578 GROWTH IN TISSUE CULTURE 6 



total protein nitrogen, Gerarde, Jones and Winnick (1952a) in an otherwise 

 similar method, isolated the protein and weighed it. They used relatively large 

 amounts of tissue in their cultures (20-40 mg wet weight of tissue per roller tube), 

 and pooled the contents of three roller tubes in a tared centrifuge tube for precipita- 

 tion with trichloroacetic acid, extraction with ethanol-ether, drying at 105 °C 

 and final weighing. Similar protein residues, dissolved in 3% sodium hydroxide, 

 were used by Stewart and Kirk (1952) for micro-Kjeldahl measurements and by 

 Boyer and Kirk (1952) for a biuret estimation. Good agreement was reported 

 between these two methods. 



A method has been reported (Oyama and Eagle, 1956) based on the Folin-Cio- 

 calteau reagent for phenols. The cells from four T-15 flasks (Earle and Highhouse, 

 1954) were pooled, dissolved in an alkaline copper reagent and treated with the 

 Folin-Ciocalteau solution. Crystalline bovine albumin was used as a protein 

 standard and results expressed in terms of a "bovine albumin equivalent". With 

 each of three strains of cells (Osgood's No. 1 1 1 human leukemia, Gey's HeLa and 

 Henle's intestinal epithelium), the increases of protein measured between the 

 first and fourth days after explantation were approximately three-fold. 



VI. RECORDING GROWTH AND BEHAVIOUR OF CELLS IN 

 TISSUE CULTURE 



The technique of time-lapse cinephotomicrography has been applied to both 

 quantitative and descriptive studies of cells in tissue culture. Moving pictures of 

 organ rudiments were first made by Braus (191 1) and of living cells by Comandon 

 (Comandon, Levaditi and Mutermilch, 191 3; Comandon and Jolly, 191 3). 

 Since that time a great deal has been learned from cinephotomicrographs about 

 the behaviour and growth of both cells and tissue rvidiments ; about changes in 

 cell form and the mode of locomotion of the whole cell; about differentiation and 

 cellular interactions; and about many intracellular activities particularly those 

 concerned in the mitotic cycle. 



The early observations of Comandon and his group were upon migration and 

 "growth" (Comandon, Levaditi and Mutermilch, 191 3). Levaditi and Mutermilch 

 (1913a) recorded contractions in cultures of chick heart fragments. The first 

 films of mitosis in living cells were made by Comandon and Jolly (1913; 19 17-18). 



The locomotion of chick fibroblasts was the subject of a study by Carrel and 

 Ebeling (1926). They concluded that the cells move in straight lines with a uni- 

 form speed of approximately 33.3 [j./h. Lymphocytes progress much more rapidly, 

 at an average rate of 19 [j,/min (Lewis, 1930). More recently, Enterline and Coman 

 (1950) studied the amoeboid movements of various normal and neoplastic cells. 

 All were found to be actively amoeboid, but these authors recorded that the move- 

 ments are intermittent and vary in speed from fractions of i [j,/min. to 6 [ji/min. 

 (average 1-2 [ji/min. i.e. 60-120 [x/h.). Characteristic movements were des- 

 cribed for the different types of blood leucocyte (Rich, Wintrobe and Lewis, 1939; 

 Lewis and Lewis, 1941a), An important characteristic of living cells, the process 

 by which large droplets of fluid are enfolded and assimilated, was discovered 



