STUDIES ON THE BEHAVIOR OF FUNGI IN THE PRESENCE OF RADIOACTIVE ISOTOPES' I. A. PEARSON, J. M. HAMMER, K. E. CORRIGAN, AND H. S. HAYDEN Department of Research, The Harper Hospital, Detroit, Michigan Received for publication June 26, 1948 Early in 1946 it was found by the authors that certain shipments of radioactive isotopes (P'2 and I'1l) were contaminated with fungus spores. At that time these spores were observed under the microscope and cultures of them were iden- tified. As a result of this observation, the authors decided to test the sensitivity of numerous stock cultures of fungi available in this laboratory. The preliminary results of this work have been published (1948). Since that time we have re- peated and expanded the work and altered the technique originally used. MATERALS AND METHODS In this study 43 fungi were exposed to radioactive iodine, radioactive phos- phorus, and high voltage X-ray. The cultures were grown on Sabouraud's medium until there was a good growth and spores were visible microscopically. Enough sterile saline was added to each tube to bring the water level to the top of the agar slant-approximately 3 to 5 ml. The cultures were taken to the isotope laboratory where 20 microcuries of radioactive phosphorus or iodine were added to each tube.2 They were left in the isotope laboratory at room tem- perature for 48 hours; subcultures were then made in the bacteriological labora- tory and incubated at room temperature. Cultures of the same fungi were ex- posed to an equivalent amount of high voltage X-ray (1,100 Roentgen units) and then subcultured. Table 1 shows the effects on the growth of the fungi as compared with the growth of control cultures. Because distance and adequate filtering are the important factors in protecting the worker from the beta rays emitted by radioactive phosphorus and the gamma rays emitted by radioactive iodine, the workers wore heavy rubber gloves and lead-impregnated aprons, and the isotope solutions were kept behind a lead shield. The solutions were dispensed from pipettes with a special isotope- dispensing bulb attached, thereby eliminating the necessity of pipetting the radio- active solutions with the mouth. It was found to be necessary that two people negotiate the transfer of the radioactive isotopes to the culture tubes. The calculation of the dosage, the pipetting, and the transferring of the radioactive isotopes to the culture tubes were done by a physicist. A second person manipu- lated the culture tubes with a pair of tongs during the inoculation. The tubes were exposed to the radioactive solutions for 48 hours in the isotope laboratory. 1 Supported in part by a grant from the American Cancer Society, Committee on Growth, through the National Research Council. 2 The chemical quantities of these elements have no bearing on the chemistry of the medium: 1 microcurie of radioactive iodine is 8 X 10-1" grams of iodine; 1 microcurie of radio- active phosphorus is 3.5 X 10-1" grams of phosphorus. 397 on August 3, 2019 by guest http://jb.asm.org/ Downloaded from
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STUDIES ON THE BEHAVIOR OF FUNGI IN THE PRESENCEOF RADIOACTIVE ISOTOPES'
I. A. PEARSON, J. M. HAMMER, K. E. CORRIGAN, AND H. S. HAYDENDepartment of Research, The Harper Hospital, Detroit, Michigan
Received for publication June 26, 1948
Early in 1946 it was found by the authors that certain shipments of radioactiveisotopes (P'2 and I'1l) were contaminated with fungus spores. At that timethese spores were observed under the microscope and cultures of them were iden-tified. As a result of this observation, the authors decided to test the sensitivityof numerous stock cultures of fungi available in this laboratory. The preliminaryresults of this work have been published (1948). Since that time we have re-peated and expanded the work and altered the technique originally used.
MATERALS AND METHODS
In this study 43 fungi were exposed to radioactive iodine, radioactive phos-phorus, and high voltage X-ray. The cultures were grown on Sabouraud'smedium until there was a good growth and spores were visible microscopically.Enough sterile saline was added to each tube to bring the water level to the topof the agar slant-approximately 3 to 5 ml. The cultures were taken to theisotope laboratory where 20 microcuries of radioactive phosphorus or iodinewere added to each tube.2 They were left in the isotope laboratory at room tem-perature for 48 hours; subcultures were then made in the bacteriological labora-tory and incubated at room temperature. Cultures of the same fungi were ex-posed to an equivalent amount of high voltage X-ray (1,100 Roentgen units)and then subcultured. Table 1 shows the effects on the growth of the fungi ascompared with the growth of control cultures.
