V METHODS OF ASSESSING GROWTH 569 



merits in particular circumstances and choice has to be made in the light of these. 

 The methods discussed below are broadly divided into the physical methods, 

 many of which involve little or no disturbance of the living cells; and chemical 

 methods, most of which require the death of the cells before the estimation can 

 be made. 



[a) Linear or areal measurements 



The outgrowth from an explanted fragment in a hanging drop or a flask culture 

 can be estimated by measuring one or more diameters of the roughly circular zone 

 of cells (Ebeling 1914), or by measuring the superficial area covered by the cells 

 (Ebeling, 192 1). Ebeling himself recognized some of the limitations of his method, 

 and stated his belief that "the only means of measuring the growth of a fragment 

 of tissue accurately would be to measure the increase in its weight". Ebeling 

 emphasized the necessity of ensuring that the thickness of the cultures was as 

 uniform as possible, so that the increases of area might be approximately propor- 

 tional to increases in mass. However, this condition is extremely difficult to fulfil 

 in practice, and moreover, even if equal thickness could be achieved at the outset, 

 the thickness seldom remains constant. Cultures thin out at the centre as migration 

 takes place (Davidson, Leslie and Waymouth, 1949), and the rate at which this 

 occurs may not be constant or comparable in pairs of cultures set up for compari- 

 son. The limitations of area measurement for assessing growth have been very 

 fully discussed, e.g. by Parker (1950) and by Willmer (1954), but still need con- 

 stant emphasis if the method, which has its uses under certain strictly defined 

 conditions, is not to be abused. Ebeling adopted the measurement of radius (by 

 ocular micrometer) or area (by projectoscope and planimetric measurement) 

 because he regarded weight measurements as impossible. Two fragments can be 

 compared by means of area measurements, and various forms of "relative growth 

 index" derived. \^ A^ and A^ are the initial areas of the fragments of tissue, and 

 B^ and B^ the areas of the cultures at the end of the growth period, the ratio 



Be A, 



Be A, 



may be derived. This is the growth index used by Ebeling. A somewhat different 

 relative growth index was introduced by Fischer (1923), namely: 



{B-A,)A, 



{B-A,)A, 



Both of these methods of comparison were regarded by Mayer (1939) as unsatis- 

 factory, because they gave a distorted picture of growth if, as is usually the case, 

 there is a difference in size between the original fragments. In Mayer's (1939) com- 

 prehensive survey of data up to that time on the growth of cell colonies in vitro, 

 attention is drawn to the fundamental differences between fresh explants of fibro- 

 blasts and passage cultures, particularly the greater uniformity of growth of the 

 latter. Six factors contributing to the "balance of growth" which is estimated by 

 increase in area are named : 



Literature p. ^Si 



