Coolidge-type molybdenum-target x-ray tube, the x-rays being

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1 THE EFFECT OF CERTAIN X-RAYS ON THE ELECTRO- PHORETIC MOBILITY OF ESCHERICHIA COLI1 MARGARET E. SMITH AND MARTIN W. LISSE Department of Agricultural and Biological Chemistry AND WHEELER P. DAVEY Department of Chemistry, The Pennsylvania State College, State College, Pennsylvania Received for publication, November 11, 1935 Recent researches concerned with the effects of x-rays on bacteria have for the most part considered rates of killing and the theory of action on a sensitive zone. The mechanism of action is not clear, however; certain studies seem to indicate that factors other than direct action on a sensitive zone are involved. In the elucidation of this problem, changes at the surfaces of the bacteria need to be considered. It is evident that x-rays may affect the distribution of ions at the surface, with a probable resultant change in surface charge and electrokinetic potential. A means of approach to the problem is through the study of electrophoretic mobilities of the particles. Reports of such studies in the literature have been few in number. Lisse and Tittsler (1931) found that irradiation of Escherichia coli with ultraviolet light resulted in death with an accompanying decrease in electrophoretic velocity. Dozois et al. (1932) reported no change either in viability or in electrophoretic velocity of Escherichia coli after irradiation in an aqueous suspension using a Coolidge-type molybdenum-target x-ray tube, the x-rays being 1 The data in this paper are taken from a thesis submitted by Margaret E. Smith in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Agricultural and Biological Chemistry, The Pennsylvania State College. Authorized for publication on July 27, 1935, as paper No. 699 in the Journal Series of the Pennsylvania Agricultural Experiment Station. 275

2 276 M. E. SMITH, M. W. LISSE AND W. P. DAVEY filtered only by the lime-glass window of the tube and the wall of the celluloid vessel containing the suspension. As a general rule, x-rays seem to be lethal to bacteria. In apparent contradiction to the absence of lethal effects noted by Dozois et al., Wyckoff (1930) had found that Escherichia coli spread on the surface of nutrient agar were killed by direct exposure to x-rays from a molybdenum target, the only filter being the Lindemann glass window of the tube. With these studies in mind and the object of determining whether death through the agency of x-rays was accompanied by a change in the electrophoretic velocity, the procedure determined upon in the present research was to irradiate the bacteria on the surface of nutrient agar, noting the lethal action, and observing any changes in electrophoretic mobilities resulting from such irradiation. APPARATUS AND TECHNIQUE Bacteria. The bacteria used were from a strain of Escherichia coli grown on nutrient agar slants. For purposes of experimentation some bacteria were removed from these slants into sterile water and thence to the surface of nutrient agar in Petri dishes, and incubated at 370C. for 18 to 24 hours. For irradiation the glass cover of the Petri dish was replaced by a lead cover with a hole for the admission of the x-rays. A section of the irradiated portion was used subsequently for the electrophoretic experiments on irradiated bacteria, and a similar section of the part protected by the lead cover was used for the experiments on non-irradiated bacteria, thus ensuring like conditions of growth for the two samples. X-ray apparatus. Two different x-ray tubes were used: (1) A Coolidge-type water-cooled molybdenum-target tube, having a lime-glass window; (2) a copper target "Phillips Structure Research Tube," the window of this tube being of Lindemann glass. Voltage in each case, supplied by means of alternating current and transformer with the tube itself acting as rectifier, was 30 KV. (root mean square) as determined by a voltmeter-coil on the core of the high voltage transformer; the tube current was maintained at 21 to 22 ma. The distance from the target to the bacteria on

