THE EFFECT OF RADIOACTIVE PHOSPHORUS UPON A SUSPENSION OF ESCHERICHIA COLI CLARENCE F. SCHMIDT Research Department, Continental Can Company, Chicago, Illinois Received for publication February 4, 1948 The effect of radioactive emanations upon microorganisms was apparently first studied by Pacinnoti and Porcelli (1899), who exposed several organisms to uranium powder and stated that they were killed after 24 hours' exposure. Strebel (1900), Aschkinass and Caspari (1901), Crooks (1903), and Pfeiffer and Friedberger (1903) found the exposure of a variety of organisms to radium emanations to be toxic or to inhibit growth. Van Beuran and Zinsser (1903) and Prescott (1904) found no toxic or inhibiting effects of radium emanation on various species under the test conditions used. Chambers and Russ (1912) exposed aqueous suspensions of various organisms to radium emanation of 0.5 millicurie per ml and found sterility of the suspension to occur in 1 to 4 hours depending upon the species tested. This appears to be the first publication showing a quantitative estimate of the intensity of radioactivity used. Spencer (1934, 1935) introduced metal-covered radium therapy needles into freshly inoculated broth cultures of Eberthella typhosa, Proteus X19, and Streptococcus pyogenes and incubated the tubes. Growth was retarded for a short time but equaled the control tubes in 24 hours. When the tubes were held at 0 C, at which multiplication could not occur, the organisms appeared to die more rapidly in the irradiated tubes than in the control tubes. Lea, Haines, and Coulson (1936, 1937) and Lea, Haines, and Bretscher (1941) studied the effects of alpha, beta, and gamma rays on several species of organisms and found the organisms to be killed in an exponential manner. In general, the results of these experiments with radioactive emanations indicate that some lethal effect upon microorganisms may be expected. The degree of the effect appears to depend upon the intensity and time of radiation, the type of the exposed organism, the conditions of exposure, and the nature of the radiation employed. It would be difficult, therefore, to predict at the present time whether any given radiation would have sufficient lethal effect to produce sterility in a food product. In order to preserve food products in a sterile condition over a long period of time with safety to the consumer the application of a heating process is necessary. This is often accompanied by some degree of change in flavor, texture, and vitamin content of the product. For this reason many efforts are being made in the industry today to develop short-time high-temperature processes to minimize these effects. If such products could be sterilized by the incorporation of radioactive materials, the effects of heat could be eliminated. The development in recent years of methods for producing artificially radioactive isotopes in quantity has made available radioactive materials that might be used for 705
706 CLARENCE F. SCHMIDT [VOL. 55 such purposes. The experiment to be reported was therefore designed to test the sterilizing properties of radioactive phosphorus upon vegetative cells in a buffer solution containing no extraneous organic substances that could interfere with the maximum sterilizing effect of the radiation. The premise upon which this experiment was designed was that if complete sterility of a suspension could TABLE 1 Arrangement of test to determine the survival of Escherichia coli suspended in phosphate buffer in the presence of radioactive phosphorus (P32) TUBE A B C D E Radioactivity in final cell suspension, microcurie/ml...0... 1,000 500 100 50 ml radioactive phosphate 2,000 microcurie/ml... 0 10 5 1 0.5 ml phosphate buffer 0.075M... 10 0 5 9 9.5 ml bacterial suspension 110,000,000 cells/ ml in 0.075 M phosphate buffer... 