V METHODS OF ASSESSING GROWTH 571 



chick intestine to be proportional to the glucose concentration, up to about 

 looo mg/ioo ml. It is evident from this kind of consideration that the nature of the 

 entire system (size and origin of tissue, type and concentration of medium) must 

 be carefully taken into account in evaluating area measurements, which are by no 

 means simple to interpret. 



Meaningful measurements of length can be taken of certain kinds of organ 

 culture, e.g. the chick limb-bone rudiments of Fell and Robison (1929) and 

 the rat femora of Ray, Mosiman and Schmidt (1954), since migration is here 

 limited to connective tissue cells surrounding the bone rudiment itself, and 

 increase in linear dimensions of the bone represent a useful measure of total 

 tissue. 



[b) Mitotic counts 



Apart from area measurements, the commonest technique employed over the 

 years for evaluating growth in unorganized cultures, e.g. hanging drop and flask 

 cultures of fibroblasts, epithelial cells and macrophages, has been the mitotic 

 count. This method can, like area measurements, be applied repeatedly to indi- 

 vidual cultures without disturbing their growth. 



The presence in a culture of cells in mitosis would appear to be incontrovertible 

 evidence of proliferative increase. It is, however, by no means easy to convert this 

 evidence into a quantitative criterion of growth. Cunningham and Kirk (1942) 

 and Tompkins, Cunningham and Kirk (1947) analyzed the use of mitotic counts 

 in growth measurements, and they stressed the many variables which make it 

 nearly impossible to estimate the true rate of increase of a cell population from 

 such counts. For example, the rate of cell division varies widely with time (Will- 

 mer, 1933a, b), so that there may be cycles of high and low mitotic activity. And it 

 is necessary, in order to convert mitotic counts into accurate information on the rate 

 of formation of new cells, to know the mitotic rhythm, and the duration of mitosis 

 and of intermitosis, all of which may fluctuate. These factors have been studied, 

 e.g. by Gaillard (1935) and by Neukomm and Richard (1952), and serve chiefly to 

 emphasize that in interpreting growth measurements based on mitotic counts, the 

 multiple factors involved must be kept in mind. Nevertheless, many studies of growth 

 have been based on the use of some type of "mitotic index" (Champy, 1914), 

 usually a comparative figure relating the proportion of mitoses in a total popu- 

 lation of cells to a similar ratio in a control culture. Perhaps the most satisfactory 

 type of index of this sort is based on the kind of evidence obtained from serial 

 photography, at short intervals, of selected areas, as practiced by Willmer (1933a) 

 and Willmer and Jacoby (1936) on colonies of fibroblasts and by Jacoby (1937) on 

 chicken macrophages. This method involves taking photographs at 5 or 6 min. 

 intervals for periods of several days of areas containing initially 100 or more cells. 

 A growth index is derived, which takes into account the fact that the cell population 

 increases as a result of both cell division and emigration of cells from the explant. 

 The number of cells X which underwent mitosis during the time t in hours is 

 subtracted from the number of cells b in the field at the end of the experiment. 

 The mean is taken between this number and the number of cells a in the field at 

 the start of the period. The number of mitoses is expressed as a percentage of this 



Literature p. 581 



