THE MICROMANIPULATION OF LIVING CELLS 



21 



tribution was the observation that the 

 nucleus need not lose its structural integ- 

 rity when removed from the cell. 



Nuclei of ovarian eggs of fish and of 

 Amphibia are proving to be excellent 

 material for micromanipulation. W. R. 

 Duryee (1937) is studying the effect of 

 chemicals on the isolated nuclei of the frog 

 and of Triturus. Chambers and Sands 

 (1923) had shown that the chromosomes of 

 spermatocytes of the grasshopper are 

 highly tenacious and elastic. Duryee 's dis- 

 sections confirm this conclusion on isolated 

 amphibian chromosomes, which are tenuous 

 filaments, often as long as 500 |j. 



The incentive for the micromanipulative 

 technique we owe to bacteriologists. We 

 are indebted to George Lester Kite for the 

 adaptation of the bacteriological technique 

 to the microdissection of living cells. 



Kite was an enthusiastic pioneer inspired 

 by A. P. Mathews who was at that time at 

 the University of Chicago. Kite's graphic 

 account of his first attempts at micro- 

 manipulation was given in a lecture at 

 Woods Hole during the summer of 1911 

 (never published). One of the operations 

 which astonished his hearers was the inter- 

 position of the tip of a microneedle between 

 the male and female pronuclei of a recently 

 fertilized Toxopneustes egg. He stated 

 that he could push one of the nuclei about 

 but that it would persist in slipping off the 

 needle to advance toward its mate.^ 



Kite used exclusively the so-called Bar- 

 ber Pipette Holder. At that time, 1911 and 

 1912, there were two instruments used to 

 isolate bacteria in the field of the oil-im- 

 mersion objective, one devised by S. L. 

 Schouten, in Holland, and the other by 

 M. A. Barber, in America. Two features 

 made it possible to extend the technique to 

 cellular research. They are the use of glass 

 needles with microscopically fine tips on 

 relatively rigid shafts, and the bending of 

 the shaft to permit the tips to be operated 

 in a hanging drop. This arrangement al- 

 lows the use of the highest available magni- 

 fications during the operations since there 

 is no obstacle between the lens of the ob- 



2 Kite 's early death was a tragic loss but the 

 work he initiated is still being carried on. 



jective and the coverslip from which the 

 hanging drop is suspended. The instru- 

 ments of Schouten and Barber had com- 

 paratively good vertical movements, which 

 are essential for picking up bacteria, but 

 the lateral movements left much to be de- 

 sired. A mechanical device for more ade- 

 quate control was necessary. Such a device 

 and the development of a micro-injection 

 apparatus have made possible astonishingly 

 delicate operations on cells. With the years 

 the technique has been progressively so 

 improved that now the investigator's in- 

 genuity is no longer being expended on the 

 technique of performance but rather on 

 the problems involved. 



Micromanipulation, exclusive of the iso- 

 lation technique, has been devoted to three 

 general forms of procedure: the dissection 

 of cells and cell aggregates, the removal of 

 materials from within cells, and the injec- 

 tion of solutions into cells. 



An outstanding dissection experiment 

 was that performed by C. V. Taylor (1920) 

 to ascertain the significance of certain 

 fibrils in the ciliate, Eiiplotes. Deep cuts 

 were made into the cortex of various 

 regions of the Euplotes. It was found that 

 recovery of normal coordination of the 

 locomotor organellae occurred in all cases 

 except those in which certain internal fibers 

 had been cut through or in which a gran- 

 ular body, the so-called motorium, where 

 the fibers converge, had been injured. 



Many of the considerable number of 

 structural components of cells have since 

 been investigated. A striking feature is the 

 difference in the degree of their stability. 

 Fibrous strands, vesicles, and rod-shaped 

 mitochondria may be moved about and 

 distorted with no apparent loss of their 

 integrity. On the other hand, other struc- 

 tures, such as the aster, readily disappear. 



The physical state of much of the proto- 

 plasm resembles that of reversible sol-gel 

 colloidal systems. The reversibility from 

 the gel to the sol state, with a subsequent 

 disappearance of structural characteristics, 

 can be induced by various experimental 

 methods, particularly those which involve 

 the use of pressure or sudden mechanical 

 agitation. By mechanical means, it has 