10 10 10 10 10 TABLE 2 Survival of Escherichia coli suspended in phosphate buffer in the presence of various concentrations of radioactive phosphorus TIME OP Ex- POSURZ kr 0 8 24 48 72 96 120 168 240 288 0 Surviving cells millions per ml (%) m I % 47.0 50.0 55.3 53.0 63.0 50.0 54.0 53.7 85.5 91.0 96.5 114.0 91.0 98.0 95.0 IITIAL RADIOACTIVITY, HICROCURIE PER XL 1,000 Surviving cells Surviving cells millions per ml (%) millions per ml (%) ml 11.5 6.6 3.6 1.6 0.43 0.12 0.25 0.51 0.30 21.0 12.0 6.5 2.9 0.78 0.22 0.45 0.94 0.54 ml 22.8 16.7 7.3 5.2 4.2 0.96 0.50 0.68 500 41.5 30.0 13.3 9.5 7.7 1.75 0.90 1.23 100 Surviving cells millions per ml (%: ml 37.0 30.6 23.0 15.0 12.2 10.7 8.6 6.0 6.2 67.2 54.5 42.0 27.3 22.2 19.5 15.6 10.9 11.2 SO Surviving cells millions per ml (%) ml % 39.0 71.0 34.0 62.0 24.0 43.5 25.0 45.5 24.0 43.5 16.0 29.0 12.4 22.6 12.6 22.9 not be obtained under these most favorable conditions, the application of radioactive materials to the much more complicated conditions of a food product could not at present be considered practical. EXPERIMENTAL METHODS AND RESULTS A solution of radioactive phosphorus (P32 emitting beta radiation) in the form of phosphoric acid was secured from the Radiation Laboratory of the Mas-
19481 EFFECT OF RADIOACTIVE PHOSPIIORUS 707 sachusetts Institute of Technology. The solution was titrated electrometrically with NaOH to a ph of 6.95 and carefully evaporated. It was transferred to a 25-ml pyrex graduate cylinder and adjusted to a volume of 18.1 ml to give a solution 0.075 M in phosphate which would contain 2,000 microcuries of radioactivity per ml at the time the experiment was to be started. The solution was then sterilized at 15 pounds steam pressure for 15 minutes, and the slight loss in volume was restored aspetically with sterile water. A suspension of Escherichia coli was prepared by washing 18-hour agar slants with sterile M/15 phosphate buffer, shaking with beads, and filtering through 100 LLI Lii 0. 100 MC TIME- HOURS FIG. 1. TEHB SURVIVAL OF ESCHIERICHIA COLI SUSPENDED IN PHOSPHATE BUFFER IN THE PRESENCE OF VARIOUS CONCENTRATIONS OF RADIOACTIVE PHOSPHORUS (P32) cotton. The suspension was held at room temperature overnight while a plate count was made. The final stock suspension was made the following day by diluting with an equal volume of sterile water to give a suspension containing 110,000,000 cells per ml in 0.075 M phosphate buffer. The experiment was started by combining radioactive phosphate solution, phosphate buffer, and bacterial suspension in the quantities shown in table 1, using 8-by-i-inch test tubes. After thorough agitation and mixing, 1-ml samples were removed from each tube for serial dilution and plate count. At least three dilutions were plated in triplicate for each sample. Further samples were taken at 8, 24, 48, 96, 120, 168, 240, and 288 hours for dilution and plating
708 CLARENCE F. SCHMIDT [VOL. 55 to determine the number of surviving cells. All plate counts were made after 48 hours' incubation at 98 F on Difco nutrient agar. The suspensions were held at 80 to 84 F during the experiment and were thoroughly shaken before each sampling. The results of the experiment are shown in table 2 and illustrated in figure 1 in which the logarithms of the percentage of survivors are plotted against time of exposure. DISCUSSION It is evident from the data in table 2 that a very definite lethal effect is exerted on this organism by the presence of radioactive phosphorus in the solution. From figure 1 it is apparent that a uniform logarithmic rate of death did not prevail throughout the experiment, but that in each case the rate of death is markedly greater during the first 8-hour period than in the succeeding time intervals. Although no attempt has been made to draw smooth curves through TABLE 3 Calculated death rate for different time intervals for Escherichia coli in the presence of radioactive phosphorus (P32) TIM INTERVAL INITIA RADIOACTIVITY, KICROCURIES PER ML 1,000 500 100 so hours K K K K 0-8 0.085 0.047 0.022-8-24 0.015 0.008 0.0045 0.0037 24-240 0.016* 0.005 0.0037 0.0023 * From 24 to 96 hours. the experimental points, it would appear that during the time interval from 8 hours to the end of the experiment an approximately logarithmic rate was maintained. These conclusions would seem to be supported by the calculations of death rate from the usual formula, K = (log A - log B) t where t = time interval in hours A = number of organisms present at beginning of the interval B = number of organisms present at the end of the interval. Table 3 shows the death rate calculated for different intervals and emphasizes the difference in death rate between the initial period and the subsequent periods. These results indicate that the suspension was not uniform in resistance but contained cells of differing susceptibility to the radiation such that the more sensitive cells were killed more rapidly at the beginning of the experiment. From the graph and from table 3, it will be noted that the death rate decreases
