446 C. C. MACKLIN. 



presented by the living cells, are easily made from such cultures. 

 These favorable circumstances plainly indicated the course 

 to be followed in an attempt to gain some knowledge of direct 

 cell division, and accordingly continuous observations of living 

 cells, and supplemental studies of fixed and stained cells of the 

 same type, were carried out. 1 



METHOD 



The cells upon which observations were made were growing 

 at a temperature of 39 to 40 Cent, from the tissue of embryo 

 chicks from two to ten days old. Cultures were prepared by the 

 method of Lewis and Lewis ('15), Locke solution being used as a 

 medium. Activation of the growth was accomplished by the 

 addition to the medium of a small quantity of autogenous embryo 

 extract or bouillon. Thus the medium contained, besides the 

 various salts, small quantities of dextrose (.25 to I per cent.) 

 and protein. To offset the concentration due to evaporation 

 during planting and in the moist chamber it was found to be of 

 advantage to dilute the medium by adding 20 to 25 per cent, of 

 freshly distilled sterile water. Heart tissue was most frequently 

 used, and gave growths which were most serviceable for observa- 

 tion during the second twenty-four hour period. 



By arranging the microscope within the incubator where the 

 tissues were cultivated it was not necessary to expose them to a 

 changed temperature during observation. A ray screen of copper 

 sulphate solution was found to be advantageous when artificial 

 light was used. Evaporation of the drop, with condensation 

 about the walls of the moist chamber, was lessened by placing a 

 small drop of distilled water in the cavity of the depressed slide, 

 and by eliminating air currents from the vicinity of the culture. 



Light seemed to have a deleterious effect upon the living 

 cultures, so that lengthy continuous observation was not found 

 to be practicable. Accordingly inspections were made as short 



1 The procedure of studying amitosis by the tissue culture method was suggested 

 by M. R. and W. H. Lewis, and I desire to record my appreciation of their kind 

 assistance and the use of their large collection of fixed and stained cultures, which 

 was utilized in the investigation. To Prof. F. R. Lillie I also am indebted for his 

 courtesy in placing a room at the Marine Biological Laboratory at my disposal, 

 where some of the work was carried on. 



