IV DEFINITIONS OF GROWTH 567 



quantity. As Lewis and Lewis (1924) reported of the hanging drop type of culture, 

 "If the cukure is successful the cells migrate from the explant... The mass of 

 migrant cells, which may or may not increase in number by division, are termed 

 growth or outgrowth." This statement draws attention to a general problem which 

 will appear in the discussion below of the different methods of measuring "growth" 

 — namely that "growth" sometimes refers to the end and overall result of a number 

 of distinct processes. On the other hand, the same term is sometimes explicitly 

 stated to apply to the measurement of a single variable. In the zone of cell out- 

 growth from a hanging drop culture, the relative contributions of migration and 

 of cell division cannot be judged from an area measurement alone, and in fact 

 many such cultures increase markedly in area without undergoing any appreciable 

 increase in total cell population. Willmer and Jacoby ( 1 936) showed that increasing 

 concentrations of embryonic extract raised the rate of migration as well as the rate 

 of mitosis of fibroblasts in hanging drop cultures. A most important experiment 

 was made by Doljanski and Goldhaber (1945) to demonstrate the effect of 

 embryonic extract on migratory, as distinct from mitotic, activity. The separate 

 effects of such extracts upon the two cell functions were studied by allowing cells 

 which had been made incapable of mitosis to migrate. Chicken fibroblasts, in 

 hanging drop cultures, were irradiated with X-rays, and then transplanted to 

 Carrel flasks. Growth curves were made, using the surface area method. The 

 dose of X-rays applied was 25,000 r, i.e. 2.5 times the amount thought necessary 

 to inhibit cell multiplication completely. The irradiated culture was divided into 

 two equal halves. The fragment placed in chicken plasma diluted with Tyrode's 

 solution increased in three days to about 8 times the initial area and then remained 

 constant up to about 6 days. The sister half placed in a similar medium supple- 

 mented with embryonic extract to 30% had increased to about 14 times at 3 days, 

 and continued to increase steadily to about 20 times at 6 days. Expressed another 

 way "the surface of cultures grown in a medium to which embryo extract was 

 added exceeded by 200% (average of 17 experiments) the growth area of cultures 

 developed in plasma alone". Besides being a clear demonstration of the separability 

 of the processes of migration and cell division, this was also the first unequivocal 

 refutation of the previously widely accepted idea that the principal effect of 

 embryo extract in a tissue culture medium was upon cell division. 



For each type of experiment, the appropriate method of assessing some aspect 

 of the growth complex must be chosen. A great diversity of cell and tissue types 

 can be cultivated, so it is not surprising that particular procedures demand 

 special methods. Some of these methods measure one parameter, some the result- 

 ant of several. The principal criteria of growth are: (i) linear change, change in 

 surface area, mass or volume of cell population; (2) change in number of cells or of 

 cell nuclei; (3) number of mitoses per unit area, per unit volume or per 1000 cells; 

 (4) ability to subdivide the culture at regular intervals without diminution of 

 population density, from which growth can be inferred; and (5) change in some 

 component of the cells, e.g. total N, nucleic acid P, rate of oxygen consumption 

 or glucose utilization. 



Possible methods of measuring growth will be discussed below under the general 

 heading of (i) physical methods and (2) chemical methods. 



Literature p. §8i 



