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The irradiation of bacterial spores with low-voltage electrons
The Irradiation of Bacterial Spores with Low-Voltage Electrons Marguerite Davis’ Prom the Biophysics
Division, Sloane Ph,ysics Luborator!y, New Haven, (‘onnecticul
Yale University,J
R,eceived ilugust 6, 1958 INTRODUCTION
The interpretation of biological effects of ionizing radiation as a function of the dose and the physical distribution of ionizing events through the application of target theory (I) has led t)o estimates of the size, shape, and multiplicity of units of the system which are sensitive t,o radiation. Deut.eron and high-energy electron bombardment of many enzymes irl the dry st,ate has shown that the sensitive volume can he ident,ified with the actual physical size of the molecule (2). IIowever, when the effec+ of radiation o11 more complex systems is st.udied, the tar@ size folltld does riot correspond t,o the whole molecule, hut to only a fractiotl of it. Highenergy radiation studies of the spores of the ha&rium KwiLZn~sSubtiZis have indicated a total target area to he nt)out onr-tetith ctf the a.rea of that portion of the spore \vhich shows nu4ear staining. ?I’othing is known as to the actual location of the sensitive units wit,hin the organism, or of the manner of organization of these units t,o form the complete viable system. Since it has been observed that. the mitot,ic* processes of a cell are drastically influencaed by radiation whereas gl~vcwlysis, respirat)ion, and other cell functions are only altcretl irt minor wys, it has been assumed that the rell nucleus and, more spwific~ally, the genetic material caanbe identified w&h the radiation-sensit,ive ut1it.s. This does tmt exclude the possibility of surface effects and extranuc*lear tlamage as a factor in ionizing radiation action, hut the part that it plays in the inactivation 1 Absl.ract.ed from a dissertation versity.
presented for t.hr l’h.1). degree in Yale Uni-
470
MARGUERITE DAVIS
of bacteria has not been separated from the effect of radiation on the organism as a whole. The depth of penetration of an ionizing particle into matter is determined by its rate of energy loss in the material; by control of the energy of an incident particle, the range of the particle can be controlled. If the energy is sufficiently low so that the entire range of the bombarding particle is less than the dimensions of the organism that is irradiated, the effect that is observed can be correlated with the depth to which the particle has penetrated into the organism. If the most sensitive portion of the cell is organized into a nucleus the effect observed when this nuclear portion is hit should be different than when only the surface or the cytoplasmic region can be reached. Low-voltage electrons can be used in this way as probes into the structure of organisms. Previous work of this type has been done by Wells (3), Haskins (4), Petrova (5), and Moos (6) using various low-energy particles on several different organisms; in no case was the found differential sensitivity correlated with the cell structure in an effort to determine the internal organization of the cell. Moos found a maximum killing effect for Pseudomonas omoginosa when the penetration depth of the particle corresponded to the cell diameter. A preliminary account of some of the work given in this paper has already appeared (12). BIOLOGICAL
MATERIALS
The spores of the bacterium, Bacillus subtilis, Marburg strain, were chosen for these experiments. B.y working with the spore of the organism instead of the vegetative cell, it was possible to carry out all of the experiments on a stock of spores that had been isolated, purified, and dried at the same time, insuring a more homogeneous population throughout the duration of the experiments. The use of the spores also made irradiation of dry material feasible as desiccation and exposure to high vacuum did not cause inactivation of the BpOre8. All the radiations were done on the material in the dry state to eliminate the indirect effeCtB of radiation due to short-lived radicals and their recombination products formed when water is present. The spores are ellipsoidal in shape, with a major axis of 1.5 p and minor axes of 0.9 p. Staining procedures have led to considering the spore as containing an organized nucleus located centrally and occupying about one-fifth of the total spore area. This nucleus appears to be imbedded in dense cytoplasm, which in turn is surrounded by a tough, highly impermeable spore wall (7). The spores are extremely thermostable and resistant to deleterious agents, factors which are attributed to the low permeability of the spore wall and to the low content of free water in the spore (8). It has been shown that about 70% of the water in the spore is present as bound water.
IRRADIATION
471
OF BACTERIAL SPORES
,,-Grid ond ~ull~oilng
To Golvonome+er~
1
]
FIG. 1. Schematic view of electron gun and bombardment EXPERIMENTAL
chamber.
PROCEDURE
The bombardments in these experiments were carried out with a simple electron gun which consisted of a heated tungsten filament and an accelerating grid. The bombardment chamber is shown in Fig. 1. The samples, placed on a stainless steel bombardment plate were located a few millimeters below the collimating tube; the geometry of the system was such that the area subtended by the beam on the irradiation plate was equal to the area of the exit hole of the collimating tube. The entire system was evacuated to a pressure of IO-6 mm. Hg to keep the scattering of the electrons to a minimum. The irradiation dose given to the samples in electrons/sq. cm. was measured from the known area of the electron beam and from the product of the electron current times the time of irradiation. The electron current was picked up directly from the irradiated samples and read on either a microammeter or d.c. amplifier, depending on the order of magnitude of the current; both meters were standardized with potentiometer and standard resistors. The accelerating high voltage from d.c. power supplies was applied to the filament and the voltage difference between filament and ground was measured on a standardized voltmeter. When irradiations were carried out at voltages greater than 1500 v., a copper shield was placed between the filament and the grid to reduce the field and prevent arcing from the filament. This system was adequate up to 3500 v., above which arcing in other parts of the system limited the high voltage range of the apparatus. Samples of the stock preparation of the Iyophilized spores suspended in quartzdistilled water were pipetted in 0.05-m]. droplets on 4% in. diameter stainless steel disks and were dried in a desiccator by pumping through a liquid-air trap.
