568 GROWTH IN TISSUE CULTURE 6 



V. METHODS OF ASSESSING GROWTH IN TISSUE CULTURE 



A great amount of effort and ingenuity has been put into the task of designing 

 methods for following quantitatively the course of a growing culture. Different 

 methods, based on different criteria of "growth", have been devised for cells cul- 

 tivated in different ways. One of the technical limitations restricting the kinds of 

 methods available to the earlier students of growth in tissue culture was the 

 almost universal use of a plasma coagulum as a support for the cells. Methods of 

 growth measurement had therefore to be contrived to take this into account, 

 that is, to be suitable for application to cells embedded in a plasma substrate. 

 Much less frequently, separation of the cells from the clot was attempted. This 

 was generally considered impracticable, and the majority of methods was there- 

 fore based on acceptance of the inseparability of the cells from the medium. 



It is significant that a textbook on tissue culture published in 1931 (Craciun, 

 1 931), when the technique was being widely practiced, particularly for studies on 

 proliferation, mentions only three methods of growth measurement (by area 

 measurement, cell counts and tissue weight). Craciun points out that only strains 

 of cells maintained for a long time in standard conditions give replicate area 

 measurements reproducible within 10%; that errors can occur because growth 

 is not planar in the plasma-clot cultures, and because the thickness of the culture 

 and the degree of cell crowding in the peripheral zone are not taken into account. 



Nevertheless, area measurement was apparently the only method regarded 

 as practical by Craciun; cell counts, and weighing the newly migrated cells, 

 were dismissed as too difficult. Even as recently as 1950, the same three methods 

 were still the only ones considered feasible by one authority (Cameron, 1950), 

 though she also mentions the possible use of estimations of the physiological 

 activity of the living cells. At the same time (Signorotti, Hull and Kirk, 1950) it 

 was still being lamented that none of the customary methods of tissue culture 

 fulfilled the criteria necessary for strictly quantitative work. These authors con- 

 sidered reducing the plasma clot, still generally thought to be essential for sup- 

 porting fibroblast cultures, but they did not entertain the possibility of dispensing 

 with it altogether, though this had already become a fairly usvial practice (White, 

 1946; Evans and Earle, 1947). Earle and his collaborators were amongst the 

 first to make determined attempts to improve conditions for quantitative study 

 by employing a flviid medium and an inert substrate, either perforated cellophane 

 or simply the glass of the culture vessel (Evans and Earle, 1947; Earle, Evans and 

 Schilling, 1950; Schilling, Earle and Evans, 1950). Once the fluid nutrient could 

 be easily and completely separated from the cells, several good methods of evalu- 

 ating growth became possible. Many cell types have now been grown in this way, 

 in fluid media directly on glass (White, 1946, 1949, 1955; Earle, Evans, Sanford, 

 Shannon and Waltz, 1951 ; Ehrmann and Gey, 1953) or in fluid suspension (Earle, 

 Schilling, Bryant and Evans, 1954; Owens, Gey and Gey, 1954), and the range 

 of quantitative methods has been correspondingly extended. 



Some methods of growth measurement can be used upon the undisturbed living 

 cultures; some require disturbance of the system or even its total destruction, so 

 that serial measurements upon a single culture are not possible. Each method has its 