3 EFFECT OF X-RAYS ON ESC1ERICHIA COLI 277 the surface of the agar was approximately 13 cm. By means of the two tubes and the additional use of a filter three types of radiation were produced, theoretically of the following quality: (1) Mo characteristic K radiation together with general radiation of wave lengths longer than 0.3 A (approx.), with the exception that the longer wave lengths were cut out to some extent by the lime-glass window of the tube; (2) Cu characteristic K radiation with general radiation longer than 0.3 A.; (3) essentially Cu Ka radiation, obtained from the copper target tube with the interposition of a nickel filter approximately 0.5 mil. (0.01 mm.) in thickness and two celluloid filters each approximately 1 mil. (0.02 mm.) in thickness. The intensity of radiation from the molybdenum target tube was not determined; intensities from the copper target tube without and with the filters as measured with an open air ionization chamber2 placed at the level used for irradiation of bacteria were approximately 950 and 440 r per minute respectively. Suspensions. Bacteria, either from an irradiated or from a non-irradiated section of the agar plate, were removed by a small stream of distilled water from a wash-bottle. The suspension was shaken up with glass beads, filtered through cotton to remove clumps, and diluted with distilled water to the desired density for electrophoretic studies. The suspension was stored in a rubber stoppered Pyrex glass flask in the laboratory until use, the end of the experimentation period being not more than three hours after preparation of the suspension. Determinations of ph were made by the LaMotte method using bromthymol blue as an indicator. Since values so obtained were within the range in which Winslow and Upton (1926) found velocity to be independent of ph, and since no correlation was found in the present studies, data concerning the ph of suspensions are not reported. Electrophoresis cells. A cell of the Northrop-Kunitz type was used in part of the research; the theory and use of such cells has recently been reviewed by Abramson (1934). After sufficient readings had been made at both the upper and lower "stationary 2 The ionization chamber was kindly loaned by Dr. G. Failla of the Memorial Hospital, New York City.

4 -278 M. E. SMITH, M. W. LISSE AND W. P. DAVEY levels" to make certain that the correct depth was found, readings were made only at the upper level. One hundred observations of velocities were obtained for each experiment, reversing the polarity after each set of 5, refocusing the microscope after 20, and refilling the cell with fresh suspension after 40 or 60 readings. The electrophoretic velocity of a particle is customarily recorded as mobility-i.e., velocity per unit potential gradient. Since the cell used did not have the auxiliary electrodes needed to measure the fall of potential through the cell itself, an estimate of this value was obtained from the dimensions of the apparatus. Hence, mobilities as reported with this cell are in reality only approximate mobilities. A new "double tube" cell was developed and used for most of the experimental work; the description, theory and advantages of this cell are presented elsewhere (Smith and Lisse, 1936). Briefly, it consists of two tubes in parallel between the electrodes, the dimensions of the tubes having a relation to each other such that no flow of liquid occurs along the axis of the tube of smaller radius, and therefore velocities at that depth are the actual electrophoretic velocities of the particles. The procedure of reversing polarity, refocusing the microscope and refilling with fresh suspension as used in the work with the Northrop-Kunitz cell was followed. In some cases, however, a total of only 60 or 80 readings were taken in one experiment; a smaller number is permissible, since there is less variation in the readings taken with this cell, due to the fact that observations are made at the level where the velocity gradient is minimum. From these observations values designated as actual mobilities were calculated, the potential gradient through the cell being given with sufficient accuracy by EIL, where E is the fall of potential between the electrodes as measured with a voltmeter, and L is the length of the tube of smaller radius. RESULTS OF EXPERIMENTS ON LETHAL ACTION OF X-RAYS A series of experiments were carried out primarily to determine whether the bacteria as used in the electrophoresis studies were killed through the action of x-rays. The technique used in most

5 EFFECT OF X-RAYS ON ESCHERICHIA COLI of the experiments was similar to that used by Wyckoff (1930), the bacteria being spread on the surface of nutrient agar, a section irradiated, and counts made of the number of colonies developing on an irradiated section and on a control section of equal area. Table 1 gives the results of these experiments, each number being the average of percentages obtained from 10 or more counts from irradiated sections and from an equal number of control sections. Conclusions concerning rates of killing cannot be made from the data, due to insufficient number of experiments and also to the fact that voltage and current were not sufficiently well controlled to allow for quantitative results, particularly in the cases of short- TABLE 1 Percentage of surviving organisms (Esch. coli) after exposure to x-rays of different nature for various periods of time, from a to 120 minutes PERIOD OF IRRADIATION TYPE OF RADIATION Mo K characteristic Cu K characteristic CU K + general + general a minutes per cent per cent per cent Intensity in r per minute time irradiation. However, allowing for the differences in intensities used, the results are of the same order as those given by Wyckoff. Five experiments were concerned with the lethal effects of copper characteristic K and general radiation on Escherichia coli in aqueous suspension. These showed that while the rays were lethal, the rate of killing was much less than when the bacteria were exposed directly. Since it is apparent (see table 1) that x-rays from the molybdenum target are far less lethal than are those from the copper target, an explanation of the absence of lethal effects noted by Dozois et al. (1932) may be that insufficient dosages were given to make the effects observable.