472
MARGTJERITE
Electrons
FIG. 2. Survival
per square
DAVIS
centimeter
curve for the spores of BacilZus subtilis irradiated of energies from 200 to 1200 v.
with electrons
The titer of the spore suspension was adjusted so that each 0.05-ml. drop contained about 50 spores; since each sample was spread over an area of 1 sq. cm., the chance of the spores drying on top of each other or of clumping was very small. After drying, the plate was placed in the irradiation chamber and each sample was successively irradiated by rotating the plate into the electron beam. The samples were aligned under the beam through use of a window 180” from the beam. When one sample was directly under the window, the sample diametrically opposite it was under the electron beam. The bombarded samples were assayed on the same disk as was used for irradiation by layering over with agar the entire stainless steel plate in a large Petri dish and incubating at 37°C. for 16 hr.’ Each viable spore would give rise to a single bacterial colony; the number of such colonies in the area of an irradiated droplet compared with the nonirradiated controls determined the survival ratio. Samples exposed to the glowing filament in vacuum with no accelerating voltage applied showed no inactivation; any decrease in survival observed could therefore be attributed to the action of the electrons incident on the spore. Each experimental point on the survival curve was determined by the irradiation of at least three different samples. It, can be shown through a calculation similar to that used in Rutherford scattering that the spore will not accumulate a charge, and therefore repel the elec4 Thanks of assay.
are due to Dr. Harold
J. Morowitz,
who first developed
this method
trons, if a conductivity equal to that of celluloid is assumed for t,he spore. Since the composition of the spore is similar to that of relluloid, being composed of carbon, hydrogen, oxygen, and nitrogen, the assumption is probably valid. Only if the spore were composed of material of the conductivity of glass would charge accumulat? on the surface.
There is IN) observable effwt with electrolls t~elow 200 V. isee Fig. 3). .\t, 200 v., a slight amount of inactivat~ion OCCIII’S, hut thr effect, is tw\‘er greater than 10 y0 with doses as high as 10’: cle~trons,,‘sq. cm. .Uwve this energy, the effwt of the incident eler*tr.ons illcreases rapidly \vith voltage; the CI~WW are all of t.he multihit typca \vith a trail& up to
1
007
1
1200 volts *- -
\’
70
‘h
50.
20.
001 70. 50.
20.
IOElectrons FIG. 3. Survival
VUrvRs
per square
centimeter
for the spores of Haciltfrs sublilis irradiated of energies from 1200 to 3500 V.
~1iI 11c]ectrorls
474
MARGUERITE
DAVIS
900 v., where the trail-off vanishes and the curve appears to be either single hit or of low multiplicity, possibly marking the transition between two different inactivation processes.At all energies greater than 900 v., the curves are logarithmic (seeFig. 3). The slope of the curve is a rapidly increasing function of voltage up to 3000 v., beyond which it changes very little with further increase in voltage. This is more clearly seen if the 37 % dose is plotted against voltage (see Fig. 4). The values for the doses below 900 v. were found from extrapolation of the straight portion
of the survival curve for that voltage; the exact significance of these points is therefore somewhat questionable. Between 900 and 3000 v. the 37 % dose changes by a factor of 4 X 104.The total energy expended in
5310”:310”“E 7:: 5 c” 2 3‘0 f lo”.E~ p
23d
753-
$ , , I , ( , ., , 3
300
900
1500 2100 Voltoge
2700
3300
3900
FIG. 4. Dose necessary to cause 37% inactivation of spores of Bacillus subtilis M a function of voltage (!&M-3500v.). The values for the dosesbelow 900v. were found by extrapolation of the straight portion of the survival curve for these voltages.
IRRADIATION
OF
BACTERIAL
475
SPORES
producing the same inactivation at 3000 and 3500 v., respectively, is 1.65 x 1015and 1.57 X 1Ol6 e.v./sq. cm. These values can be compared with those found by Morowitz (9) as a result of high-energy radiation of the spores, e.v./sq. cm. for 37% survival
Radiation used
2.03 x 10’4 6.53 x 10” 8.7 x 10’6
X-rays (55-kv. peak) 3.8 m.e.v. deuterons 2650 A. ultraviolet light
A difference in the reaction of the spores to the different energies of incident electrons is also seen mhen the dose rate and the intensity of the irradiation is varied, the same total dose being given in each case; the total amount of irradiation in electrons per square centimeter per second t,imes the length of time of irradiation is held constant, the variKm MlTS toa lo* CUC-~I .
100 ‘--.C
%
Jl -
75
____--
,Al
;-------
a-- 3
Suwval 5. ‘i
I 0
I
I I I 8 4 6 2 Time of wadlotion in minutes
I IO
I
I
I
1
I
2
4
6 mmutes -
8
IO
1 12
12
minutes FIG,
5. Variation in dose rate at 600,900, and 1200 v.; in each case the same amount of irradiation was administered but the rate was varied over a factor of 20.
476
MARGUERITE
DhVIS
ables being changed over a factor of 20. As seen in Fig. 5, it was found that a beam of electrons of high intensity given over a short period of time is more effective than the same number of electrons incident at a slower rate. Variation in dose rate has no effect at higher voltages. DISCUSSION
These data indicate that the spore can be divided into three regions of different radiation sensitivity. Correlation of this information with the ranges of the electrons as determined by experiment, (10) leads to a concept of the internal structure of the spore. I. Energies Less thall ZOOVolts No damage is apparent when the spore is irradiated with electrons of very low energy. At 200 v. where inactivation is first observed, the electron has a range of 30 A. This can be taken as a minimum value of the thickness of an outer inert layer surrounding the spore as only a small fraction of the organisms can be inactivated with electrons of this range. The energy at which t)he trail-off vanishes marks the maximum thickness of the outer inert layer; this is somewhere between 600 and 900 v. The maximum thickness is less than 230 A., the range of a 900-v. electron. II.
