NEUTRON EFFECTS ON ANIMALS 45 



enormously resistant. In one instance, 5000 n units were applied to a 

 young culture of euglena without diminishing the number of active in- 

 dividuals to be found in a microscopic field, and without giving rise to a 

 greater number of inactive or distorted specimens. It is true, however, 

 that in this sample as well as in others exposed to 2500 n and 1250 n units 

 respectively, there were macroscopic changes which became apparent on 

 standing 48 hours. In unirradiated controls, free-SAnmming organisms 

 were seen as a fairly uniform haze in the liquid, while in the bombarded 

 samples a water-clear zone roughly proportional to the intensity of bom- 

 bardment appeared at the top of the medium, and a dark green la3'er of 

 sediment collected on the bottom of the tube. Repeated microscopic 

 examination still failed to establish any visible differences between exposed 

 and unexposed samples. 



The difficulty of obtaining representative samples from euglena cultures 

 is considerable, owing to their tendency to congregate in corners, such as 

 the angle formed b\^ a meniscus and the wall of a small vessel, and to 

 various tropisms. The same factors render even direct counting methods 

 extremely inaccurate. Such considerations led us to abandon attempts to 

 decide whether the phenomenon observed represented actual killing of the 

 cells or perhaps a sort of auto-agglutination due to a change in acidity or 

 tonicity of the medium in which the cells were bombarded. 



Culture Media. Bacteria were gro^^^l in beef heart infusion broth 

 (Difco), usually in 250 cc. Erlenmeyer flasks containing 100 cc. of medium. 

 Plate counts were made by placing measured volumes of appropriate dilu- 

 tions in sterile Petri dishes, then pouring in and thoroughly mixing a thin 

 layer of the clear potato medium used by Hollaender and Claus (2). Plates 

 and primary cultures Avere incubated at 37°C. When frequent counts 

 were to be made on a subculture, however, the medium was maintained at 

 room temperature both before and after planting in order to avoid frequent 

 changes resulting from opening and closing the door of an incubator. 



Occasionally, experiments were made with cultures which were con- 

 siderably diluted in sterile saline before bombardment, in order to eliminate 

 errors which might arise as a result of the effect of the neutrons on the 

 nutrient medium rather than the cells themselves. Such dilutions, as well 

 as those required in making counts, were made in sterile saline which was 

 measured aseptically into sterile vessels immediately before use. 



Neutrons. It was known at the outset that the available intensity of 

 neutron bombardment was likely to prove inadequate to bring about 

 readily observed responses in bacteria. We wished, therefore, to make 

 maximum use of the neutrons available to us, and if possible, to increase 

 their efficiency in some way. This might be possible, for instance, if the 

 organisms proved to be particularly sensitive to neutrons possessed of a 



