576 GROWTH IN TISSUE CULTURE 6 



(?) Glucose utilization 



Closely related to determinations of glycolysis depending upon respironaetric 

 techniques are the methods involving estimation of glucose utilization (or lactate 

 production) in the medium from growing cultures. Such measurements have 

 been made by Gemmill, Gey and Austrian (1940), by Wilson, Jackson and 

 Brues (1942) and by Willmer (1942). Lactate production is, by and large, corre- 

 lated with utilization of glucose, but neither is a function of growth alone. 



(j) Nucleic acids 



On the assumption of proportionality between the nucleic acid content of the 

 cell and the amount of cellular protoplasm (Berenblum, Chain and Heatley, 1939), 

 Willmer (1942) introduced the method of determining the nucleoprotein phos- 

 phorus content of tissue cultures, as a measure of the "total number of cells 

 present, together with some estimate of their size". The method of Willmer was 

 adopted by Davidson and Waymouth (1943, 1944, 1945, 1946) and later extended 

 (Davidson, Leslie and Waymouth, 1949) to measurement of both ribonucleic 

 acid phosphorus (RNAP) and deoxyribonucleic acid phosphorus (DNAP) in 

 the same cultures (Waymouth, 1951). Hull and Kirk (1950a), taking as a working 

 definition of growth the "increase in metabolizing protoplasm over the amount 

 implanted", made RNAP and DNAP estimations, after Schmidt-Thannhauser 

 fractionation, on roller tube cultures of chick heart tissues, in a manner essentially 

 similar to that of Davidson, Leslie and Waymouth (1949). They found also (Hull 

 and Kirk, 1950b) that incorporation of ^^P into DNA was most active at the time of 

 maximum mitotic rate and that -^^P uptake into the cultures as a whole was similar 

 to the uptake into the nucleic acid fractions (Hull and Kirk, 1950c). Gerarde, 

 Jones and W^innick (1952b) studied -^^P uptake and turnover in the DNA and 

 RNA of growing cultures, and reported high turnover of DNA during growth. 

 Incorporation of adenine-8-''*C, free or in labelled nucleic acids, into growing 

 tissue cultures was studied by Lu and Winnick (1954a). The labelled adenine was 

 used for synthesis of both adenine and guanine, of both RNA and DNA. In a sub- 

 sequent report, Lu and Winnick (1954b) found that incorporation of adenine 

 into the nucleic acids could be abolished by respiratory inhibitors, and they 

 claimed that conditions which stimulate active growth also stimulate active uptake 

 of adenine and thymidine. However, it cannot be excluded that conditions of 

 active metabolism without growth might favour uptake of adenine and thymidine. 

 In the system of Lu and Winnick, the two appear not be to separated. 



Barski and Manigault (1951) used a photometric method for measuring the 

 amounts of nucleoprotein in cultures stained in a quantitative manner with 

 toluidine blue. The method as described estimates total nucleoprotein, thovigh 

 the avithors suggest that the principle could be extended to differential deter- 

 mination of RNA and DNA, using specific stains for each. Variations between 

 replicates were high [^ 25%), but the method was nevertheless applicable to 

 comparison of the effects of various media on growth (Barski, Mavirin, Wielgosz 

 and Lepine, 195 1). 



Ribonucleic acid is known to be present in most cells in larger amounts than 

 DNA, and to be subject to variations under different physiological conditions. 



