252 HYDROGEN 



were given 20 mc injected intraperitoneally weekly for 5 weeks; a sheep 

 was given about 3 c intraperitoneally (H-8). To study the comparative 

 metabolism of hydrogen, deuterium, and tritium, bean plants were grown 

 from germination in water containing 100 ^c/ml of H^ as the oxide and 

 0.99 per cent deuterium oxide (H-8). After harvest the plant water for 

 each tissue was removed by azeotropic distillation with benzene. The 

 benzene was removed from the residue, which was set aside for determina- 

 tion of ratios of tissue-bound isotopes. The incorporation of H2O into 

 algae was studied by growing the organisms in nutrient solution contain- 

 ing about 1 mc/ml (H-9). At termination the cells were washed twice in 

 distilled water, which removed less than 6 per cent of the total tritium, 

 then vacuum-dried and combusted over copper oxide in a conventional 

 macro furnace. The combustion water was collected and analyzed for 

 tritium. 



Advantage was taken of the soft-beta energy to visualize the intra- 

 cellular localization of H^-labeled compounds by paramecia and yeast 

 (H-10, H-11). The paramecia were grown in a medium containing 

 sodium acetate labeled in the methyl groups with H^. The acetate had 

 an activity of 4 X 10" dis/min/mg, and the water of the medium, 2 X 10^ 

 dis/min/mg. After growth, the paramecia were fixed in 2 per cent 

 aqueous osmic acid, centrifuged, washed, fixed in Formalin, washed, 

 dehydrated in alcohol, embedded in paraffin, and cut in l-/x sections. 

 Nuclear-track types of emulsion were used. The sections were transferred 

 to a water bath, floated onto photographic emulsions, deparaffinized, 

 dried, and exposed for 1 to 4 weeks at about 5°C. After photographic 

 development and processing, the specimens were viewed unstained. 

 Yeast was grown also in labeled acetate, and a drop of distilled water con- 

 taining the radioactive yeast was placed on the emulsion or placed on a 

 glass slide, dried, and apposed to emulsion surfaces for exposure and sub- 

 sefjuent processing. Control experiments to rule out pseudophotographic 

 effects were run with nonradioactive organisms or with Formvar-coated 

 emulsions to prevent contact of specimen and emulsion. 



H-1. Johnston, W. H., R. L. Wolfgang, and W. F. Libby: Tritium in Nature, Science, 



113: 1-2 (1951). 

 H-2. Brues, Austin M., Agnes M. Stroud, and Leola Rietz: Toxicity of Tritium 



Oxide to Mice, Proc. Soc. Exptl. Biol. Med., 79 : 174-176 (1952). 

 H-3. Farmer, Earl C, and Irving A. Berstein: Determination of Specific Activities of 



Tritium-labeled Compounds with Liquid Scintillators, Science, 117: 279-280 



(1953). 

 H-4. Hayes, F. N., and R. Gordon Gould: Liquid Scintillation Counting of Tritium- 

 labeled Water and Organic Compounds, Science, 117: 480-482 (1953). 

 H-5. Glascock, R. F.: A Combustion Technique for the Assay of Tritium, '^C and 



i''C in a Single 10 Mg Sample of Biological Material, Biochem. J. London, 62: 



699-704 (1952). 



