572 GROWTH IN TISSUE CULTURE 6 



mean figure, and the whole divided by the duration t. The "growth index" is thus: 



X I GO 



X 



a + [b — - x) t 



2 



According to Willmer (1933a, b), the concentration of embryonic extract affects 

 migration rate, but appears to have little influence on the mitotic index of heart 

 fibroblasts. The mitotic index of cultures of periosteal fibroblasts, on the other 

 hand, is more affected by the concentration of embryo extract. It is important to 

 bear in mind that treatments which arrest mitosis {e.g. radiation, colchicine) may 

 increase the mitotic index at a particular time, without this increase being in any 

 way correlated with growth by increase in the cell population {cf. Stroud and 

 Brues, 1954). 



An attempt to achieve a more complete picture of the growth of explants in 

 hanging drop cultures than is given by mitotic counts alone was made by Tomp- 

 kins, Cunningham and Kirk (1947), who measured, as well as the number of 

 dividing cells and the number of resting cells per unit area, the culture thickness, 

 average cell size and number of cells per unit volume of the culture. Their most 

 important observations were that (a) mitosis is more prevalent near the periphery 

 in young (24-48 h.) cultures; (b) average cell size in the interior may be 1/2- 1/4 

 that at the periphery; (c) the maximum number of cells per unit volume occurs 

 half way between the centre and the periphery and (d) thickness increases 

 markedly from periphery to centre. 



{c) Possibility of subdivision 



A culture which can be divided into two or more parts, each of which is capable 

 of reaching the size of the parent part within a given period, is evidently growing. 

 A quantitative measure can be derived from information on the time intervals 

 between subdivisions, because a rapidly growing culture, unless the rate of cell 

 destruction is unusually high, can be subdivided more frequently than a slowly 

 growing one {cf. Perry, Evans and Earle, 1955). Over fairly long periods of time 

 {i.e. of the order of months) this can be a very accurate measure of the nett rate 

 of growth of cells in tissue culture. 



{d) Cell and nuclear counts 



Where the size of the cell population is taken as the basis for growth measure- 

 ments, the method of counting whole cells or cell nuclei is applicable. It is not 

 often technically possible to count the total number of cells in a culture, though 

 this has been done by Puck, Marcus and Cieciura (1956) as a means of evaluating 

 the rate of growth of clonal colonies derived from single cells. Usually, sampling 

 from a uniform cell suspension is necessary. Success therefore depends upon 

 being able to make such a suspension, and the recent technical advances have led 

 to the now common routine practice of harvesting, suspending and sampling 

 cells for inoculating new cultures as well as for enumeration (Evans, 1951 ; Evans, 

 Earle, Sanford, Shannon and Waltz, 1951). 



The method of isolating cell nuclei by treatment with citric acid of cells from a 



