the results obtained can be extremely valu- 

 able in the identification of enzyme systems. 



Microchemical analysis of biological materials. — Lowry and Bessey 

 (1946), through the use of a specially designed curvette and the 

 preparation of materials at low temperatures ( — 20° C.), adapted the 

 Beckman spectrophotometer for the measurement of the chemical 

 components of extremely minute sections of cellular material. They 

 were able to obtain quantitative measurements on volumes as low as 

 25 cmm. (0.025 ml.). With their adaptations, for example, (hey 

 have measured ascorbic acid in 0.01 ml. of serum, vitamin A and caro- 

 tene in 0.035 to 0.06 ml. of serum, and ascorbic acid in the blood 

 cells and platelets of 0.1 ml. of blood. With this technique it is con- 

 ceivably possible to make quantitative analyses of whole nuclei 

 cleaved from frozen tissues. 



Microdissection. — One of the outstanding techniques of recent years 

 has been that of microdissection. There are four or five different 

 types of microdissecting instruments, but the one used by Chambers 

 probably leads the field. In this approach to the living cell, small 

 needles or knives are made by drawing out glass tubes or glass rods in- 

 to very fine points and edges. These tubes are then placed in holders 

 which can be maneuvered most gently and accurately within ranges of 

 1 or 2 microns or fractions thereof ; nuclei of cells can be removed ; 

 chromosomes can be pulled out ; myof ibrilla and neurof ibrilla can be 

 teased and pulled out of their respective fibers by this ingenious 

 method. By this application scientists are agreed now that these struc- 

 tures can no longer be considered artifacts, but actual organoids of 

 the living cell. 



Another advantage of the microdissecting techniques is that small 

 quantities of material can be injected into a living cell, thus enabling 

 the student to study experimentally the functions of various proto- 

 plasmic constituents. 