6 280 M. E. SMITH, M. W. LISSE AND W. P. DAVEY Further experiments showed that irradiation of the medium and subsequent covering of the surface of the agar with Escherichia coli did not result in death of the bacteria; and also that the lethal effects of copper radiation are cut off, or at least greatly diminished, by the interposition of the glass cover of the Petri dish between the x-rays and the bacteria. For the electrophoresis studies, the surface of the agar was covered with a more dense suspension than for the above studies; the bacteria were grown for 24 hours at 370C. before irradiation, thus being in colony form. Sub-cultures were also made from these irradiated sections and incubated to determine the presence of viable cells. Conclusions from the lethal studies which are of particular interest in the electrophoresis studies are: 1. Twenty-minute exposure of Escherichia coli to Cu characteristic K and general radiation, or to Cu K. radiation resulted in death of all of the bacteria in the irradiated section on an agar plate, either in the case where the bacteria were spread out on the plate, or where colony formation was present. 2. Twenty-minute exposure to Mo characteristic K and general radiation did not result in death of all of the bacteria in the irradiated section. 3. Long-time irradiation, with any of the three types of radiation used, resulted in death of all of the bacteria on the irradiated section. EXPERIMENTAL RESULTS OF ELECTROPHORESIS STUDIES Five series of experiments concerned with electrophoretic mobilities of irradiated and non-irradiated bacteria were performed. The irradiated bacteria were suspended in distilled water in the manner previously described; conditions of irradiation for the five series were as follows: in series I, the bacteria were exposed for 20 minutes to radiation from the molybdenum target tube, and in series II to unfiltered radiation from the copper target tube for 20 minutes; in series III and IV, the same two types of radiation were used respectively, but the period of exposure was 120 minutes; series V consisted of 240-minute exposure to Cu K. radiation.

7 EFFECT OF X-RAYS ON ESCHERICHIA COLI Each series comprised 10 pairs of experiments, the Northrop- Kunitz cell being used for 8 pairs of experiments in series I and 7 pairs in series II, and the new cell for the remainder. A pair of experiments consisted of the 60 to 100 observations on bacteria taken from the irradiated section of the plate, and also of the same number of observations on the non-irradiated bacteria obtained from a protected portion of the same plate. In each pair of experiments, conditions of growth of bacteria, suspension medium, the cell used and temperature were practically identical. The mobility in the case of the new cell, or the approximate mobility in the case of the Northrop-Kunitz cell corresponding to each observed velocity, was calculated, and the mean mobility-i. e., arithmetic mean (A.M.) of the 60 to 100 values was determined. Frequency distribution diagrams of the mobilities obtained in the experiments indicated that the data for an experiment tend to conform to the normal distribution curve; it appeared that the calculation of standard deviations (S.D.) might be of value. Two advantages of such determinations were made use of in the accumulation of data. The value obtained serves as a criterion for data which need to be examined critically and perhaps discarded, since an unusually high value might result from errors such as focusing on different levels, or drifting in one direction. Furthermore, use of the standard deviation gives information as to the number of values which should be determined in any experiment for the desired amount of accuracy. Since variation in readings with the new cell tended to be less than with the Northrop-Kunitz cell, a smaller number of observations was justified. Table 2 gives the values of the mean mobilities and the standard deviations calculated for the 10 pairs of experiments in series I. The per cent change in mobility resulting from irradiation in series I, calculated from the values of the arithmetic means of the 3Standard deviation is defined by: S.D. = v 2(X-A.M.)2 281 where X - A.M. is the difference between a value X and the mean, and N is the number of values of X.

