V METHODS OF ASSESSING GROWTH 573 



culture grown in a fluid medium, was devised by Sanford et al. (Sanford, Earle, 

 Evans, Waltz and Shannon, 1951; Sanford, 1951). The culture is incubated for 

 I h. at 37 °C with o. i A/ citric acid. This releases the cells from the glass and strips 

 the cytoplasm from the nuclei. The nuclei are then collected by centrifugation, 

 washed, and resuspended in a fluid whose viscosity is increased by addition of 

 methylcellulose. The suspension is well shaken to ensure uniform distribution 

 during sampling, and the nuclei, stained with crystal violet, are counted in a 

 haemocytometer. This method is admirable for rapidly growing and healthy 

 cultures, but has its disadvantage : nuclei from dead as well as living cells may be 

 included in the count. The Sanford method has been widely applied, occasionally 

 with some modifications. For example. Eagle (1955a, b) cut down the treatment 

 with citric acid to 10 or 15 min., and further shortened the method by omitting 

 washing and centrifugation of the cells after treatment with citric acid. Parker, 

 Healy and Fisher (1954) improved the yield of nuclei by a brief pre-treatment 

 with 1% tannic acid, to coagulate the nuclei before adding the citric acid. This 

 group of workers (Healy, Fisher and Parker, 1954a, b) found excellent agreement 

 between nuclear count (Earle's strain L cells) and deoxyribonucleoprotein 

 phosphorus measurements in growing cultures. Eagle, Oyama, Levy, Horton and 

 Fleischman (1956) later adopted the principle of Zwilling's (1954) technique for 

 facilitating dispersal of the cells by use of sodium versenate (disodium ethylene- 

 diamine tetraacetate). The cells so dispersed were suspended in citric acid 

 containing crystal violet and samples taken for counting in a haemocytometer. 

 In a similar way, Ginsberg, Gold and Jordan (1955) have used versene acid 

 (ethylenediamine tetraacetic acid) as an aid to dispersing cells from roller tube 

 cultures, in preparation for counting in a haemocytometer. 



A number of investigators has made use of the observation of Rous and Jones 

 (191 6) that living cells are relatively resistant to digestion by trypsin, to obtain 

 suspensions of cells, free from dead cells and from intercellular protein. Among 

 those who have recently published data depending on cell counts from trypsin- 

 dispersed cultures are Rinaldini (1953, 1954) ; Scherer, Syverton and Gey (1953) ; 

 Syverton and Scherer (1954); Cailleau and Kirk (1954); Neuman and McCoy 

 (1955) and Puck, Marcus and Cieciura (1956). Harris (1955) has made a critical 

 study of the effect of trypsin upon living connective tissue cells from the rat. He 

 concluded that the resistance of this type of cell to trypsin was relatively low, and 

 found that considerable damage could be caused by prolonged treatment with 

 trypsin. The changes produced were reversible in the early stages, but continued 

 action produced irreversible effects, culminating in complete cell disintegration. 

 The method should therefore be applied with caution and not indiscriminately; 

 the resistance of particular cell types should be studied carefully. Harris suggests 

 that the conditions used by some investigators might result in 50% of non-viable 

 cells. In his own experiments, he limited the concentration (50 (J-g/ml of crystalline 

 trypsin) and the time for which the enzyme acted (10 min.), to avoid damage to 

 the rat fibroblasts. He counted freshly isolated samples of the cells from trypsinized 

 suspensions in a haemocytometer, cultivated them in special small culture 

 chambers permitting oxygenation of the cells over a period of several days, and 

 made serial counts of all the cells in several marked areas of the cultures as a 



Literature p, ^8i 



