X-rayed culture of the human type of Mycobacterium tuberculosis. They

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1 THE EFFECTS OF X-RAYS ON A STRAIN OF EBERTHELLA TYPHOSA THOMAS H. GRAINGER, JR. Department of Biology, Lehigh University, Bethlehem, Pennsylvania Received for publication October 9, 1946 The study of the effect of X-rays on bacteria is suggested from the success which geneticists have had in using X-rays on multicellular forms as a means of inducing genetic changes. If it may be postulated that bacteria have the same genetic setup as higher forms, then it is conceivable that bacteria might be affected in the same way by X-rays. This would follow along the same line of work as Muller (1927), who produced mutations in many types of reprodu'ctive and somatic cells with the use of X-rays. The present report is concerned with the effect of X-rays on a strain of Eberthella typhosa. Most of the literature concerning the effect of X-rays on bacteria relates only to the lethal action. Progress has been reviewed at intervals by Clark (1934), Duggar (1936), McCulloch (1945), and Rahn (1945). There is evidence to indicate that there is a distinct difference between killing a bacterium by X-rays and killing a bacterium by heat or by disinfectants. Thus, according to Lea (1946), "after irradiation, the bacterium which is rendered incapable of giving rise to a colony may still be motile (Bruynoghe and Mund, 1935), may still be capable of respiration (Bonet-Maury, Perault and Erichsen, 1944) and may, when cultured and examined microscopically, show some growth (Luria, 1939)."' Thus, the killing action of X-rays on bacteria is possibly the result of the production of a lethal mutation. Whether bacteria have the same hereditary mechanism as higher plants is still a matter of conjecture, but it is quite probable that they do. Thus, according to Lewis (1941), a nucleus of a bacterium might be considered as "a naked gene string encrusted with chromatin, or a single naked gene string." If the bacterium is a haploid (which is most probable since it is asexually produced), then there is a great possibility of a lethal mutation having immediate effect. There have been few publications concerning the changes that may occur in X-rayed cultures. The first to study the effect of X-rays on bacteria was Minch (1896). He exposed a strain of E. typhosa on agar to the X-rays produced from a Hittorf tube at a distance of 10 cm for a period of 8 hours. EIis only observation was that there appeared to be no gross change in the development of the colony. Haberland and Klein (1921) exposed a human strain of the tubercle bacillus to X-rays and found no change in its biochemical or physiological activities. On the other hand, Lange and Fraenkel (1923) noted that the infectivity for guinea pigs was greatly diminished when they used a 33-dayold X-rayed culture of the human type of Mycobacterium tuberculosis. They demonstrated that younger cultures were more resistant to the X-rays. Klovekorn (1925) exposed Escherichia coli and Staphylococcus aureus to X-rays and 1 The references given in this quotation are not specifically cited in this paper. 165

2 166 THOMAS H. GRAINGER, JR. noted that there were certain modifications in the cultural characteristics only when the cultures were 28 to 30 days old. Bertrand (1929) found that X-rays of 2 A had no effect on the virulence or rapidity of growth of S. aureus or Microsporon audouini. Rice and Guilford (1931) showed that X-ray treatment of a bovine strain of M. tuberculosis increased the dissociation from "rough" to "smooth" colonies in a rapidly growing culture. Smith, Lisse, and Davey (1936) noted that there was no significant change in the electrophoretic mobility of Escherichia coli after exposure to X-rays. Forfota and Hatmori (1937) claim that the antigenic structure of E. typhosa undergoes change under the effect of hard X-rays. Lea, Haines, and Coulson (1937) found that occasionally long filamentous rods would be formed from an X-rayed culture of E. coli. This was explained as due to the interference with the fission mechanism. Drea (1938, 1940) showed that the virulence of a culture of human tubercle bacilli could be considerably attenuated by successive irradiations with X-rays over a long period of time. Haberman and Ellsworth (1940) noted the increase in dissociation of S. aureus and Serratia marcescens in actively proliferating cells. Their work seems to indicate that hard rays were more effective in producing dissociants. Haberman (1941) showed that a culture of staphylococci exposed to X-rays lost the lethal factors, and those of dermoneurosis and hemolysis. There was no observable association in colony types. It appears from the work of Lea, Haines, and Bretscher (1941) that X-rays of various lengths on E. coli and spores of Bacillus mesentericus produced