Received for publication August 25, fresh broth immediately after the exposure. The. of ozone and oxides of nitrogen.

Transcription

1 DEVELOPMENT OF BACTERIOPHAGE IN X-RAY INACTIVATED BACTERIA LOUIS W. LABAW, VERNON M. MOSLEY, AND RALPH W. G. WYCKOFF National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, Public Health Service, Federal Secrity Agency, Bethesda, Maryland Received for publication August 25, 1952 In previous studies (Labaw, Mosley, and Wyckoff, 195a, b) from this laboratory we have shown that large yields of bacteriophage can be obtained from cultures of Escherichia coli which have been exposed to enough ultraviolet radiation to prevent their subsequent growth to form observable colonies when spread on fresh nutrient agar. In this work it was found that though the phosphorus metabolism of these bacteria is greatly depressed by the irradiation, it does not cease for two hours following exposure to the ultraviolet light; and it is during this time that bacteriophage can develop. It was observed also that during this interval of diminishing phosphorus uptake, the bacteria continue to grow and may even undergo one or two divisions. Infection with bacteriophage brings this growth to a prompt end though it may accelerate for a time the rate at which phosphorus is used by the infected bacterial system. The present paper records somewhat imilar observations made on systems consisting of X-rayed E. coli infected with bacteriophage. They are of interest in showing that bacteriophage grows even better on X- rayed than on ultraviolet irradiated bacteria and in bringing out certain other differences between bacteria "killed" by the two types of radiation. no more than 1 mm thick. The bacteria were kept in saline for as short a time as possible, the suspension was kept cool, and care was taken to ensure that it did not become heated during irradiation. The doses of radiation applied varied from 67,5 r up to at least 75, r. For most experiments involving the larger doses the bacteria were spread, before irradiation, as a thin mat on an agar surface and washed off with fresh broth immediately after the exposure. The maximum time of exposure (to achieve 75, r) was three hours, and during this and all other were kept iced prolonged irradiations the plates and air was circulated to prevent accumulation of ozone and oxides of nitrogen. Suitable control experiments in which the bacteria were shielded from the X-irradiation, while still being exposed to the chemical products produced by the air ionization, showed that the bacteria were not damaged perceptibly by the accumulation of these chemical by-products or by the several steps taken in handling the preparations. Doses of radiation were estimated with a Victoreen dosimeter fitted with a thin nylon chamber; the usual corrections were made for absorption in the walls of this chamber. It should be pointed out that these corrections are probably inadequate for estimatig the unfiltered output of the tube used here, and that the recorded METHODS dqses are too low. Evidence for this lies in the The strain of bacteria and of bacteriophage low values for 5 per cent killing of irradiated and the methods of their cultivation are the same E. coli compared with previous work (ILAcas. as those that have been used for previous work sae and Holweck, 1928, 1929; Wyckoff, 193) in this laboratory. All irradiations have been and in the far greater kiling per roentgen of made on young and rapidly multiplying organisms (from one hour broth cultures grown in organisms spread on agar surfaces. a tryptose phosphate broth). The methods of labeling with radio phosphorus The X-radiation was the total unfiltered output of a water-cooled tungsten target, beryllium uptake of the bacteria-bacteriophage system are and of measuring with its help the phosphorus window Machlett type OEG-5 tube operated those standard in this laboratory (Labaw, Mosley, and Wyckoff, 195b). Samples of the ir- at 4 KV and 3 MA from the high voltage supply of a North American Philips X-ray Diffraction Unit. For the smaller dosages the with bacteriophage, were prepared also in the radiated bacteria, before and after incubation bacteria were irradiated as a suspension in saline usual way for electron microscopy; then they 33