200 to 900 volts
Electrons of energy greater than 200 v. are able to penetrate through the outer spore coat into a region which shows low radiation sensitivity. The inactivation curves in this region are multiple hit,, the degree of multiplicity decreasing wit,h increasing energy. At 900 v. the trail-off has disappeared, and the curve can be interpretfed as either single hit or very low multiple hit. At 1200 v., the logarithmic nat’ure of the curves is definite. The degree of mult’iplicity of the curves from 200 t’o 900 v. cannot be determined since t.he energy incident on the sensitive region is not homogeneous due to t,he statistical variation in energy loss and scattering of electrons in the outer inert spore coat. The change in degree of multiplicity can be taken as a measurement of the relative efficiency of the electrons of the different energies in causing inactivation. If 900 v. is taken as the transition from a multiple to a single hit inactivation process, the maximum t,hickness of this layer can only be 200 A., with a minimum value unknown. In this case the total thickness of the two outer coats cannot he greater than 230 A. If 900 v. is con-
IRR.\DT.lTION
OF B.1C”TERT.\T, SPORES
177
xideretl as a low multiple-hit curve, the transit ion to a single hit bking place between 900 and 1200 v., then the maximum thickness of the inner spore mall is less than 320 A. and the minimum less than 120 A. (350 A., the range at which the inactivation cwrves have hecome logarithmic minus 230 X., the range \vherr the it-ail-of’f has disappeared.1 Sittce the very rapid change in 37 y0 dose with cnerpy does not appear at energies less than 1200 v., this latter ittterpretatiotl is favored. ‘I’he data cit,ed t,htts far do not indicate \vhc~tlrer this spore \~a11thic*kttess varies from spore to spore in a gi\-etr popttlation or if e:rc+ individual spore h:is a tiottutiiform lager surroutidittg it. If the latter c*ase\vere true, the amottttt of inactivation ohser\wl \vould he due to random orietttjatiott of t.he spores. If a given populatiott of spores tvcrc reorient ccl after ratliat ion and reirradiat,ed, the same sort, of wt.\-i\T:tl curve \~wld 1~ seen, \vith a ttxil-off at the same per cent. ittartivatiott; if, on the other hand, each spore Ivere surrounded hy a uniformly t hit. 1, Lva.11, hut this thickntw varied from spore to spore, reirradiation aft.er rwrietttat~iot~ of the spores would h:t\,c no further effect on the survival: t hcl spores \\.hic*h had sttrvived the first irradiation would again sttrvivr the aecotttl. :\ largtl number of spores were irradiated at riO0 v. I\-it h a dose \\ell ottt ott the trail-off portion of the sttrvival cttrve. .1Rer irradiation, the spores \vtlrf’ \vashed off the irradiation pIatje, titered. attcl again dried down on t,he plate in the same manner as twfore and wirradiated with elect,rons of the same energy, l’he surviwl cttrves found after such a procedure \vrre identical with l,he first], showing that the sport’ coab is nonunjform ott each individual spore, and t hu.i the \\h& population is essent,ialIy homogeneous in t,his respect. ‘I’hiti esperimettt also ewludes the possiMity of t,he trail& portion of the crtr\:e twirrg due to the presettw of’ :I protective hyer of broth or to the organic8 stthstjattcSespresent in the spore suspension as SII& ma,terial \\-ould hewrnt~ diluted in the washing prowdure and a greater tiwl ittactivat.iotr \vould twttlt. ‘l’he present, data do not allow 5 more dcfittit.e atrni,vsis of the mrchattixm of the a.otion of tbe radiation in this regiott. ‘l’he cumulative nature of the inactivation and the constant energy wclttitwrtetit ttecessary slipports t’he hypothesis of severa hit,s itt otw target as f he inactivation prowss in the inner spore coat. Tothittg wtt lw said atwttt, the ttttmber or size of the targets.
In this range, the survival curves are all fq~otwtttial and the cross s&ion ittweases rapidly with increase in voltage top to 3000 v. whew :t
478
MARGUERITE
DAVIS
point of near maximum sensitivity seems to have been reached, since the curve levels off at this point to an almost constant value. This increase in cross section to a maximum value is not an ion density effect because in no case is the range of the electron comparable to the celldimensions; the total energy of the electron is dissipated inside the spore. Although the ionizations of a 3000-v. electron are more widely separated at the beginning of its path, as the electron loses energy it travels more slowly and the number of ionizations per unit path length increases. At the end of the track the ion density is the same as it was for the lowervoltage electron; the only difference is its ability to penetrate more deeply into the spore. If a sensitive volume is calculated when the target dimensions are greater than the mean separation of ionizations, the dose found for a given biological effect will be larger than that necessary to achieve inactivation on the basis of a single event occurring in one target. In the case of the very-low-voltage electrons, the whole track acts as a unit; for the higher voltages, the end of the track where the electron has been considerably slowed down also acts as a single unit. The cross section calculated at 900 v. (0.5 so. A.) cannot be interpreted as having any physical significance in demonstrating the structure of the spore but can only be viewed as an expression of the probability that an electron will have a range greater than the minimum value, and once within the protective layer surrounding the spore will hit a sensitive unit and therefore inactivate the spore. The probability of such an event occurring increases very rapidly with energy due to the combination of two factors. As the energy is increased, the fraction of electrons with a range greater than some minimum range gets through to the sensitive region of the spore. At 1200 v. a greater number have this range and by 3000 v. it is only the very rare electron which has been completely deflected out of the initial direction of travel that does not penetrate this far. ’ The increase in cross section is also due to the fact that the chance of an electron, once inside the spore, of hitting a sensitive unit increases as the distance which it travels in the area increases. When an electron has an initial energy of 3500 v. it has a maximum path length of 3100 A. An insignificant portion of the electrons do not penetrate the outer coat of the spore; once inside, the average electron travels a great enough distance, making enough collisions so that nearly every electron encounters a sensitive unit. The cross section at this point is 2.22 X lO+ so. cm. If we consider the electron as losing energy through primary ionizations, each primary ionization expending an average of 110 e.v., a 3500-v.