lethal mutations. The striking effect was that the bacteria continued to grow, in the sense of increasing in size, but failed to divide. Gray and Tatum (1944) produced, by means of X-rays, mutant strains of E. coli and Acetobacter melanogenum characterized by their inability to produce specific biochemical reactions. Tatum (1945) likewise produced mutant strains of E. coli by exposing two mutant strains to a second X-ray treatment. Some of the effects of X-rays upon bacteria which have been noted are the production of lethal action, permanent changes in colony form and color, and the loss of certain biochemical reactions. MATERIALS AND METHODS [VOL. 53 X-ray apparatus. A General Electric crystal diffraction X-ray unit, type VWC, form E, was used. It was operated at 30,000 volts with a filament current of 18 milliamperes. A molybdenum target Coolidge type with unfiltered radiation was used. The maximum characteristic radiation was A. These rays were hard, but, because of the presence of continuous radiation and the use of unfiltered radiation, some soft rays were present. In general, the rays are spoken of as hard or soft on the basis of their wave length. Those which have wave lengths greater than 1 A are classified as soft rays and those which have wave lengths less than 1 A as hard rays. The soft rays are more readily absorbed by living material, but the hard rays are more penetrable. The baqteria that were exposed to the rays from this machine were placed at a

3 19471 EFFECTS OF X-RAYS ON EBERTHELLA TYPHIOSA 167 distance of 61 inches from the X-ray tube in front of the X-ray window. The bacteria, in a distilled water suspension, were placed in a small pyrex glass tube and were accurately placed in the path of the X-rays by means of a fluorescent screen directly behind the tubes. Culture. The strain of Eberthella typhosa used had been recently isolated from the blood of a typhoid patient. The strain was characteristic of the species in respect to all the biochemical and physiological characteristics as described in Bergey's Manual (1939). Neither its antigenic formula nor its phage specificity was determined. For the purpose of this study a single cell of this culture was isolated and put into nutrient broth. It was incubated at 37 C for 18 hours and was streaked on agar slants. These agar slants were incubated for 18 hours, and the growth was harvested by means of gentle washing with sterile, distilled water. The suspension was then diluted with sterile, distilled water to give a concentration by the nephelcmeter method of McFarland the no. 10 tube, which corresponds to about 3 billion organisms per ml as determined by plate counts. One ml of this suspension was then placed in a chemically clean, sterile pyrex glass test tube. The suspension was then ready for exposure. Lethal studies. The tubes containing the suspension of the organisms were held securely in a clamp against the window of the X-ray machine. The organisms were exposed for periods of, 1, 11, 2, 21, 3, 31, 4, and 41 hours. Immediately after exposure, the suspension was diluted in a tenfold series to a dilution of 1:1,000,000,000. Sterile distilled water in 9-ml amounts was used for the dilutions, and nutrient agar pour plates were made in duplicate from each of the dilutions. All plate counts were made after an incubation period of 48 hours. Only those plates that contained from 30 to 300 colonies per plate were used to determine the number of bacteria that survived. A control consisting of 1 ml of the same suspension that was unexposed to the X-rays was used in all cases. The temperature during exposure was 25 C. It was found that the killing of the organisms resulted in a logarithmic order of death. The results were plotted on semilogarithmic paper (figure 1). After i-hour exposure, 50 per cent of the organisms had been killed; at the period of 4 hours, only 0.05 per cent survived; and at 41 hours, none survived. It has been shown by several workers (Lea, 1946) that what is described as a lethal effect of radiation upon a bacterium is the inability to give rise to a colony on appropiate laboratory media. The bacterium may still be capable of performing some of its life processes. Thus it was of interest to observe whether or not the actively motile culture of E. typhosa was still motile after 41 hours of irradiation. After this period of exposure the organisms were examined for motility by the hanging drop method. The great majority of bacteria appeared to be nonmotile, but a small percentage showed motility. However, when these organisms were placed in suitable media, they failed to develop colonies. Anomalous variation. The modifications of bacteria have been called mutations, variations, dissociations, saltations, discontinuous variations, etc. There is little real justification for the use of these terms as applied to bacteria.

