388 ENERGY LOSS AND BIOLOGICAL EFFECTS 



were used in these studies of the earliest demonstrable biological effects of 

 x-rays.* The results to be reported were obtained with the fluorochronie acri- 

 dine orange (3,6-tetraniethyl diamino acridine).t This dye is most unusual in 

 that it exhibits a fluorescence concentration effect; that is, its characteristic 

 fluorescence color depends on its concentration. If the concentration is high {ca. 

 1:500), then the fluorescence color is red, and if the concentration is low, then 

 the fluorescence color is green. Fortunately, living protoplasm can accumulate 

 only a small amount of dye and will fluoresce green; when damaged or destroyed 

 its dye-binding capacity is increased, and it will fluoresce red. 



Soft x-rays, unfiltered, from a beryllium-window x-ray tube were used. The 

 tube was always operated at 50 kvp and at 10 ma. Dosage was varied by the 

 time of exposure. Intensity measurements were made with a 250-r nylon and a 

 100-r standard Victoreen chamber at 10 cm. The dose was then calculated for 

 3.8 cm, the distance at which the sections were irradiated. The calculated dose 

 was of the order of 150,000 r per min in air. In this study the upper epidermis 

 of the onion, Allium cepa, was used. The sections of about 1 scj cm were floated 

 in a tap-water dye bath of 1 : 10,000 dilution immediately after irradiation. After 

 staining for 10 min, the sections were momentarily rinsed in tap water and placed 

 on a shde for examination in the fluorescence microscope. Figures 1-4 show 

 the characteristic appearance of control and irradiated specimens. 



Ordinary diachromes lend themselves to biological radiation-effect studies 

 also. One that received the most attention was neutral red. It was desirable to 

 see how control and irradiated onion sections would appear with neutral red 

 staining. Therefore, a neutral red dye bath of 1:5000 concentration was pre- 

 pared, and in this solution sections were stained for 5 min and then examined in 

 an ordinary bright microscope. A section of irradiated epidermis that had been 

 stained with neutral red tap-water solution of pH 7.4 demonstrated the damaged 

 "spots" as bright cherry-red areas. While this preparation was still on the mi- 

 croscope stage, Ho N NaOH was slowly added to one edge and the excess fluid 

 absorbed on the opposite edge with bits of filter paper. The base penetrated 

 first into the injured cells and eventually into the others. As the solution pene- 

 trated, the neutral red was precipitated out, and what were earlier the cherry-red 

 spots now became the foci of needle-like crystal growths. In the "spotless" cells 

 the neutral red was thrown out of solution into a diffuse aggregate pattern. 



The same section was then reacidified with a phosphate buffer of pH 5. The 

 crystals redissolved, and the section returned to its original appearance with the 

 cherry-red spots. Figures 5-7 demonstrate graphically the above-described 

 phenomena. These spots are taken to represent local pH changes in the altered 

 cytoplasm. 



* Krebs, A. T., and Z. S. Gierlach, vital staining with the fluorochrome acridine 

 orange and its application to radiobiology. I. Alpha-ray effects, AiJi. J. Roentgenol. 

 Radium Therapy, 65: 93, 1951. 



t Strugger, S., Fluoreszenzmikroskopie und Mikrobiologie, M. and H. Schaper, 

 Hannover. 