IRRADIATION
OF
BACTERIAL
SPORES
479
electron will form 3‘2 primary ionizations along its total path length. These will not be distributed evenly along the path, but will cluster at the end of the path where the velocity of the particle is small. If we consider as an estimate that one-half of the events takes place in the last quarter of the path length, the events will be on the average 150 A. apart in the first three-fourths of the track, and only ahout 40 A. apart in the final 800 A. If a sensitive volume is calculated on the assumption that the primary ionizations are evenly distributed along the path, a volume of 4.16 X IO-l8 cc. is found. Because this does not’ take into account the unequal distribution of the events, the volume is underestimated as the total number of effective events is less than the number of primary ionizations. By considering the distribution of events assumed above, t)he volume is increased by a factor of 2. As the energy is increased, a direct volume calculation will become more nearly applicable, since t,he distance hetween ionizations, on the average, becomes greater than the target. dimensions, and a minimum value for the 37 y0 dose will he found when this is true. In order to find the true volume at this point certain assumptions will still have to be made about the distribution of energy loss. Experimental difficulties prevented data from being taken at voltages greater than 3500 v. At this energy the cross section has not as yet reached a maximum. The sensitive volume as calculated by Morowitz (9) from x-ray data on the basis of two hit curves was 6.15 X 10-17 cc. The entire spore is vulnerable to radiation when bombarded by the high-energy particles, and yet the cross section does not change in a discontinuous manner once the electrons have energy enough to penetrate the spore wall. From these data it can be postulated that the radiation-sensitive units are dispersed uniformly throughout the internal region of the spore, and not concentrated in a compact nucleus. If the sensitive elements of the spore were grouped in a compact unit located asymmetrically inside the protecting coats, unless there is reason to assume an orientation of this unit other than random, the logarithmic survival curves would trail off at different survivals depending on the energy of the incident electron. The spores with the nucleus located beyond the range of the electron would survive. No trail-off is seen even with the relatively low-energy electrons with ranges of the order of onetenth of the diameter of the spore. The sensitive units are greater than 4 X lo-‘* cc. but less than 6 X 10-I’ cc. in size. The moIecuIar weight of these units is therefore about 2 X lo’, which perhaps identifies it with
480
MARGUERITE DAVIS
the desoxyribonucleic acid (DNA) of the cell. The molecular weight of DNA as found by irradiating pneumococcus-transforming factor with high-energy particles (11) is 6.5 X 106. Each unit in the spore which is sensitive to radiation may therefore be an aggregate of several DNA molecules. Contrary to cytological studies which have presented evidence for an organized nucleus in the spore from various staining procedures, the data from these experiments do not indicate such a region to be present. If all the nuclear material of the cell were concentrated in one large unit, a change in radiation sensitivity would have been observed when this portion came within the range of the electrons. Since such a response is not found, the only conclusion that can be made is that the units of the spore which show maximum sensitivity to radiation are dispersed throughout the interior in units of high molecular weight. The data do not give any estimate of t’he number of such ur~its. ACKNOWLEDGMENTS I would like to express my sincere appreciation to Dr. Franklin Hutchinson who suggested ‘this problem, and under whose guidance the work was done. I would also like to thank Dr. E. C. Pollard for his advice and suggestions throughout the course of the experiments.
SUMMARY
Three regions of radiation sensitivity are found in the spores of Bacillus .subliEis. The first two form the spore wall, which is variable in thickness on each spore. The outer layer is completely inert, showing no reaction to radiation; this might correspond to a dried slime layer which is said to surround the spore. An inner spore wall lies within this completely inert layer which shows low sensitivity to ionizing radiation, the effect being cumulative. The total thickness of the two spore coats is less than 350 A. The portion of the spore most sensitive t’o radiation is organized into units of large molecular weight which are dispersed at random throughout the interior of the spore. No evidence of a single organized nucleus containing the particles of maximum radiation sensitivity is found by this method of investigation. REFERENCES 1. LEA, D. E., The Action of Radiation Press, Cambridge, 1947. 2.
POLLARD,
E.
C.,
Advances
in
Biol.
on Living and
Cells.
Cambridge
Med. Phys. 3, 153 (1953).
University
IRR,~DIATCON
OF
Ii \VTI:RI.\l,
-1s I
SPORES
3. WELLS, D, A., h’ature 124, 983 (1929). 4. HASKINS, C. P., J, Appl. Phys, 9, 553 (1938) 5. h~RnV.4 J , ~ota-rt. Cent?. Beih. A61, 397 11942). 6. Moos, W. S., ~Vuclcon~rs 6. 50 (1951). i.
~VII,I,IAIW,
0.
Is.,
I,AMANN.4,
(,:.,
IiN.\YSJ,
(;.,
ET AL.,
kd.?riO~.
h!SVS.
16, x!+
(1952). 8. FRIEDMAX, (:., ANL, HENRY, D. 8., J. Bacterial. 36, 99 (1938). 9. M~ROWITZ, ,J., Ph.D. Thesis, Yale Univrrsity, 1951. 10. D.t\.rs, M., Physical Rev., (1951), irl press. 11. FI.I.KE:, I). J., DREW, It., ASI) P~LLAJW, 13:. C., 1'13~. :\-all. iicad. Sci. U. S. 38, 180 (1952). 12. [)A\ IS, 1I., AND HCTCEIIXSOX, F., Arch. Biochew and Biophys. 39, 459 (I%%?).
