I EFFECTS OF ENVIRONMENT 553 



arguable that the period of "several hours" contact of medium with multiplying 

 cells was too short to produce the hypothetical critical changes in the medium. But 

 the most plausible kind of change would appear to consist of the production by the 

 cells at their surface of a diffusible material depending for its effectiveness upon 

 high concentration. This is compatible with Harris' observation that inocula 

 smaller than 16-18 cells/mm^ could grow if the distribution was uneven. Small 

 clusters with a density of 20 cells/mm2 or greater would proliferate. From this 

 finding Harris concluded that the "minimum viable inoculum size appeared to 

 be more closely related to the density of the cell population than to the ratio of 

 the total population to the volume of medium". The hypothesis that "adaptation"' 

 occurs at the cell surface rather than in the medium is further supported by Harris' 

 observations on the effect of repeated changes of the nutrient upon rapidly 

 growing cultures. No interruption of growth occurred, either by suspension of 

 cells already in the course of division, or by prevention of others from entering 

 upon the process. The growth curve (cell number with time) was unchanged. 

 This, as Harris states, "would appear to provide strong evidence against the view 

 that when a growing culture of connective tissue cells is transferred to fresh medium 

 the medium has to be "adapted ""before growth can continue". Harris did, moreover, 

 succeed in obtaining growth from single cells, either in "adapted" or fresh medium, 

 by reducing the volume of fluid surrounding the cell to capillary dimensions, 

 similar to those in the capillary tubes used by Sanford. Single, or a few, cells were 

 placed between a coverslip and a serum-agar slab (resembling the arrangement 

 described by Pulvertaft, 1952, for microscopic study of bacteria) and colonies 

 were successfully grown. 



The first isolation of a clone, made by Sanford, Earle and Likely (1948), was 

 a considerable technical feat. The proportion of successful isolations was low, but 

 was nevertheless followed by isolations from several other strains. More recently 

 it has been reported (Puck and Marcus, 1955; Puck, Marcus and Cieciura, 1956), 

 that clones of cells, from certain cell strains, can be obtained in i03% yield from 

 single cells. The methods used are technically relatively simple. In the first method, 

 a suspension of cells, prepared by the use of trypsin as a disaggregating agent in 

 essentially the manner of Rous and Jones (1916), is allowed to settle in a layer on 

 the bottom of a Petri dish. This layer of cells is irradiated with X-rays in a dose 

 sufficient to render the cells incapable of multiplication, but not sufficient seriously 

 to affect their metabolism. Into this layer of immobilized "feeder" cells, the cells 

 for clonal isolation are seeded. They are found to divide and give rise to colonies 

 with (in the case of strain HeLa human cervical carcinoma) 100% efficiency. The 

 second method dispenses with the "feeder" cell layer, and is similar in principle 

 to the Pulvertaft-Harris method. The cells are allowed to settle on glass and are 

 overlaid with a soft agar gel. The agar acts in two ways (i) by increasing the 

 viscosity of the medium and so decreasing loss of metabolites from the cells 

 by diffusion and (ii) by depressing the tendency of the cells to migrate. These 

 techniques open up for the future possibilities of isolating "mutant" clones from 

 amongst a large population of cells, and for obtaining easily cultures of known cell 

 lineage hitherto available only to those with much patience and elaborately 

 equipped laboratories. 



Literature p. 581 