Because distance and adequate filtering are the important factors in protectingthe worker from the beta rays emitted by radioactive phosphorus and the gammarays emitted by radioactive iodine, the workers wore heavy rubber gloves andlead-impregnated aprons, and the isotope solutions were kept behind a leadshield. The solutions were dispensed from pipettes with a special isotope-dispensing bulb attached, thereby eliminating the necessity of pipetting the radio-active solutions with the mouth. It was found to be necessary that two peoplenegotiate the transfer of the radioactive isotopes to the culture tubes. Thecalculation of the dosage, the pipetting, and the transferring of the radioactiveisotopes to the culture tubes were done by a physicist. A second person manipu-lated the culture tubes with a pair of tongs during the inoculation. The tubeswere exposed to the radioactive solutions for 48 hours in the isotope laboratory.
1 Supported in part by a grant from the American Cancer Society, Committee on Growth,through the National Research Council.
2 The chemical quantities of these elements have no bearing on the chemistry of themedium: 1 microcurie of radioactive iodine is 8 X 10-1" grams of iodine; 1 microcurie of radio-active phosphorus is 3.5 X 10-1" grams of phosphorus.
The + signs indicate the amount of growth as compared with growth of control cultures.The amount of growth was graded from 0 (no growth) to ++++ (normal growth).
The racks holding the cultures were taken to the bacteriological laboratory on ametal tray and placed behind a protective shield. Sterile 12-inch drawn-out
Figure 1. A section of dog liver showing several miliary abscesses, one with an organism,Blastomyces dermatitidis, in the center. X 200. H and E and gentian violet stain.
Figure 2. Section through a small bronchus of the same animal, showing purulent exu-date which contains several Blastomyces dermatitidis organisms. X 200. H and E andgentian violet stain.
4Figure 4. (a) A Fusarium colony after a 48-hour exposure to 100
X 125. Dilute methylene blue. (b) Autoradiograph of (a). X 125.microcuries of p32,
glass pipettes with rubber bulbs attached were used to agitate the solution in thetest tubes until suspended particles of spores and mycelia were visible; several
drops of this suspension were withdrawn with the pipette and inoculated onSabouraud's slants. During this procedure the workers wore heavy rubbergloves and rubber aprons. Again one person handled the pipetting and a secondhandled the subcultures. Following each step in the procedure, Geiger countswere done on the laboratories and equipment used and on the personnel in-volved. The cultures receiving the X-ray wvere handled in the routine mannerw-ithout radioactive precautions.
RESULTS
Since the amounts of P32, 1131, and X-ray used in this experiment were notlethal and no gross morphological changes in the fungi wN-ere noted, a pathogenand a nonpathogen, after exposure to the radioactive solutions, wvere inocuilatedintravenously into animals to check any alteration in pathogenicity. 4l1ternaria,the nonpathogen, could be followedI with the Geiger counter after injection;when the animal was sacrificed after 15 days no lesions were found and the or-ganism could not be cultured from post-mortem material. The animals re-ceiving Blastomyces dermatitidis, the pathogen, died in 24 days or less of gen-eralized blastomycosis. The distribution of these organism could also be fol-lowved, after injection, with a Geiger counter, and positive cultures of B. dermati-tidis wvere obtained from the post-mortem material. (See figures 1 and 2 forphotomicrographs of lesions.) The cultures used for injection were filteredthrough a Seitz filter and washed until the filtrate3 was radionegative to theGeiger counter. The radioactive spores and mycelia on the filter were resus-pended in sterile physiological saline before injection. Preparations made ofthis material and of Fusarium were placed on unexposed lantern slides and auto-radiographs obtained, showing that the fungi themselves were radioactive.(See figures 3 and 4.) It was not possible to obtain autoradiographs of singlespores or mycelia because the silver granules in the emulsion were too large andthere was a considerable amount of scattering. This technique is being studliedfuirther and developed.
SUMMARY
A proce(lure for handling cultures containing radioactive mater ials is described.Numerous pathogenic and nonpathogenic fungi survived exposure to radio-
active phosphorus, iodine, and high voltage X-ray wvithout alteration in theirmorphology or pathogenicity.
Solutions of radioactive iodine and phosphorus are not self-sterilizing.Several autoradiographs of fungi are presented.
The authors wish to express their gratitude to the Department of Bacteri-ology, Duke University, wsho supplied some of the cultures used in this study.
REFERENCE
PEARSON, I. A., HAMNIMIER, J. A., AND HILL, E. J. 1948 The behavior of funigi in the pres-ence of radioactive tracers. Harper Hosp. Bull., 6, 46-55.
3A filtrate that gave not more than 10 C/M/ml was considered to be radionegative.