Division, Sloane Ph,ysics Luborator!y, New Haven, (‘onnecticul
Yale University,J
R,eceived ilugust 6, 1958 INTRODUCTION
The interpretation of biological effects of ionizing radiation as a function of the dose and the physical distribution of ionizing events through the application of target theory (I) has led t)o estimates of the size, shape, and multiplicity of units of the system which are sensitive t,o radiation. Deut.eron and high-energy electron bombardment of many enzymes irl the dry st,ate has shown that the sensitive volume can he ident,ified with the actual physical size of the molecule (2). IIowever, when the effec+ of radiation o11 more complex systems is st.udied, the tar@ size folltld does riot correspond t,o the whole molecule, hut to only a fractiotl of it. Highenergy radiation studies of the spores of the ha&rium KwiLZn~sSubtiZis have indicated a total target area to he nt)out onr-tetith ctf the a.rea of that portion of the spore \vhich shows nu4ear staining. ?I’othing is known as to the actual location of the sensitive units wit,hin the organism, or of the manner of organization of these units t,o form the complete viable system. Since it has been observed that. the mitot,ic* processes of a cell are drastically influencaed by radiation whereas gl~vcwlysis, respirat)ion, and other cell functions are only altcretl irt minor wys, it has been assumed that the rell nucleus and, more spwific~ally, the genetic material caanbe identified w&h the radiation-sensit,ive ut1it.s. This does tmt exclude the possibility of surface effects and extranuc*lear tlamage as a factor in ionizing radiation action, hut the part that it plays in the inactivation 1 Absl.ract.ed from a dissertation versity.
presented for t.hr l’h.1). degree in Yale Uni-
470
MARGUERITE DAVIS
of bacteria has not been separated from the effect of radiation on the organism as a whole. The depth of penetration of an ionizing particle into matter is determined by its rate of energy loss in the material; by control of the energy of an incident particle, the range of the particle can be controlled. If the energy is sufficiently low so that the entire range of the bombarding particle is less than the dimensions of the organism that is irradiated, the effect that is observed can be correlated with the depth to which the particle has penetrated into the organism. If the most sensitive portion of the cell is organized into a nucleus the effect observed when this nuclear portion is hit should be different than when only the surface or the cytoplasmic region can be reached. Low-voltage electrons can be used in this way as probes into the structure of organisms. Previous work of this type has been done by Wells (3), Haskins (4), Petrova (5), and Moos (6) using various low-energy particles on several different organisms; in no case was the found differential sensitivity correlated with the cell structure in an effort to determine the internal organization of the cell. Moos found a maximum killing effect for Pseudomonas omoginosa when the penetration depth of the particle corresponded to the cell diameter. A preliminary account of some of the work given in this paper has already appeared (12). BIOLOGICAL
MATERIALS
The spores of the bacterium, Bacillus subtilis, Marburg strain, were chosen for these experiments. B.y working with the spore of the organism instead of the vegetative cell, it was possible to carry out all of the experiments on a stock of spores that had been isolated, purified, and dried at the same time, insuring a more homogeneous population throughout the duration of the experiments. The use of the spores also made irradiation of dry material feasible as desiccation and exposure to high vacuum did not cause inactivation of the BpOre8. All the radiations were done on the material in the dry state to eliminate the indirect effeCtB of radiation due to short-lived radicals and their recombination products formed when water is present. The spores are ellipsoidal in shape, with a major axis of 1.5 p and minor axes of 0.9 p. Staining procedures have led to considering the spore as containing an organized nucleus located centrally and occupying about one-fifth of the total spore area. This nucleus appears to be imbedded in dense cytoplasm, which in turn is surrounded by a tough, highly impermeable spore wall (7). The spores are extremely thermostable and resistant to deleterious agents, factors which are attributed to the low permeability of the spore wall and to the low content of free water in the spore (8). It has been shown that about 70% of the water in the spore is present as bound water.
IRRADIATION
471
OF BACTERIAL SPORES
,,-Grid ond ~ull~oilng
To Golvonome+er~
1
]
FIG. 1. Schematic view of electron gun and bombardment EXPERIMENTAL
chamber.
PROCEDURE
The bombardments in these experiments were carried out with a simple electron gun which consisted of a heated tungsten filament and an accelerating grid. The bombardment chamber is shown in Fig. 1. The samples, placed on a stainless steel bombardment plate were located a few millimeters below the collimating tube; the geometry of the system was such that the area subtended by the beam on the irradiation plate was equal to the area of the exit hole of the collimating tube. The entire system was evacuated to a pressure of IO-6 mm. Hg to keep the scattering of the electrons to a minimum. The irradiation dose given to the samples in electrons/sq. cm. was measured from the known area of the electron beam and from the product of the electron current times the time of irradiation. The electron current was picked up directly from the irradiated samples and read on either a microammeter or d.c. amplifier, depending on the order of magnitude of the current; both meters were standardized with potentiometer and standard resistors. The accelerating high voltage from d.c. power supplies was applied to the filament and the voltage difference between filament and ground was measured on a standardized voltmeter. When irradiations were carried out at voltages greater than 1500 v., a copper shield was placed between the filament and the grid to reduce the field and prevent arcing from the filament. This system was adequate up to 3500 v., above which arcing in other parts of the system limited the high voltage range of the apparatus. Samples of the stock preparation of the Iyophilized spores suspended in quartzdistilled water were pipetted in 0.05-m]. droplets on 4% in. diameter stainless steel disks and were dried in a desiccator by pumping through a liquid-air trap.