4 168 THOMA H. GRAINGER, JR. Our conceptions of heredity have been derived from living things which pass through a sexual cycle and which have a nuclear mechanism. We do not have adequate knowledge concerning the hereditary mechanism of bacteria to interpret them in similar terms. The views concerning the existence of nuclei in bacteria range from the supposition that bacteria have no nuclei to the view that the entire cell is a nucleus (Lewis, 1941). We may postulate that the bacterium is a haploid since it is asexually produced, but it may be a diploid or even a polyploid. Even when we are dealing with a single bacterial cell, we cannot be sure that we are dealing with only one nuclear unit (Wilson and Miles, 1946). FIG. 1. Af 2 0r 2' ' 6 f 727,oe o7~ f o /e [vol. 53 THE EFFECTS OF VARIOUS EXPOSURE TIMES ON THE SURVIVAL RATES OF EBERTHELLA TYPHOSA Bacteria may also be considered as premitotic. Their hereditary constitution might thus be conceived as not being differentiated into specialized functions and parts. Thus the hereditary processes in bacteria may be quite different from those of multicellular organisms. According to Huxley (1942), "One guess may be hazarded; that the specificity of their constitution is maintained by a purely chemical equilibrium, without any of the mechanical control superposed by the mitotic (and meiotic) arrangements of higher forms." He implies that it is unreasonable to expect bacteria to have fully developed mitotic mechanisms. The mitotic mechanism of higher organisms is a complicated, highly developed process. It must have resulted from long and gradual evolution. It seems reasonable to assume that bacteria as a group may exhibit numerous steps in the evolution of the mitotic process. Some may be entirely premitotic and depend upon some quite unknown mechanism for the transmission of hereditary

5 1947] EFFECTS OF X-RAYS ON EBERTHELLA TYPHOSA 169 tendencies. Others may have partly developed or fairly well developed mitotic mechanism. Since it is desirable to avoid any implication concerning the hereditary mechanism of bacteria (until we have adequate knowledge), the term anomalous variation is suggested. Anomalous connotes the idea of a deviation from normal order that refuses to submit to an explanation or classification. Variations in organisms are known to be due to the following causes: (1) changes in the environment, (2) gene mutation, (3) changes in chromosome complexes, (4) gene recombination or hybridization, and (5) a combination of any of these four. A sixth category may be added for convenience to include all causes in which the causative mechanism of the variation is unknown, namely, anomalous variation. It is noted that this is a classification based on causes of variation. Thus, for example, the modification of a bacterial culture that may occur after exposure to X-rays is an anomalous variation, as well as any other variation the causative mechanism of which is unexplained at the present time. The plates used above in determining the number of organisms killed also served as material for the study of anomalous variation immediately after X-ray treatment. Ten exposures to X-rays were made at different times, and the colonies surviving the 4-hour period of exposure were studied by means of a colony microscope lens (3 X )to note any changes in morphology. At random 100 colonies that survived this period of exposure were picked each time. Approximately 0.05 per cent of the organisms had survived this length of time. A total of 1,000 colonies picked were transferred to semisolid agar to determine motility, by the method of Tittsler and Sandholzer (1936), and incubated for 24 hours. After this period of time transfers were made to various other media and studied from the following aspects: colonial character on agar and gelatin; morphology and stain by Gram's method; fermentation of lactose, glucose, and