2 19531 BACTERIOPHAGE IN X-RAY INACTIVATED BACTERIA 331 were examined under a type EMU microscope as manufactured by RCA. Electron micrographs were made of the irradiated E. coli after incubation for various Yield of T6r+ TABLE 1 bacteriophage from X-rayed and control cuiltures of Escherichia coli BACTERIAL TITERS BACTERIOPHAGE TITERS EXPERIMENT SURFACE DOSE MEDIUM (COLI PER ML) (T6r+ PIER ML) NO. SURFACE DOSE MEDIUM Coli before Coli after From x-rayed coli From control coli 1 16, Saline 1. X X X X , Saline.9 X X X 1' 5.7 X 11' 3 735, Broth 1.2 X X X X , Agar ca 1 X X , Agar ca 1 X X , Agar ca 1 X X 11 EXPERIMENTAL RESULTS Experiments of three general sorts were carried out. One was arranged to ascertain the yields of bacteriophage from E. coli receiving increasing doses of X-rays. A second series was designed to show the influence of increasing amounts of X- rays on the phosphorus uptake by uninfected cultures of E. coli and by such cultures after infection with T6r + bacteriophage. In another group of experiments a comparison was made between the reduction in yields of several types of bacteriophage developing in cultures of E. coli that had been subjected to the same dose of X-rays. In order better to relate these results to earlier work involving ultraviolet irradiation of host bacteria, a parallel set of observations of yields was made using ultraviolet light and X- rays to irradiate the host bacteria. A general idea of the effect of increasing X-ray dose on the yield of bacteriophage when the X- rayed bacteria were infected immediately after irradiation is given by the typical results of table 1. They bring out a number of points. Chief among these is the very high yield of bacteriophage per irradiated bacterium. Though this yield is diminished after great doses of X-rays, its decline is slow. Even after perfect "sterilization" of the culture, as in experiments 4, 5, and 6, the yield per organism is not less than a hundred. The data of this table emphasize the statement already made that the killing action of the X-rays is very much greater on bacteria spread on the surface of agar than on bacteria in suspension in saline. Figure 1. An electron micrograph of cells of Escherichia coli immediately after exposure to a moderate (ca 16, r) dose of X-rays. Except for the beginnings of a polar accumulation of their protoplasm, these bacteria appear normal. The preparations from which this and the following micrographs have been made are pseudo-replicas obtained by pouring the bacterial suspension on agar and removing the surface layer in a collodion film formed on it. The ringed depressions around the bacteria have resulted from stretching of the thin substrate that envelops them. Palladium shadowing. Magnification = 15, X. lengths of time. Immediately following irradiation with doses up to ca 2, r, the bacteria appear normal (figure 1). After a subsequent incubation of two hours, many of the irradiated organisms have grown into long filaments (figure 2) apparently as a result of loss of the ability to

3 332 L. W. LABAW, V. M. MOSLEY, AND R. W. WYCKOFF [VOL. 65 divide. These long filaments ultimately disintegrate. This is a striking illustration of the fact, long known, that the division mechanism of many cells is more sensitive to damaging radiation than is the capacity for immediate growth. Heavier doses of X-rays arrest more and more, likely to give swollen forms (figure 3) which soon shrivel and fragment (figure 4). A conspicuous feature of the heavily irradiated organisms, not seen in normal E. coli, has been the central pits so obvious in figure 5. Some pictures suggest that these may represent a first, and only, step to- Figure 2. Bacteria incubated for 12 minutes in fresh medium after receiving the same dose of X- rays (16, r). Many filaments such as these characterize - ~ such ~ a ~ preparation. ~ 9,WX. Figure 4. After 12 minutes' incubation many of the cells of a similarly heavily irradiated culture disintegrate in the fashion illustrated in this picture. 9, X. Figusre 3. Cells in a. culture subjected to a heavy dose of X-rays (ca 75, r) and incubated for 3 m~inutes. These swollen irregular forms commonly show the abnormal polar accumulations of their protoplasm seen here. 9, X. however, the capacity for any growth, and therefore these long forms are absent from the more heavily ir-radiated cultures. WThat little growth there is in the strongly ir-radiated bacteria is Figure 5. Before disintegrating many of these heavily irradiated, incubated cells exhibit the central pits evident here. Incubation of 6 minutes. 9, X. wards division. If the irradiated cultures, whether they have been subjected to heavy or light doses of X-rays, are seeded with bacteriophage immediately after irradiation, electron micrographs indicate that bacteriophage develops in them in an apparently normal fashion (figure 6). In