472
MARGTJERITE
Electrons
FIG. 2. Survival
per square
DAVIS
centimeter
curve for the spores of BacilZus subtilis irradiated of energies from 200 to 1200 v.
with electrons
The titer of the spore suspension was adjusted so that each 0.05-ml. drop contained about 50 spores; since each sample was spread over an area of 1 sq. cm., the chance of the spores drying on top of each other or of clumping was very small. After drying, the plate was placed in the irradiation chamber and each sample was successively irradiated by rotating the plate into the electron beam. The samples were aligned under the beam through use of a window 180” from the beam. When one sample was directly under the window, the sample diametrically opposite it was under the electron beam. The bombarded samples were assayed on the same disk as was used for irradiation by layering over with agar the entire stainless steel plate in a large Petri dish and incubating at 37°C. for 16 hr.’ Each viable spore would give rise to a single bacterial colony; the number of such colonies in the area of an irradiated droplet compared with the nonirradiated controls determined the survival ratio. Samples exposed to the glowing filament in vacuum with no accelerating voltage applied showed no inactivation; any decrease in survival observed could therefore be attributed to the action of the electrons incident on the spore. Each experimental point on the survival curve was determined by the irradiation of at least three different samples. It, can be shown through a calculation similar to that used in Rutherford scattering that the spore will not accumulate a charge, and therefore repel the elec4 Thanks of assay.
are due to Dr. Harold
J. Morowitz,
who first developed
this method
trons, if a conductivity equal to that of celluloid is assumed for t,he spore. Since the composition of the spore is similar to that of relluloid, being composed of carbon, hydrogen, oxygen, and nitrogen, the assumption is probably valid. Only if the spore were composed of material of the conductivity of glass would charge accumulat? on the surface.
There is IN) observable effwt with electrolls t~elow 200 V. isee Fig. 3). .\t, 200 v., a slight amount of inactivat~ion OCCIII’S, hut thr effect, is tw\‘er greater than 10 y0 with doses as high as 10’: cle~trons,,‘sq. cm. .Uwve this energy, the effwt of the incident eler*tr.ons illcreases rapidly \vith voltage; the CI~WW are all of t.he multihit typca \vith a trail& up to
1
007
1
1200 volts *- -
\’
70
‘h
50.
20.
001 70. 50.
20.
IOElectrons FIG. 3. Survival
VUrvRs
per square
centimeter
for the spores of Haciltfrs sublilis irradiated of energies from 1200 to 3500 V.
~1iI 11c]ectrorls
474
MARGUERITE
DAVIS
900 v., where the trail-off vanishes and the curve appears to be either single hit or of low multiplicity, possibly marking the transition between two different inactivation processes.At all energies greater than 900 v., the curves are logarithmic (seeFig. 3). The slope of the curve is a rapidly increasing function of voltage up to 3000 v., beyond which it changes very little with further increase in voltage. This is more clearly seen if the 37 % dose is plotted against voltage (see Fig. 4). The values for the doses below 900 v. were found from extrapolation of the straight portion
of the survival curve for that voltage; the exact significance of these points is therefore somewhat questionable. Between 900 and 3000 v. the 37 % dose changes by a factor of 4 X 104.The total energy expended in
5310”:310”“E 7:: 5 c” 2 3‘0 f lo”.E~ p
23d
753-
$ , , I , ( , ., , 3
300
900
1500 2100 Voltoge
2700
3300
3900
FIG. 4. Dose necessary to cause 37% inactivation of spores of Bacillus subtilis M a function of voltage (!&M-3500v.). The values for the dosesbelow 900v. were found by extrapolation of the straight portion of the survival curve for these voltages.
IRRADIATION
OF
BACTERIAL
475
SPORES
producing the same inactivation at 3000 and 3500 v., respectively, is 1.65 x 1015and 1.57 X 1Ol6 e.v./sq. cm. These values can be compared with those found by Morowitz (9) as a result of high-energy radiation of the spores, e.v./sq. cm. for 37% survival
Radiation used
2.03 x 10’4 6.53 x 10” 8.7 x 10’6
X-rays (55-kv. peak) 3.8 m.e.v. deuterons 2650 A. ultraviolet light
A difference in the reaction of the spores to the different energies of incident electrons is also seen mhen the dose rate and the intensity of the irradiation is varied, the same total dose being given in each case; the total amount of irradiation in electrons per square centimeter per second t,imes the length of time of irradiation is held constant, the variKm MlTS toa lo* CUC-~I .
100 ‘--.C
%
Jl -
75
____--
,Al
;-------
a-- 3
Suwval 5. ‘i
I 0
I
I I I 8 4 6 2 Time of wadlotion in minutes
I IO
I
I
I
1
I
2
4
6 mmutes -
8
IO
1 12
12
minutes FIG,
5. Variation in dose rate at 600,900, and 1200 v.; in each case the same amount of irradiation was administered but the rate was varied over a factor of 20.
476
MARGUERITE
DhVIS
ables being changed over a factor of 20. As seen in Fig. 5, it was found that a beam of electrons of high intensity given over a short period of time is more effective than the same number of electrons incident at a slower rate. Variation in dose rate has no effect at higher voltages. DISCUSSION
These data indicate that the spore can be divided into three regions of different radiation sensitivity. Correlation of this information with the ranges of the electrons as determined by experiment, (10) leads to a concept of the internal structure of the spore. I. Energies Less thall ZOOVolts No damage is apparent when the spore is irradiated with electrons of very low energy. At 200 v. where inactivation is first observed, the electron has a range of 30 A. This can be taken as a minimum value of the thickness of an outer inert layer surrounding the spore as only a small fraction of the organisms can be inactivated with electrons of this range. The energy at which t)he trail-off vanishes marks the maximum thickness of the outer inert layer; this is somewhere between 600 and 900 v. The maximum thickness is less than 230 A., the range of a 900-v. electron. II.
