V METHODS OF ASSESSING GROWTH 575 



their cultures, by centrifuging them in haematocrit tubes. This method has many 

 advantages for cells which can be freely obtained in suspension, either by 

 simple mechanical means, or by treatment with trypsin, versene or other aids to 

 dispersion. It has been found particularly suitable for estimating increases in cell 

 population in rapidly growing cells in fluid media (Waymouth, 1956a) and for 

 comparing the effects of different nutrient media on cell proliferation (Waymouth, 

 1956b). The VanAllen microhaematocrit (VanAllen, 1925) is of suitable dimen- 

 sions for measviring the volume of the cell population from a D3.5 Carrel flask 

 culture. 



(g) Nephelometry 



Where cells can be kept in a state of uniform suspension, nephelometry can be 

 used, as with bacterial cultures. This method has been applied to embryonic 

 chick heart cells by Rinaldini (1954), and to monkey kidney cells by Youngner 

 (1954). Comparison between optical density and counts of cell nuclei by the 

 Sanford method, made by Youngner, revealed that the optical density values 

 were in general below those expected. This was attributed to varying degrees of 

 aggregation. A correction could be applied for this. 



{h) Metabolic measurements 



Attempts have been made to use respiration and glycolysis as measures of 

 growth. The principal difficulty encountered in trying to correlate metabolism 

 with growth is that the maintenance metabolism forms a large and variable 

 proportion of the total, and to make proper corrections for it is extremely difficult. 

 Major fluctuations in metabolic activities may result from a number of variables 

 which may or may not be independent of growth, e.g. in response to changes in 

 available oxygen, in concentrations of certain enzymes and coenzymes, or concen- 

 tration of glucose or other substrates. Since isolated particles from cell cytoplasm 

 are capable of respiring, the existence of measurable activity is not even a reliable 

 indication that viable cells are responsible for the process measured. Metabolic 

 measurements cannot therefore be recommended as a means of assessing prolifer- 

 ative activity. Early attempts to correlate glycolysis with growth (Meier, 1931; 

 Laser, 1932; Lipmann, 1933; Lipmann and Fischer, 1932) are of historic interest 

 only. However, these and later measurements of the metabolism of cells in tissue 

 cultures {e.g. by Gemmill, Gey and Austrian, 1940; Danes and Leinfelder, 1951; 

 Danes, Christiansen and Leinfelder, 1953; Danes, 1955a, b; Prop, 1954; Harris 

 and Barclay, 1955) are of interest in demonstrating differences in overall metab- 

 olism of various cell types, and the relative capacities of certain cells in vitro for 

 aerobic and anaerobic glycolysis, though they bear no valid relation to growth 

 measurements. Thus for example, Harris and Barclay (1955) compared glucose 

 utilization and lactic acid production by rabbit macrophages under aerobic and 

 anaerobic conditions. They calculated the oxygen uptake of the cells from measure- 

 ments based on a logarithmic relationship between oxygen tension in the medium 

 and the current produced between a platinum cathode and a non-polarisable 

 (lead) anode. Changes of 1-2 [i.moles/1 could be recorded without ampHfication. 

 This appears to be a sensitive means of measuring respiration in tissue cultures. 



Literature p. 58/ 



