VITAL STAINING V.ITH TH5 FLIfORO CHROME ACRIDINE ORANGE AMD ITS APPLICATION TO RADIOBIOLOGY. I. ALPHA RAY EFFECTS * by Z. S. Gierlach, Capt., M.C, and Dr. A. T. Krebs, Biophysicist from Medical Department Field Research Laboratory Fort Knox, Kentucky 2 September 1949 Sub-project under Study of Body Reactions and Requirements under Varied Environmental and Climatic Conditions. Approved 31 Hay 1946. MDFRL Project No. 6-64-12-06-(14). Project No. 6-64-12-06 Sub-project MDFRL 06-(l4) MEDEA 2 September 1949 ABSTRACT VITAL STAINING WITH THE FLUOROCHROME ACRIDINE ORANGE AND I'K APPLICATION TO RADIOBIOLOGY. I. ALPHA RAY EFFECTS OBJECT It was demonstrated in 1940 by Bukatsch and Haitinger, as well as Strugger, that the modern fluorochrome acridine orange can be used to stain protoplasm and can be used as a tool to study cellular viability. As the study of radiation effects on single cells is important for the proper understanding of the mechanism of radiation effects, it was thought possible to apply this method for that purpose. The first observations have been made with alpha particles from polonium prepara- tions. RESULTS AND CONCLUSIONS Single cells and cell groups of yeast and onion epidermis irradiated with alpha rays demonstrate through changes in acridine fluorescence the radiation effect. When normal the cells have a green fluorescence and when completely destroyed the fluorescence color is red. It is even possible to detect degrees of radiation damage by various changes of the fluorescent color, that is, from green to yellow to orange and finally to red. With this method, it has been possible to estimate the amount of radiation necessary to kill 50% of the cells. RECOMMENDATIONS It is proposed to apply this method to the study of other forms of radiation—such as x-rays, beta rays, etc. Further quantitative work is indicated so that similarities or dif- ferences in effects with different ionizing sources may be studied. Submitted byJ Z. S, Gierlach, Captain, M.C, A. T, Krebs, Ph.D., Biophysicist Approved RAY GJ MGGS(J U Director of Research Approved FREDERICK J. KNOBLAUCH Lt. Col., M.C. Commanding VITAL STAINING VJITH THE FLUOROCHROME ACRIDINE ORANGE AND ITS APPLICATION TO RADIOBIOLOGY. I. ALPHA RAY EFFECTS I. INTRODUCTION In 1940, Bukatsch and Haitinger (1) and Strugger U) introduced the modern fluorochrone acridine orange (Figure l) for the vital staining of protoplasm. Compared to other techniques, it has the advantages that the protein component of living protoplasm is stained without a noticeable toxic reaction; the dye in dilutions of 1:10,000 or greater has sufficient "optical density" to be detected in the stained cell; in aqueous solutions, the dye exhibits a "concentration effect", i.e., the characteristic fluo- rescence of the solution depends on the dye concentration. Dilutions approx- imating 1:10,000 exhibit a green fluorescence whereas dilutions approximating 1:500 have a red color. The concentration characteristic of the dye has been used extensively to test the viability of tissue, for dead cells take up more dye and fluo- resce red, whereas living cells take up less dye and fluoresce green. Using other methods adequate to determine the viability of cells, this difference in fluorescence in life versus death was observed with yeast, Allium cepa, Tradescantia, milk sediments, spermatozoa and various pathogenic and non- pathogenic bacteria (3)• In 1944* the acridine orange staining technique was proposed as a tool to study damage to cells by radiation (4) • The present report comirms and extends the original preliminary investigations. FIG. 1. 