98 E. L. POWERS 



of the values at saturation is 1-25; that is, in the presence of oxygen, 

 spores exhibit 35 per cent more radiation sensitivity than they do when 

 irradiated in the presence of nitrogen and exposed to oxygen imniedi- 

 atel}^ after. This is a small oxygen effect compared with the oxygen 

 effects of about 300 per cent usually observed in wet systems (Gray, 

 1957-8). This, however, is not the total oxygen effect that can be 

 demonstrated in the bacterial spore. As shown in Fig. 2, the radiation 

 sensitivity in the presence of oxygen increases more rapidly with in- 

 creasing temperature than it does in its absence. At room temperature 

 the ratio observed is about 1 -25 as noted on the jDrevious figure. Above 

 30°, the dramatic decrease in radiation sensitivity observed in the 

 absence of oxygen cannot be demonstrated when oxygen is present 

 (Powers et al., 1959). This indicates that oxygen prevents the thermal 

 reversion of the free radicals we observed in the other experiments. 

 Verification of this can be obtained by irradiating the spores in nitrogen 

 at any one of the temperatures below 30°, exposing them to oxygen 

 briefly, removing the oxygen, and then treating with nitric oxide or 

 heat. In these instances, the radiation sensitivity is not affected by 

 heating or by the gas. Removal of the free radicals is possible, then, 

 only if they are manipulated prior to their exposure to oxygen. These 

 facts mean that the free radicals and oxygen form complexes that are 

 irreversible, and that are damaging to the cell. This oxyradical or per- 

 oxyradical should be very strongly oxidizing, and should undergo a 

 secondary reaction to produce damage to the cell. If the free radical is 

 disposed of before oxygen is admitted, the damaging complex cannot 

 be produced. 



The i)hysical evidence 



We have physical evidence in the form of electron spin paramagnetic 

 resonance (ERR) analysis that supports our interpretation that part of 

 the radiation damage is brought al)out bj^ long-lived free radicals to- 

 gether with oxygen (Ehret et al.. 1960; Powers et al., 1961). When the 

 spores are irradiated at low tem])erature and an ESR spectrum is re- 

 corded, we observe a second derivative tracing with three peaks with 

 30 (t spacings (Fig. 4). If these spores are l)rought to room temperature 

 for brief periods, and then returned to low temperatures for reading, 

 twin ])eaks 10 G on each side of the centre line grow in at the expense 

 of the central peak. After about 20 minutes at room temperature, the 

 fully developed spectrum can be seen: it consists of the originally 

 observed triplet with a 1:2:1 configuration, and a newly developed 

 doublet with a 1:1 configuration. 