8 282 M. E. SMITH, M. W. LISSE AND W. P. DAVEY mean mobilities for the irradiated and non-irradiated samples (see table 2) is -2.8 per cent. The customary method of treating data of electrophoretic mobilities has been to select a more or less arbitrary per cent change as the limit of experimental error to be allowed. In selecting this value, factors such as the number of experiments performed and the amount of variation of values TABLE 2 The mobilities of irradiated and non-irradiated Esch. coli in aqueous solutions Conditions of irradiation: Mo x-rays from Coolidge type tube operating at 30 KVr.m.c 22 ma., impinging for 20 minutes on bacteria spread on surface of agar, distance 13 cm. A.M. AND S.D. OF MOBILITIES IN p/8ec./volt/cm. PAIR NUMBER CELL USED Non-irradiated Iradiated A.M. S.D. A.M. S.D. (1) (2) (3) (4) (5) (6) 1 N.K.* N.K N.K N.K Newt New N.K o N.K N.K N.K Sum A.M * N.K. = Northrop-Kunitz cell. t New = new type cell. from the mean should be considered; that is, the use of statistical methods is indicated. In the present study a means of determining whether changes to the extent found are significant was sought through the use of "Student's" method of analysis for paired data. This method is given by Pearson (1914) together with the table of probabilities. Love and Brunson (1924) discuss the applicability of this method, and Love (1924) gives a table

9 EFFECT OF X-RAYS ON ESCHERICHIA COLI of the "odds," indicating whether values determined are significant. Analysis of the data given in columns (3) and (5) of table 2 with reference to Love's table shows that the odds are less than 12:1 against the difference in mean mobilities of the non-irradiated and irradiated samples being due to chance alone. Odds greater than 20:1 are usually termed significant; odds greater than 100:1 are very significant. Thus, analysis shows that no significant change in electrophoretic mobility was brought about through irradiation of Escherichia coli for 20 minutes with x-rays from the molybdenum target tube. A similar analysis of the data given in columns (4) and (6) of table 2 shows that the odds are TABLE 3 Summary of changes in mobility and in variation in mobilities of Esch. coli with exposure to x-rays of different wave lengths CT INTHNSITY SERIES OF IRIATION M ANGE IN CHANGE IN NUMBER TYEO RAITO NEST IE MOBILITY VARIATION r/minute minutes per cent I Mo K + general 20 0* 0 II Cu K + general III Mo K + general IV Cu K + general V Cu Ka *0 indicates that changes were not significant. 283 less than 10: 1 against the differences between the standard deviations of the non-irradiated and irradiated samples being due to chance alone; therefore we would conclude that no significant change in the variation of mobilities has resulted from irradiation. Analyses by "Student's" method of the data obtained in the other four series show that very significant changes in the mobility resulted from irradiation in the experiments of series IV and V, the odds in these 2 cases being greater than 10,000:1 and 300:1 respectively; in no other cases were the changes significant. Table 3 gives a summary of the results obtained in the electrophoretic studies for the five series. Changes which are not of sufficient amount to be significant are indicated in the table by 0; in the 2 cases where significant changes occurred the per cent of

10 284 M. E. SMITH, M. W. LISSE AND W. P. DAVEY change, calculated from the values of the arithmetic means of the mean mobilities, is given. DISCUSSION OF RESULTS The lethal studies showed that 20-minute exposure to copper characteristic and general x-radiation resulted in death of all of the bacteria in the irradiated section, whereas exposure for the same length of time to molybdenum characteristic and general radiation resulted in death of only a certain percentage of the bacteria. In either case no significant change in electrophoretic mobility was observed. This clearly indicates that change in electrophoretic mobility is not a criterion of death of the organism, or at least that any change produced is not of sufficient magnitude to be observed by the methods used. A change in mobility can be brought about through the agency of x-rays as is evidenced by the decreased mobility resulting from long-time irradiation either with copper characteristic and general radiation, or with Cu K. radiation. It is difficult to state with certainty the cause for the decreased mobility. As noted previously, Lisse and Tittsler (1931) found that irradiation of Escherichia coli with ultraviolet light resulted in decreased mobility; it is possible that wave lengths in this range are effective in the present case. Such wave lengths would be present in appreciable amounts in the unfiltered copper radiation, and might result from fluorescent radiation of the Cu Ka radiation, and probably to a very small extent from the molybdenum radiation. Experimental evidence is in accord with this view, since the copper general and characteristic radiation was the most effective, and the molybdenum radiation the least effective in changing the mobilities. Another explanation might be that death of the bacteria brings about conditions, such as changes in permeability or adsorption, which may result after some time in the decrease in mobility noted. From the studies it appears that there is a difference in action of different wave lengths. The dosages of Cu characteristic and general radiation and of Cu Ka in the experiments of series IV and V were 11.4 X 104 and 10.6 X 104 r respectively (see table 3

