L. H. GRAY 



samples, irrespective of the conditions of irradiation. Subsequent to irradia- 

 tion, cells were treated in precisely the same way, whether they had been 

 irradiated in vitro or in vivo. 



A careful preliminary investigation established that, provided the dose was 

 adjusted to give roughly equal amounts of damage, the presence or absence 

 of oxygen at the time of irradiation had no influence on the percentage of 

 abnormal anaphases seen at different times after irradiation. This was true 

 whether the irradiation was at 18°C or at 37 °C. When the inoculum was 

 0-2 ml. containing roughly 2 x 10^ cells, the percentage of abnormal 

 anaphases rose slowly with increasing interval between irradiation and 

 fixation to a flat maximum at 14 to 15 hours, and then fell. As a standard 

 procedure, cells were therefore fixed at 14 hours after inoculation. Since the 

 problem of ascertaining the precise oxygen tension of the cells at the time of 

 irradiation was approached somewhat differently in the case of /// vitro and 

 in vivo irradiations, these two aspects of the investigation will be described 

 separately. 



IN VITRO IRRADIATIONS 



In the case of in vitro irradiation, the diflftculty hinges essentially about the 

 fact that radio-sensitivity is varying most rapidly with oxygen tension at very 

 low oxygen tensions, namely those corresponding to concentration of dis- 

 solved oxygen of about 5 [xM/l. Fluid containing this amount of dissolved 

 oxygen is in equihbrium with a gas phase which contains about • 3 per cent 

 of oxygen at room temperature, and about 0-5 per cent at 37 °C. Those 

 familiar with Warburg manometric techniques will appreciate the difficulty 

 of maintaining an adequate oxygen concentration in a fluid phase containing 

 respiring tissue, even when the gas phases consist of almost pure oxygen. 

 The difficulties are obviously increased several hundred-fold in the present 

 experiments on account of the very low oxygen tensions which are of interest. 

 These were satisfactorily overcome in the case of irradiations at room 

 temperature by the use of low tumour cell concentrations, and an irradiation 

 vessel which allowed gas to be blown at high speed obliquely and eccentri- 

 cally on to the surface of a small depth of ffuid contained in a gas-tight 

 enclosure, {see Figure 1). The gas inflow^ at about 60 c.c./sec agitated and 

 vigorously stirred the fluid. The all-glass syringe at the base of the irradia- 

 tion vessel was used once a minute for 15 min before irradiation to withdraw 

 the fluid and reintroduce it into the irradiation vessel as a small fountain. 

 This counteracted any tendency to sedimentation of cells and broke up 

 small clumps into individual cells. In order that oxygen shall pass from the 

 gas into the fluid phase at a rate which keeps pace with cellular respiration, 

 there must inevitably be a pressure differential. Our objective was to keep 

 this differential small compared with that corresponding to a concentration 

 of 5 [J.M 1. in the fluid phase. A calculation based on Qq^ ~ 2 \j\. of oxygen 

 per minute per 10^ cells at IS'C, kindly measured for us by Dr. D. L. 

 Dewey, and a measured rate of exchange of oxygen between the gas phase 

 and a fluid which did not contain cells, indicated that the concentration 

 of dissolved oxygen in the irradiation experiments, at room temperature, 

 would be known with the desired accuracy if we worked with concentrations 

 not exceeding 4 x 10" tumour cells per ml. The margin of uncertainty in 



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