200 to 900 volts
Electrons of energy greater than 200 v. are able to penetrate through the outer spore coat into a region which shows low radiation sensitivity. The inactivation curves in this region are multiple hit,, the degree of multiplicity decreasing wit,h increasing energy. At 900 v. the trail-off has disappeared, and the curve can be interpretfed as either single hit or very low multiple hit. At 1200 v., the logarithmic nat’ure of the curves is definite. The degree of mult’iplicity of the curves from 200 t’o 900 v. cannot be determined since t.he energy incident on the sensitive region is not homogeneous due to t,he statistical variation in energy loss and scattering of electrons in the outer inert spore coat. The change in degree of multiplicity can be taken as a measurement of the relative efficiency of the electrons of the different energies in causing inactivation. If 900 v. is taken as the transition from a multiple to a single hit inactivation process, the maximum t,hickness of this layer can only be 200 A., with a minimum value unknown. In this case the total thickness of the two outer coats cannot he greater than 230 A. If 900 v. is con-
IRR.\DT.lTION
OF B.1C”TERT.\T, SPORES
177
xideretl as a low multiple-hit curve, the transit ion to a single hit bking place between 900 and 1200 v., then the maximum thickness of the inner spore mall is less than 320 A. and the minimum less than 120 A. (350 A., the range at which the inactivation cwrves have hecome logarithmic minus 230 X., the range \vherr the it-ail-of’f has disappeared.1 Sittce the very rapid change in 37 y0 dose with cnerpy does not appear at energies less than 1200 v., this latter ittterpretatiotl is favored. ‘I’he data cit,ed t,htts far do not indicate \vhc~tlrer this spore \~a11thic*kttess varies from spore to spore in a gi\-etr popttlation or if e:rc+ individual spore h:is a tiottutiiform lager surroutidittg it. If the latter c*ase\vere true, the amottttt of inactivation ohser\wl \vould he due to random orietttjatiott of t.he spores. If a given populatiott of spores tvcrc reorient ccl after ratliat ion and reirradiat,ed, the same sort, of wt.\-i\T:tl curve \~wld 1~ seen, \vith a ttxil-off at the same per cent. ittartivatiott; if, on the other hand, each spore Ivere surrounded hy a uniformly t hit. 1, Lva.11, hut this thickntw varied from spore to spore, reirradiation aft.er rwrietttat~iot~ of the spores would h:t\,c no further effect on the survival: t hcl spores \\.hic*h had sttrvived the first irradiation would again sttrvivr the aecotttl. :\ largtl number of spores were irradiated at riO0 v. I\-it h a dose \\ell ottt ott the trail-off portion of the sttrvival cttrve. .1Rer irradiation, the spores \vtlrf’ \vashed off the irradiation pIatje, titered. attcl again dried down on t,he plate in the same manner as twfore and wirradiated with elect,rons of the same energy, l’he surviwl cttrves found after such a procedure \vrre identical with l,he first], showing that the sport’ coab is nonunjform ott each individual spore, and t hu.i the \\h& population is essent,ialIy homogeneous in t,his respect. ‘I’hiti esperimettt also ewludes the possiMity of t,he trail& portion of the crtr\:e twirrg due to the presettw of’ :I protective hyer of broth or to the organic8 stthstjattcSespresent in the spore suspension as SII& ma,terial \\-ould hewrnt~ diluted in the washing prowdure and a greater tiwl ittactivat.iotr \vould twttlt. ‘l’he present, data do not allow 5 more dcfittit.e atrni,vsis of the mrchattixm of the a.otion of tbe radiation in this regiott. ‘l’he cumulative nature of the inactivation and the constant energy wclttitwrtetit ttecessary slipports t’he hypothesis of severa hit,s itt otw target as f he inactivation prowss in the inner spore coat. Tothittg wtt lw said atwttt, the ttttmber or size of the targets.
In this range, the survival curves are all fq~otwtttial and the cross s&ion ittweases rapidly with increase in voltage top to 3000 v. whew :t
478
MARGUERITE
DAVIS
point of near maximum sensitivity seems to have been reached, since the curve levels off at this point to an almost constant value. This increase in cross section to a maximum value is not an ion density effect because in no case is the range of the electron comparable to the celldimensions; the total energy of the electron is dissipated inside the spore. Although the ionizations of a 3000-v. electron are more widely separated at the beginning of its path, as the electron loses energy it travels more slowly and the number of ionizations per unit path length increases. At the end of the track the ion density is the same as it was for the lowervoltage electron; the only difference is its ability to penetrate more deeply into the spore. If a sensitive volume is calculated when the target dimensions are greater than the mean separation of ionizations, the dose found for a given biological effect will be larger than that necessary to achieve inactivation on the basis of a single event occurring in one target. In the case of the very-low-voltage electrons, the whole track acts as a unit; for the higher voltages, the end of the track where the electron has been considerably slowed down also acts as a single unit. The cross section calculated at 900 v. (0.5 so. A.) cannot be interpreted as having any physical significance in demonstrating the structure of the spore but can only be viewed as an expression of the probability that an electron will have a range greater than the minimum value, and once within the protective layer surrounding the spore will hit a sensitive unit and therefore inactivate the spore. The probability of such an event occurring increases very rapidly with energy due to the combination of two factors. As the energy is increased, the fraction of electrons with a range greater than some minimum range gets through to the sensitive region of the spore. At 1200 v. a greater number have this range and by 3000 v. it is only the very rare electron which has been completely deflected out of the initial direction of travel that does not penetrate this far. ’ The increase in cross section is also due to the fact that the chance of an electron, once inside the spore, of hitting a sensitive unit increases as the distance which it travels in the area increases. When an electron has an initial energy of 3500 v. it has a maximum path length of 3100 A. An insignificant portion of the electrons do not penetrate the outer coat of the spore; once inside, the average electron travels a great enough distance, making enough collisions so that nearly every electron encounters a sensitive unit. The cross section at this point is 2.22 X lO+ so. cm. If we consider the electron as losing energy through primary ionizations, each primary ionization expending an average of 110 e.v., a 3500-v.