1948] EFFECT OF RADIOACTIVE PHOSPHORUS 709 with decreasing intensity of radioactivity. Attempts to relate the logarithm of the number of survivors to radioactive dosage calculated as a function of intensity and time were not particularly successful in producing a curve uniformly expressing the results of the four intensity levels. Therefore these calculations are not presented. It is evident also from table 2 that complete sterilization was not obtained even with the highest concentration of radioactive phosphorus used. In the case of concentrations equivalent to 1,000 microcuries and 500 microcuries per ml it is very evident that a small fraction, approximately 1 per cent or less, of the cells are able to survive the radiation. It seems apparent that at present successful application of radioactive materials to the sterilization of canned foodstuffs, at least, at the concentration levels tested is not indicated by this experiment. It also may be interesting to note that had sterilization been achieved the following would be the cost per no. 2 can for each of the concentrations tested: 1,000 microcuries $3,660, 500 microcuries $1,830, 100 microcuries $366, and 50 microcuries $183, based on the cost of radioactive phosphorus at the time the experiment was conducted. SUMMARY Radioactive phosphorus (P32) added to a suspension of Escherichia coli in phosphate buffer has a definitely lethal effect upon the cells that in a general way is related to the initial concentration. Complete sterilization of the suspension was not obtained in the highest concentration tested. The survivor curve indicated a more rapid rate of killing during the first time interval and the existence of a small percentage of the cells that were more resistant and survived to the end of the experiment. The present experiment does not suggest in any way the possibility of the application of radioactive materials to the sterilization of food products. REFERENCES ASCHKINASS, E., AND CASPARI, W. 1901 Ueber den Einfluss dissociirender Strahlen auf organisierte Substanzen, inbesondere uiber die bakterienschadigende Wirkung des Becquerel-Strahlen. Arch. Physiol., 86, 603-618. BucHANAN, R. E., AND FULMER, E. I. 1930 Physiology and biochemistry of bacteria. Vol. II. Williams & Wilkins Co., Baltimore, Md. CHAMBERS, H., AND Russ, S. 1912 The bactericidal action of radium emanation. Proc. Roy. Soc. Med., 5, 198-212. CROOKS, H. 1903 Emanations of radium. Chem. News, 87, 308. LEA, D. E., HAINES, R. B., AND BRETSBHER, E. 1941 The bactericidal action of X-rays, neutrons, and radioactive substances. J. Hyg., 41, 1-16. LEA, D. E., HAINES, R. B., AND COULSON, C. A. 1936 The mechanism of the bactericidal action of radioactive radiations. Proc. Roy. Soc. (London), B, 120, 47-75. LBA, D. E., HAINES, R. B., AND COULSON, C. A. 1937 The action of radiations on bacteria. Proc. Roy. Soc. (London), B, 123, 1-21. PACINNOTI, G., AND PORCELLI, V. 1898 Azione microbicida esercitata dai raggi Becquerel su alcuni microorganismi patogeni. Florence. After Index Medicus, 1899. Cited by Buchanan and Fulmer. PFEIFFER, R., AND FRIEDBERGER, E. 1903 Ueber die bacterientodtende Wirkung der Radiumstrahlen. Klin. Wochschr., 40, 640-641.
710 CLARENCE F. SCHMIDT [VOL. 55 PREScoTT, S. C. 1904 The effect of radium rays on the colon bacillus, the diphtheria bacillus and yeast. Science, 20, 248-252. SPENCER, R. R. 1934 The sensitivity, in vitro, of bacteria to the beta and gamma rays of radium. U. S. Pub. Health Service, Pub. Health Repts., 49, 183-192. SPENCER, R. R. 1935 Further studies of the effect of radium upon bacteria. U. S. Pub. Health Service, Pub. Health Repts., 50, 1642-1655. STREBEL, H. 1900 Zur Frage der lichttherapeutischen Leistungsfahigkeit des Induktionssfunkenlichtes nebst Angabe einiger Versuche uiber die bakterienfeindliche Wirkung der Becquerelstrahlen. Fortschr. Gebiete R6ntgenstrahlen, 4, 125-132. VAN BEIuAN, F., AND ZINSSER, H. 1903 Some experiments with radium on bacteria. Am. Med., 6, 1021-1022. Downloaded from http://jb.asm.org/ on September 22, 2018 by guest