3, 6 Tetramethyl diamino acridine II. EXPERIMENTAL A. Apparatus, methods and rrocedures The specimens investigated were the epidermis of the onion cepa) and yeast (Fleishmann)• The third shell of the onion was used and from it the epidermis was peeled away by the gentle suction technique described by Strugger (3). The epidermis sections (about 10 rnm. square) were then stained in the 1:10,000 dye solutions. The pH was adjusted to within physiological limits and the staining time was 10 minutes. After staining, the sections were removed and rinsed, placed on a slide, covered with a cover slip and observed under the fluorescence microscope• In working with yeast, fresh preparations were made each time. The yeast cells were stained in 1:5,000 dilutions of slightly alkaline dye, -this could be accomplished by taking either a droplet of dye and one of a yeast suspen- sion, or mixing equal parts of dye and yeast solution in a test tube— total dilution of dye about 1:10,000. Staining time was also about 10 minutes and the suspension was examined in the same manner as the onion epidermis• Irradiations were made with alpha particles from polonium was deposited on nickel foils 5 mm. The strongest prepara- tion used gave about 10' alpha Irradiation was done in small chambers (see Figure 2), so constructed that the alpha source to speci- men distance was about one millimeter, prevent the condensation of moisture on the Po preparation, a gentle stream of dry air may be blown over it. COVERGLASS TISSUE v MICA COVER CHAMBER POLONIUM PR EPARATION FIG, 2, Irradiation Chamber The dye used in this work came from two sources, A German, (Standard- isierter Farbstoff "Bayer”, Grubler-Holborn) and an American acridine orange (C.I, 788 National Acridine division). In spite of the fact that the absorp- tion curves of the two differed slightly, their staining behaviors were similar, stains were used interchangeably in this investigation. The equipment used for examination of the stained specimen consisted of a light source with proper blue or U-V filter, an ordinary microscope and an eye protective filter (Figure 3). The light source was either a Leitz self- fed carbon arc used with a Beckman B-2 and B-4 filter combination or else the Spencer 353 fluorescence equipment, The eye protective filter was a Wratten K-3 located in or on the eyepiece. To obtain a dark background, it is impor- tant to use the proper filter combination. A glance at the transmission curves of two blue U-V filters and two K-3 filters will stress immediately this importance (Figure 4). Lq obtain greater efficiency in U-V reflection, the standard microscope mirror was replaced by an aluminized front surface mirror. To record the changes going on in the preparation during the irradiation, frequent photomicrographs were taken riming and post irradiation, Tp.ese V;erc made with the aid of the Leitz Micro-Ibso attachment and a 35 mm. Leica housing. Both Ansco color and Kodachrome film proved satisfactory. Proper exposure times were determined by trial exposures first with black and white and later with color film. As a check on the adequacy of the dye as an indicator of cell viability, the technique was studied with Allium cepa epidermis that had been killed by heat in two ways (Figure 5). Obtained from the Eldorado Mining and Refining (1944) Limited. Ottawa. Canada. -•EVE PROTECTIVE FILTER EYEPIECE OBJECTIVE -PREPARATION -SLIDE -CONDENSER DIAPHRAGM CUVETTE LIGHT SOURCE FRONT SURFACE REFLECTOR BLUE-UV-FILTER CONDENSER FIG. 3* Schematic Diagram of Fluorescence Microscope In the first method, the sections were held in live steam for a few minutes and then stained along with a control section taken from the same onion shello In the control section, the membranes were red, the cytoplasm greenish- brown. while the nuclei were a brilliant green. Dead epidermis (3 minutes in steam; showed a completely different appearance. The membranes, cytoplasm, vacuoles and nuclei fluoresced red. In the second method, living onion epi- dermis which had been stained in the usual manner and had a normal appearance, was touched in a small area through the cover glass with a hot wire. Immed- iately, the cells near or in contact with the hot wire developed red cytoplasm and nuclei whereas in surrounding areas where the heat was not so intense, the cells had the original or slightly enhanced appearance of greenish cytoplasm and brilliant green nuclei. Yeast cells respond in the same manner to these tests. B. Results The same effect, as produced by heat killing, was demonstrated when tissue cells were