xylose; lead acetate production; indole formation; growth on potato; and motility as indicated by flagella staining. The colony morphology of the 1,000 colonies observed remained similar to the parent unexposed control culture. Likewise, all the other tests remained the same as the control, with one exception. After one exposure, from which 100 colonies were picked at random, 78 of the colonies demonstrated a loss of motility. The colony morphology of the 78 nonmotile forms was the same as that of the unexposed control colonies when first examined. Subsequent plating of these cultures, however, showed a change in the colony formation from a smooth to an intermediate form. All the other tests used concerning these 78 nonmotile forms remained the same as the control. The 78 nonmotile cultures were, in addition, tested in the following substances: raffinose, galactose, maltose, fructose, salicin, sorbitol, sucrose, dulcitol, inulin, inositol, and mannitol. The fermentation reactions remained the same as the unexposed parent control culture. In addition to these anomalous variation studies, suspensions in distilled water of an 18-hour culture of E. typhosa were exposed to the X-rays in exactly the same manner as described for the lethal studies. But, in addition to the same

6 170 THOMAS H. GRAINGER, JR. [vol. 53 time periods of exposure, the organisms were irradiated for 1, 5, 10, 15, 20, 25, 30, and 35 minutes. After exposure, 0.5 ml of the bacterial suspension were transferred to 9 ml of nutrient broth and incubated at 37 C. Subcultures on agar plates were made daily for a period of 20 days from the 15 cultures, as well as from the unexposed control. Observations were made on -the colonial character of the organisms after 48-hour incubation. The colony form was the only observation made since this type of variation would be easily detected. However, this character of the exposed cultures remained the same as that of the parent unexposed type. This anomalous variation is not the only one that might have occurred, and it is quite possible that other changes were overlooked. DISCUSSION The culture of E. typhosa used in this study was isolated first from the blood of a typhoid patient and then by single cell technique. Observations of the parent unexposed culture did not show any detectable changes in the colonial character. Approximately 3,000 unexposed colonies were observed. The lethal studies confirmed the findings of other investigators and showed that the rate of death is of a logarithmic order. The interesting observation is that there is a distinct difference between killing a bacterium by radiation and, for example, by heat. Some of the bacteria were still motile after 41 hours of exposure, although they failed to produce any growth on suitable laboratory media. The anomalous variation observed was the loss of motility in 78 out of 100 colonies picked at random after one of the experimental exposures. This loss of motility was not observed again even after many repeated X-ray exposures. The observations made on the X-rayed cultures were limited to a few morphological and biochemical reactions. It is quite possible that other changes may have occurred but were not observed. ACKNOWLEDGMENT The author wishes to acknowledge his appreciation to Professors Stanley Thomas and F. J. Trembley for the interest they have taken and the advice they have given throughout this work, and to Professor H. V. Anderson and his assistants, Mr. C. W. Tucker, Jr.,and Mr. G. D. Nelson, for their assistance in the use of the X-ray apparatus. SUMMARY A strain of Eberthelta typhosa, isolated from the blood of a typhoid patient and then isolated by single cell technique, was exposed to X-rays of maximum characteristic radiation of A. The so-called "lethal" effect of the X-rays resulted in a logarithmic order of death. Some of the organisms, however, were still motile but failed to grow on suitable laboratory media. After 4 hours of exposure, in which 0.05 per cent survived, the culture was

