THE MICROMANIPULATION OF LIVING CELLS 



23 



to be at variance with that of the indicator 

 in the cytoplasmic matrix. Least open to 

 criticism are the results obtained with the 

 salts of the highly dissociated acid com- 

 pounds, such as the sulfonated indicators 

 of Clark and Lubs. Living cells are rela- 

 tively impermeable to these substances. 

 Moreover, they are highly diffusible within 

 the protoplasm and they can be injected in 

 minute quantities with a minimum danger 

 of upsetting the buffering system within 

 the cell. A considerable variety of cells 

 have been injected and, with due precau- 

 tions being taken, the colorimetric pH 

 values, of the watery phase in protoplasm I 

 at least, appear to be as follows: for the 

 interior of the nucleus, a pH of 7.6 to 7.8; 

 for the cytoplasmic matrix, a pH of 6.7 to 

 6.9 and for the interior of cytoplasmic 

 vacuoles, a variable pH of 5.0 or greater. 

 When cytolysis occurs the cytoplasmic pH 

 drops to about 5.4, while that of the nucleus 

 tends to persist. 



Of considerable interest is the surface 

 boundary of protoplasm, a subject which 

 has been recently reviewed (Chambers 

 1938b). Probably the strongest argu- 

 ment for the existence of a differentiated 

 layer on the surface of protoplasm is the 

 fact that a colored solution which cannot en- 

 ter from without will, when micro-injected, 

 spread through the interior but will not 

 pass out of the cell. The surface layer can 

 be torn with a microneedle and may or may 

 not be repaired, depending upon the sud- 

 denness of the tear. The maintenance or 

 repair of this surface layer does not neces- 

 sarily depend upon the presence of specific 

 electrolytes in the environment. This is 

 indicated in the so-called "churning" ex- 

 periment on unfertilized sea-urchin eggs. 

 The extraneous coats of the eggs were re- 

 moved by several washings in a solution of 

 potassium chloride isotonic with sea water. 

 The eggs were then passed through several 

 changes of any of the experimental solu- 

 tions, viz., sodium chloride, potassium 

 chloride, calcium chloride or magnesium 

 chloride. It was found that the churning 

 effect, that is, a central or axial streaming 

 away from the needle tip and an over-all 

 superficial streaming toward it, could be 



induced in solutions of any one of the 

 several salts just mentioned. 



A main difficulty in studying the physi-1 

 cal properties of the protoplasmic surface i 

 lies in the failure to differentiate it from 

 extraneous coatings which normally sur- 

 round the egg and serve to give it mechani- 

 cal support. Recently, considerable atten- 

 tion has been given to ways and means for 

 distinguishing and establishing the exis- 

 tence of such coatings. An extremely deli- 

 cate and objective one is the oil coalescency 

 method. Oil drops exuding from the tip 

 of a micropipette are brought into contact 

 with the surface of a cell. Under certaini 

 conditions the cell and oil coalesce in such 

 a way that the oil is engulfed by the cell 

 (Chambers 1938b; Chambers and Kopac 

 1937). 



The method used up to the present is as 

 follows: The tip of a micropipette pre- 

 viously filled with oil is placed a short 

 distance from the surface of the cell which 

 is suspended in a hanging drop in view 

 under the microscope. In the case of the 

 unfertilized mature Arhacia ovum, which 

 averages 70 to 75 p in diameter, the tip of 

 the micropipette is usually placed 4 to 5 |j 

 from the cell surface. Pressure is then 

 exerted by means of the injection apparatus 

 so that the oil exudes to form a drop which 

 enlarges until its surface touches the cell 

 surface. Usually the diameter of such a 

 formed droplet will be about 10 p. The con- 

 tact with the cell, occurring while the oil 

 drop is expanding, ensures the presentation 

 of an uncontaminated surface of the oil to 

 the cell. This is essential since most oils de- 

 velop an adsorption film from impurities 

 usually present in the sea water, which pre- 

 vents the occurrence of coalescence. An oil 

 drop first allowed to exude from a pipette 

 and then brought into contact, even with 

 denuded sea-urchin eggs, rarely exhibits 

 the coalescency reaction. 



In the coalescency experiments, quantita- 

 tive results have been obtained by driving 

 the oil out of the pipette under constant 

 and known pressures. For this purpose 

 M. J. Kopac has developed a special device 

 as an accessory to the usual micro-injection 

 apparatus. A driving pressure is built up 



