V METHODS OF ASSESSING GROWTH 577 



However, much evidence has been put forward in support of the hypothesis that 

 the deoxyribonucleic acid content of the interphase nucleus is constant in amount. 

 In large samples, as in a tissue culture, the average value for a particular cell 

 type (and for all the somatic cells in any one species) appears sufficiently constant 

 that it can be used to calculate the total cell number in the sample (Davidson and 

 Leslie, 1950). Davidson and Leslie (1951) deduced cell number in roller tube 

 cultures of chick heart fibroblasts from DNAP measurements, assuming the 

 average figure of 2. 35. 10"'*^ mg DNAP per nucleus. Changes in other components 

 of the cells during growth (e.g. in protein and non-protein nitrogen, and in P 

 fractions other than nucleic acid P) were expressed in relation to the DNAP and 

 so in terms of weight per cell. 



Healy, Fisher and Parker (1954a) have successfully used DNAP measurements 

 for assessing growth in tissue cultures. They introduced a modification of the 

 Schmidt-Thannhauser technique, namely the use of insulin and MacFadyen's 

 reagent to ensure better recovery and separation of the RNA and DNA fractions. 

 With this technique, excellent correlations were obtained between DNAP and 

 nuclear counts in growing cultures. 



(k) Protein 



Measurements of total protein, usually as nitrogen, can be used to give an 

 estimate of total cellular material. There are however, two difficulties associated 

 with this procedure. The first is that of making a clear separation of cells from 

 a protein-containing nutrient solution, especially in the case of cultures grown in 

 a plasma clot. This is more easily overcome than the second and related problem, 

 which arises from the fact that extracellular protein may be associated with the 

 cells, either produced by the growing cells themselves (e.g. collagen) or adsorbed 

 to the cell surface from the medium. It may be difficult in practice to separate 

 this closely associated protein from the cells, and it is indeed sometimes open to 

 argument whether such protein ought or ought not to be regarded as "cellular 

 protein". In some cases it seems clear that it ought not; for example, collagen has 

 been found to increase even in cultures in which RNA and DNA were decreasing 

 (Gerarde and Jones, 1953), but the distinction cannot always be clearly made. 



To circumvent some of the problems associated with the use of a clot, Warner, 

 Hanawalt and Bischoff ( 1 949) grew heart tissue from chick and rat on a glasscloth 

 substrate in a fluid medium. They encountered the difficulty that nitrogen-con- 

 taining material, probably protein, accumulated on the glasscloth, even when the 

 cloth alone, in the absence of any tissue, was immersed in a protein-containing 

 nutrient solution. Moreover the accumulation started within the first hour of 

 incubation, and increased up to 2 1 days. It was therefore necessary to use a uni- 

 form and minimum amount of glasscloth, and to run control blanks without tissue 

 at each time interval. This experience draws attention to the need for similar 

 controls when other substrates are used. 



Davidson and Leslie (1951) measured protein nitrogen on the trichloroacetic 

 acid-precipitated and lipid-extracted residue from roller tube cultures. The 

 protein material was dissolved in i jV" sodium hydroxide by incubation at 37 °C 

 and the nitrogen estimated by a micro-Kjeldahl method. Instead of determining 



Literature p. 581 