killed by radiation. Depending on the strength of the radia- tion source, the effect becomes more or less quickly manifest during the irra- diation. Under the given conditions. Allium cepa preparations irradiated with polonium alpha sources that give 10' alpha particles/mmo2/min., showed within 20-30 minutes a typical change of appearance. A few nuclei appeared brighter TRANSMISSION % A = BECKMAN B 2 8 B4 COMBINED B = SPENCER U-V FILTER C = EASTMAN K~3 D = EASTMAN K-3 WAVELENGTH IN MILLIMICRONS FIG. 4. Transmission curves of two blue ultraviolet filters and two different Eastman K-3 filters. green, some others had a yellowish tinge. Others were orange and a few red. Because of the fact that alpha particle hits are a random event, the damaged nuclei show a statistical distribution in the irradiated area. (See Figure 6.) VIABLE CELLS DEAD CELLS VIABLE a DEAD CELLS FIG, 5. Acridine orange as indicator of cell viability, (Onion epidermis killed with heat). PIG, 6, 20-30 minutes irradiation FIG, 7* 10 hours irradiation 5 All the cells are red, the cytoplasm is coagulated and separated from the cell walls and lies as a small, tortuous cloud in the center of the cell. Identical effects have been produced in yeast cells. Also here, depend- ing on the irradiation strength and the time of exposure, the number of red fluorescing yeast cells gradually increases. It is a simple matter to ob- serve the process and to count the number of green or red" fluorescing cells as a function of time, Ihese experiments indicate the approximate amount of alpha radiation necessary to kill the cells. Rough estimates show that when Allium cepa epidermis was bombarded under the above specified conditions, the employed millicurie source of alpha particles from polonium kills about 50 per cent of the cells after an irradiation time of thirty minutes. Earlier, Hercik (5), studying the effects of alpha irradiation on Allium cepa epidermis and using post vital staining techniques, calculated that 25 to 30 x 10? alpha particles/rnra. are necessary to kill 50 per cent of the cells, although the sensitivity oi the cell depended somewhat on the type of onion and on seasonal variance. III. SUMMARY AND CONCLUSIONS Using the acridine orange vital staining technique, the results of earlier preliminary investigations have been confirmed and extended. The findings are: 1. Cells and cell groups irradiated before staining and also radiated stained cells demonstrate the radiation effect by a color change in the emitted fluorescent light as they become non-viable. When normal, the cells have a green fluorescence. When completely destroyed, the cells have a bright red fluorescence. 2. Intermediate stages or degrees of radiation damage are character- ized by shades of yellow or orange fluorescence, 3. These changes occurring in cells under alpha radiation have been fixed on color film, 4. Ihe amount of irradiation necessary to kill cells can be readily quantitated. By this means it is possible to study some of the details of radiation damage. The results reported together with the photomicrographs amply justify the use of the acridine orange vital staining method in radiobiology. IV. RECOmSNDATIQNS Further quantitative work is indicated so that similarities or differ- ences with different ionizing sources may be studied. V. BIBLIOGRAPHY 1* Bukatsch, r # and Haitinger. BeitrSge zur fluoreszenz— mikroskopischenDarstellungdes Zellinhaltes, insbesondere des Cyt©plasmas und des Zellkerns. Protoplasms 2k* 515, 1940. 2. Strugger, S, Fluoreszenzmikroskopische Untersuchongen ilber die Aufnahme and Speicherung des Acridinorange durch lebende and tote Pflanzenzellen. Jenaische Ztschr. f. Naturwiss. 21> 97, 1940. 3. Strugger, S. Fluoreszenzmikroskopie and Hikrobiologie, M. and H. Schaper, Hannover, Germany, 1949. This work lists 157 references to the literature, 4. Krebs, A. Uber die Vervrendbarkeit der Struggerschen \cridin-0range- Vital-FHrbung fdr strahlenbiologische Probleme. Strahlenth. 15, 346-352, 1944. V 5» Hercik, F, bie ./irkong der Alpha Bestrahlung auf Zellgruppen, Radiologies. 2, 214, 1937.