IRRADIATION
OF
BACTERIAL
SPORES
479
electron will form 3‘2 primary ionizations along its total path length. These will not be distributed evenly along the path, but will cluster at the end of the path where the velocity of the particle is small. If we consider as an estimate that one-half of the events takes place in the last quarter of the path length, the events will be on the average 150 A. apart in the first three-fourths of the track, and only ahout 40 A. apart in the final 800 A. If a sensitive volume is calculated on the assumption that the primary ionizations are evenly distributed along the path, a volume of 4.16 X IO-l8 cc. is found. Because this does not’ take into account the unequal distribution of the events, the volume is underestimated as the total number of effective events is less than the number of primary ionizations. By considering the distribution of events assumed above, t)he volume is increased by a factor of 2. As the energy is increased, a direct volume calculation will become more nearly applicable, since t,he distance hetween ionizations, on the average, becomes greater than the target. dimensions, and a minimum value for the 37 y0 dose will he found when this is true. In order to find the true volume at this point certain assumptions will still have to be made about the distribution of energy loss. Experimental difficulties prevented data from being taken at voltages greater than 3500 v. At this energy the cross section has not as yet reached a maximum. The sensitive volume as calculated by Morowitz (9) from x-ray data on the basis of two hit curves was 6.15 X 10-17 cc. The entire spore is vulnerable to radiation when bombarded by the high-energy particles, and yet the cross section does not change in a discontinuous manner once the electrons have energy enough to penetrate the spore wall. From these data it can be postulated that the radiation-sensitive units are dispersed uniformly throughout the internal region of the spore, and not concentrated in a compact nucleus. If the sensitive elements of the spore were grouped in a compact unit located asymmetrically inside the protecting coats, unless there is reason to assume an orientation of this unit other than random, the logarithmic survival curves would trail off at different survivals depending on the energy of the incident electron. The spores with the nucleus located beyond the range of the electron would survive. No trail-off is seen even with the relatively low-energy electrons with ranges of the order of onetenth of the diameter of the spore. The sensitive units are greater than 4 X lo-‘* cc. but less than 6 X 10-I’ cc. in size. The moIecuIar weight of these units is therefore about 2 X lo’, which perhaps identifies it with
480
MARGUERITE DAVIS
the desoxyribonucleic acid (DNA) of the cell. The molecular weight of DNA as found by irradiating pneumococcus-transforming factor with high-energy particles (11) is 6.5 X 106. Each unit in the spore which is sensitive to radiation may therefore be an aggregate of several DNA molecules. Contrary to cytological studies which have presented evidence for an organized nucleus in the spore from various staining procedures, the data from these experiments do not indicate such a region to be present. If all the nuclear material of the cell were concentrated in one large unit, a change in radiation sensitivity would have been observed when this portion came within the range of the electrons. Since such a response is not found, the only conclusion that can be made is that the units of the spore which show maximum sensitivity to radiation are dispersed throughout the interior in units of high molecular weight. The data do not give any estimate of t’he number of such ur~its. ACKNOWLEDGMENTS I would like to express my sincere appreciation to Dr. Franklin Hutchinson who suggested ‘this problem, and under whose guidance the work was done. I would also like to thank Dr. E. C. Pollard for his advice and suggestions throughout the course of the experiments.
SUMMARY
Three regions of radiation sensitivity are found in the spores of Bacillus .subliEis. The first two form the spore wall, which is variable in thickness on each spore. The outer layer is completely inert, showing no reaction to radiation; this might correspond to a dried slime layer which is said to surround the spore. An inner spore wall lies within this completely inert layer which shows low sensitivity to ionizing radiation, the effect being cumulative. The total thickness of the two spore coats is less than 350 A. The portion of the spore most sensitive t’o radiation is organized into units of large molecular weight which are dispersed at random throughout the interior of the spore. No evidence of a single organized nucleus containing the particles of maximum radiation sensitivity is found by this method of investigation. REFERENCES 1. LEA, D. E., The Action of Radiation Press, Cambridge, 1947. 2.
POLLARD,
E.
C.,
Advances
in
Biol.
on Living and
Cells.
Cambridge
Med. Phys. 3, 153 (1953).
University
IRR,~DIATCON
OF
Ii \VTI:RI.\l,
-1s I
SPORES
3. WELLS, D, A., h’ature 124, 983 (1929). 4. HASKINS, C. P., J, Appl. Phys, 9, 553 (1938) 5. h~RnV.4 J , ~ota-rt. Cent?. Beih. A61, 397 11942). 6. Moos, W. S., ~Vuclcon~rs 6. 50 (1951). i.
~VII,I,IAIW,
0.
Is.,
I,AMANN.4,
(,:.,
IiN.\YSJ,
(;.,
ET AL.,
kd.?riO~.
h!SVS.
16, x!+
(1952). 8. FRIEDMAX, (:., ANL, HENRY, D. 8., J. Bacterial. 36, 99 (1938). 9. M~ROWITZ, ,J., Ph.D. Thesis, Yale Univrrsity, 1951. 10. D.t\.rs, M., Physical Rev., (1951), irl press. 11. FI.I.KE:, I). J., DREW, It., ASI) P~LLAJW, 13:. C., 1'13~. :\-all. iicad. Sci. U. S. 38, 180 (1952). 12. [)A\ IS, 1I., AND HCTCEIIXSOX, F., Arch. Biochew and Biophys. 39, 459 (I%%?).