11 EFFECT OF X-RAYS ON ESCHERICHIA COLI for data), their ratio being approximately 1.1:1; the ratio of resultant changes in velocity was approximately 15:8. With due consideration of the relatively large experimental error inherent in this type of experiment, it appears that the decrease in mobility is a function of the quality of the radiation, not merely of the intensity. Wyckoff (1932) suggested that the lethal effects of x-rays and ultraviolet light might not be the same, even though semi-logarithmic curves of death were found in both cases. The experiments reported here seem to corroborate this view. Thus, while death through the action of ultraviolet light and certain other agencies may be due to action on the exterior of the organism, the lethal effect of x-rays may be due to direct action on a sensitive zone or nucleus. It is of interest to note that no change in variation of mobilities occurred, even in the 2 cases where the mean mobilities did change. That is, the values of the mobilities of the irradiated samples seem to conform to distribution curves of the same type as those of the non-irradiated samples. CONCLUSIONS 1. No significant change in electrophoretic mobility of Escherichia coli in aqueous suspension resulted from: (a) Twenty-minute exposure of the bacteria on the surface of nutrient agar to molybdenum characteristic K and general radiation (30 KVr.m.s., 22 ma., 13 cm. distance); this dosage was sufficient to result in death of only a certain per cent of the organisms in the exposed section. (b) Twenty-minute exposure of the bacteria to copper characteristic K and general radiation. The dosage of 1.9 X 104 r was more than sufficient to result in death of all of the irradiated organisms. (c) One hundred and twenty-minute exposure of the bacteria to the molybdenum characteristic K and general radiation; this dosage was greater than that required to kill all of the organisms in the exposed section. 2. Death through the agency of characteristic and general molybdenum or copper x-radiation is not accompanied by a 285

12 286 M. E. SMITH, M. W. LISSE AND W. P. DAVEY change in the electrophoretic mobility of the bacteria in aqueous suspension to an extent measurable by microscopical studies of the type made. 3. Exposure of Escherichia coli on the surface of nutrient agar to dosages of Cu characteristic K and general radiation or to Cu Ka radiation in amounts much greater than is required to bring about death of all of the organisms in the exposed section results in a significant decrease in the electrophoretic mobility of the bacteria in aqueous suspension. The Cu characteristic K and general radiation seems to be more effective in this respect than is the Cu Ka radiation. 4. Values of mobilities of irradiated samples seem to give distribution curves of the same type as those of the non-irradiated samples, no increase in the amount of variation being found as a result of irradiation. REFERENCES ABRAMSON, H. A Electrokinetic Phenomena. The Chemical Catalogue Company, New York. Dozois, K. P., TITTSLER, R. P., LissE, M. W., AND DAVEY, W. P Jour. Bact., 24, 123. LISSE, M. W., AND TITTSLER, R. P Proc. Soc. Exp. Biol. and Med., 28,811. LovE, H. H Jour. Amer. Soc. Agron., 16,68. LOVE, H. H., AND BRUNSON, A. M Jour. Amer. Soc. Agron., 16, 60. PEARSON, K Tables for Statisticians and Biometricians. University Press, Cambridge, England, xliii. SMITH, M. E., AND LIssE, M. W Jour. Phys. Chem., (in press). WINSLOW, C.-E. A., AND UPTON, M. F Jour. Bact., 11, 367. WYCKOFF, R. W. G Jour. Exp. Med., 52, 769. WYCKOFF, R. W. G Jour. Gen. Physiol., 15, 351.