4 19531 BACTERIOPHAGE IN X-RAY INACTIVATED BACTERIA 333 view of this fact experiments were made to determine what would be the effect of infecting bacteria with bacteriophage after incubation for various lengths of time following irradiation. A typical result with a suspension of E. coli subjected to 16, r is shown in figure 7. As this and similar experiments have shown, the final titer of bacteriophage declines in an approximately semilogarithmically linear fashion as the preinfection incubation is extended to about two hours. The change in slope and absence of a further decline with longer incubation are due to bacteriophage production in the bacteria that have survived the radiation and have been multi- of phosphorus, both before and after infection with bacteriophage, parallels that previously described for ultraviolet light. For all doses of X-rays, large and small, the uptake of pn by X-rayed organisms is less than that of the corresponding control culture of unirradiated E. coli. As would be expected the uptake by the lol w w 19 > _ Figure 6. Masses of T6r+ bacteriophage particles developing from X-ray irradiated (16, r) bacteria. Portions of two partly intact cells appear at the top and bottom of the photograph. Incubation following introduction of the infecting bacteriophage = 45 minutes. 15, X. plying during this incubation. Electron micrographs also show a diminished yield of bacteriophage from irradiated bacteria incubated before infection. Little, if any, response can be seen in X-rayed cells which have been incubated for about two hours before adding the bacteriophage. What has been observed is compatible with the hypothesis that the X-rayed bacteria can serve as hosts for the production of bacteriophage during the period following irradiation when they can still grow, that their efficiency as hosts diminishes with the residual capacity for growth and ceases when the bacteria have stopped growing Ṫhe effect of X-raying bacteria on their uptake 6 12 MINUTES INCUBATION TIME OF X-RAYED BACTERIA BEFORE INFECTION WITH T6r+ Figure 7. Reduction of lysate titer when T6r+ was added after different times of incubation of irradiated Escherichia coli in tryptose broth. The concentrated bacteria were irradiated in 1 mm depth of saline with a surface dose of 16, r, then resuspended in broth to a concentration of 18 per ml. The infection ratio was 8 T6r+ per bacterium. The colony count ratio of irradiated to unirradiated bacteria was 2.2 X 1-5. X-rayed bacterial culture.is less, the greater the X-ray dose. For example, the utilization of phosphorus by unirradiated bacteria and by cultures that have received a moderate and heavy dose of X-rays is shown in figure 8. All these uptake curves have the same general shape. They rise very steeply during the first half hour of growth and then flatten off to a maximum value that is commonly reached after about two hours of incubation.

5 334 L. W. LABAW, V. M. MOSLEY, AND R. W. WYCKOFF [VOL. 65 Curves of similar shape are obtained for the phosphorus uptake of both normal and X-rayed cultures after infection with T6r + bacteriophage (figure 8). The effects of X-irradiation therefore are conveniently shown by comparing maxima of the uptake curves (table 2). Dosimeter measurements of the X-rays applied are recorded in w I- Li~ -J In 16: z I tion. The data of the fourth column illustrate the stimulated phosphorus consumption that is observed in heavily irradiated cultures after inoculation with bacteriophage. It is apparent in the fact that all the ratios of this column for the stronger irradiations have values less than unity. This is the same kind of stimulation as that observed after ultraviolet irradiation (Labaw, Mosley, and Wyckoff, 195b). As can be seen from column 5 as much phosphorus is used by lightly X-rayed as by normal cultures after inoculation with bacteriophage; the utilization, TABLE 2 r Comparisons of maxima of phosphorus uptake curves of normal and X-rayed Escherichia coli cultures infected and uninfected with T6r+ bacteriophages r SURVIVORS RATIOS OF MAXIMA OF S PHOSPHORUS UPTAKES _~~~~~~~~ LL w -J. -J w a- z a-i... INFECTED UNINFECTED WITH T6r MINUTES INCUBATION TIME IN LABELED MEDIUM Figure 8. Radiophosphorus uptake by about 5 X 19 infected and uninfected X-rayed Escherichia coli which were incubated in tryptose broth containing 15 counts per minute per ml as measured with an end window geiger counter with an approximate counting geometry of 25 per cent. The bacteria were irradiated in concentrated suspensions for the total surface doses indicated on the diagram. this table, but in view. of their greater apparent effectiveness on bacteria spread on solid medium, the effective radiations for the fifth, sixth, and seventh surface doses in column 1 are all greater than the fourth as shown by the survivor ratios in column 2. Column 3 shows that irradiation always reduces the amount of phosphorus subsequently used by a culture of uninfected bacteria, the reduction being greater after heavy irradia- SURFACE X-RAY DOSE Irradiated in fluid 67,5 3.3 X , 6.6 X , 1.8 X , 8.3 X Irradiated on solid medium 39, , , however, is reduced by heavier doses of X-rays. This parallels the attainable titers of bacteriophage and is what would be expected if most of this phosphorus is employed in forming bacteriophage. An interesting feature of the uptake curves of the more heavily irradiated uninfected cultures of E. coli is the falling away from their maxima as incubation is prolonged. As figure 9 indicates, this begins after less than an hour of incubation. Comparing this with what is seen in the electron micrographs made from such cultures indicates

