574 GROWTH IN TISSUE CULTURE 6 



means of assessing growth. Cell suspensions from chick embryo tissue have been 

 prepared by simply bringing the pH to g.o-g.2 (Mookerjee, 1953); and rat liver 

 cells can be readily dispersed without trypsin by incubating the tissue at 37° for 

 10 min. in mildly acid solution (Longmuir and apRees, 1956). These methods 

 might be applicable, with suitable modifications, to the dispersal of other cells. 

 Cells grown in the suspended state, e.g. in the "tumbling tube" cultures of mouse 

 lymphosarcoma MB III (Owens, Gey and Gey, 1954, 1956) or the strain of 

 monkey kidney cells which multiplies in suspension (Graham and Siminovitch, 

 1955), can be sampled directly for enumeration. 



[e) Tissue mass 



The direct measurement of tissue mass is one of the older methods by which 

 attempts were made to assess changes in the amount of tissue in a growing culture. 

 Several investigators who made respiration and other chemical measurements on 

 growing cultures, tried weighing in order to be able to express their results in 

 terms of wet or dry weight. 



Wet weight estimations were made by Lipmann (1933), after freeing the tissue 

 from plasina by treatment with 0.25A" sodium hydroxide. Such estimations when 

 applied to tissue cultures are open to the objections which apply generally to wet 

 weight measurements — namely the difficulty on the one hand of freeing the tissue 

 from adherent fluid, and on the other of avoiding undue desiccation of the tissue. 



The determination of dry weight also involves difficulties. Because water makes 

 up 80% or more of the total tissue mass, the absolute mass of the conventional 

 type of tissue culture is rather small to give precise measurements of dry weight. 

 Moreover, precautions must be taken to maintain a moisture equilibrium during 

 weighing. 



Cultures have been separated from their plasma substrate for dry weight 

 estimations by use of dilute alcohol (Meier, 1931) or pepsin digestion (Laser, 1932). 

 The magnitude of the quantities handled may be judged from the fact that Meier 

 estimated the initial dry weight of his cultures to be 0.015 mg. Laser used the dry 

 weight for comparison with area measurements in fibroblast cultures, and showed 

 that, whereas area stopped increasing after 4 days, dry weight continued to go 

 up until at least 10 days, and that the shapes of the two types of growth curve were 

 quite different. 



Gemmill, Gey and Austrian (1940) worked with tissue in roller tubes, and 

 measured both wet and dry weights. In 45 determinations on rat sarcoma cells, 

 they found that the dry weight averaged 11.7% of the wet weight, with a range 

 of 6.1-18.2%. 



Mass cultures of the kind now regularly grown in agitated flasks by Earle, 

 Bryant, Schilling and Evans (1956), in which a single flask may contain nearly 

 6* 10^ cells (23.6 g wet weight) make it possible to apply wet weight measurements 

 to whole cultures or to samples taken from individual large flasks. 



{f) Cell or culture volume 



Because of the technical difficulties of weighing the cells, Gemmill, Gey and 

 Austrian (1940) tried substituting measurement of the total volume of cells in 