7 19471 EFFECTS OF X-RAYS ON EBERTHELLA TYPHOSA 171 studied from the following aspects: colonial character on agar and gelatin; morphology and stain by Gram's method; fermentation of lactose, glucose, and xylose; lead acetate production; indole formation; growth on potato; and motility. All the results of these tests on the organisms exposed to X-rays remained the same as those of the tests on the unexposed control, with one exception. On one occasion, a high percentage of the organisms exposed showed a loss of motility. These nonmotile forms were in addition tested in other sugars, but the fermentation reactions remained the same as in the control. Subsequent plating of these nonmotile cultures showed a change in colony formation from a smooth to an intermediate form. REFERENCES BERGEY, D. H., BREED, R. S., MURRAY, E. C. D., AND HITCHENS, A. P Manual of determinative bacteriology. 5th ed. Williams and Wilkins Co., Baltimore, Md. Refer to p BERTRAND, M Studies of the effects of long wave X-rays on Staphylococcus aureu8 and Microsporon audouini. Can. Med. Assoc. J., 20, CLARK, G. L The effects of X-radiation on cell structure and growth. A general survey of radiobiology. Cold Spring Harbor Symposia Quant. Biol., 2, DREA, W. F The attenuation by X-rays of the virulence of human tubercle bacilli. Am. Rev. Tuberc., 38, DREA, W. F Attenuation of human tubercle bacilli. Am. Rev. Tuberc., 41, DUGGAR, B. M Biological effects of radiation. 1st ed. McGraw-Hill Book Co., New York. Refer to 2, FORFOTA, E., AND HAMORI, A Ueber die Wirkung harter R6ntgenstrahlen auf Typhusbazillen. Zentr. Bakt. Parasitenk., I, Orig., 139, GRAY, C. H., AND TATUM, E. L X-ray induced growth factor requirements in bacteria. Proc. Natl. Acad. Sci. U. S., 30, HABERLAND, H. F. O., AND KLEIN, K Die Wirkung der R6ntgenstrahlen auf Tuberkelbazillen. Milnch. med. Wochschr., 68, HABERMAN, S Dissociation of staphylococci by X-rays. J. Bact., 42, HABERMAN, S., AND ELLSWORTH, L. D Lethal and dissociative effects of X-rays on bacteria. J. Bact., 40, HUXLEY, J Evolution-the modern synthesis. Harper and Brothers, New York. Refer to p KL6VEKORN, H Die Einevirkung der Rontgenstrahlen auf Bakterien. Strahlentherapie, 20, LANGE, L., AND FRAENKEL, M Die Wirkung von R6ntgenstrahlen auf Tuberkelbacillen. Klin. Wochschr., 2, LEA, D. E The action of radiations on virus and bacteria. Brit. Med. Bull., 4, LEA, D. E., HAINES, R. B., AND BRETSCHER, E Bactericidal action of X-rays, neutrons and radioactive radiations. J. Hyg., 41, LEA, D. E., HAINES, R. B., AND COULSON, C. A Action of radiation on bacteria. Proc. Roy. Soc. (London), B, 123, LEWIS, I. M The cytology of bacteria. Bact. Revs., 5, MCCOLLOCH, E. C Disinfection and sterilization. 2d ed. Lea and Febiger, Philadelphia, Pa. Refer to p MINCH, F Zur Fr,age ilber die Einwirkung der R6ntgenschen Strahlen auf Bakterien und ihre eventuelle therapeutische Verwendbarkeit. Miinch. med. Wochschr., 5,

8 172 THOMS H. GRAINGER, JR. [VOL. 3 MULLzDR, H. G Artificial transmutation of the gene. Science, 66, RAEm, Physical methods of sterilization of microorganisms. Bact. Revs., 9, RICE, C. E., AND GILFORD, R. B Studies on variability of tubercle bacilli; influence of X-rays on dissociation. Can. J. Research, 6, SMITH, M. E., LIssE, M. W., AND DAVEY, W. P The effect of certain X-rays on the electrophoretic mobility of Escherichia coli. J. Bact., 31, TATUM, E. L X-ray induced mutant strains of E. coli. Proc. Natl. Acad. $ci. U. S., 31, TiTTSLER, R. P., AND SANDHOLZER, L. A The use of semi-solid agar for the detection of bacterial motility. J. Bact., 31, WILSON, G. S., AND MaLBs, A. A Topley and Wilson's principles of bacteriology and immunity. 3d ed. Williams and Wilkins Co., Baltimore, Md. Refer to 1, Downloaded from on March 11, 2019 by guest