6 1953] BACTERIOPHAGE IN X-RAY INACTIVATED BACTERIA 335 that this falling away might be attributed to the disintegration of the irradiated bacteria and the consequent liberation into the medium of the phosphorus they contained. In a last set of experiments comparisons were made between the yields of several types of bacteriophage from aliquots of an X-rayed bacterial culture and between these yields and corresponding yields from ultraviolet irradiated bacteria. Results of two experiments are collected in table 3. After the rather mild dose of X-rays used for the first experiment (ca 1, r) there was no drastic reduction in the titer of any of the bacteriophages though the lower yields for T5, and perhaps for Ti (column 7), are significant. With the more heavily damaged ultraviolet irradiated bacteria, however, bacteriophage T6r+ stands out from all the other strains studied in the high yield it gives (column 4). Greater differences between strains appear when, as in the second experiment, more heavily irradiated bacterial hosts are used. As can be seen from column 9, T6r+ is conspicuous, as it was in the ultraviolet experiment (column 4), for its ability to use heavily damaged host cells. This is not true of its variant T6r which rather is to be grouped with Ti and T5 as strains which develop poorly o uj z l5r go X t 4L IJ O a- If lol l ~~UNINFECTED MINUTES INCUBATION TIME IN LABELED MEDIUM Figure 9. Radiophosphorus uptake by 5 X 19 infected and uninfected X-rayed Escherichia coli which had been subject to a surface dosage of 735, r when spread on cold agar. Radioactive medium was similar to that used for experiments shown in figure 8. TABLE 3 Comparisons of yields of several bacteriophages from aliquots of an X-rayed Eacherichia coli culture and between these yields and similar yields from ultraviolet irradiated bacteria EXPRIMN 1 Z"ZRnMNT~ll T EXP ER ME 2 BACTERIOPHAGE Bacteriophage titers (per ml) on Ratio Bacteriophage titers (per ml) on Ratio of -of Ultraviolet Control yields X-rayed Control yields Titers on Ratio of rayed bacteria bacteria bacteria bacteria X-rayed bacteria yields.4 Ti 3. X X X X X T3 3.2 X X X X X T5 3. X 1' 2.1 X X X X T6r+ 4.7 X 1' 5.2 X X X X T6r _ X T7 3.8 X X X X X Coli count per 4. X X 1' 1. X X X 11 ml Survivors 3.1 X 1-1. X 1~ X 1-7 irradiated unirradiated on heavily irradiated bacteria. The present data suggest further that T3 and T7 grow better on X-rayed than on ultraviolet "killed" organisms. SUMMARY Measurements of radiophosphorus uptake and titer, and electron micrographs, were made of

7 336 L. W. LABAW, V. M. MOBLEY, AND R. W. WYCKOFF [vol. 65 cultures of X-rayed Ewderid&ia coli with and without infection by T6r+ bacteriophage. They show that the yield of T6r+ bacteriophage on X-rayed bacteria, infected immediately after irradiation, is large even for completely "killed" cultures, decreases with increasing dose, and for any one dose, decreases with the time of incubation of the X-rayed E. coli before infection. The radiophosphorus uptake by both uninfected and infected irradiated bacteria also decreases with increasing dose, but the decrease in the infected cultures is less than that in the uninfected when there is T6r+ bacteriophage multiplication. This enhanced radiophosphorus uptake is similar to, but less than half, that found with ultraviolet irradiated cultures. This difference reflects the relative insensitivity to irradiation of the capacity of the cells for immediate growth compared to its division mechanism, a fact which is shown also by electron micrographs of X-rayed bacteria having different subsequent incubation times. A study of the yield of different bacteriophages on X-rayed bacteria shows that T6r+ is readily able to use heavily damad cells whereas TI, T5, and T6r develop poorly, with the yield of T3 and T7 taking an intermediate position. REFERENCES IABAW, L. W., MOsLBY, V. M., AND WYCKOFF, R. W. G. 195a Electron microscopy of ultraviolet irradiated bacteria and their interaction with bacteriophage. Biochim. et Biophys. Acta, 5, LABAW, L. W., MOsLEY, V. M., AND WYCKOFF, R. W. G. 195b Radioactive studies of the phosphorus metabolism of T2r+ bacteriophage with Escherichia coli. J. Bact., 6, LACAasGNE, A., AND HoLwwcK, F Bactericidal action of X-rays. Compt. rend., 186, LACASSAGNE, A., AND HoLWCK, F Action of X-rays of long wave length on microbes. Compt. rend., 188, WYCKOFF, R. W. G. 193 Killing of certain bacteria by X-rays. J. Exptl. Med., 52, The killing of colon bacilli by X-rays of different wave lengths. J. Exptl. Med., 52,