LABELING CROWN GALL BACTERIA WITH p32 FOR RADIOAUTOGRAPHY'

TOM STONIER2Biology Department, Brookhaven National Laboratory, Upton, New York

Received for publication February 2, 1956

Crownn gall is a disease of various woody andherbaceous plants, characterized by the formationof tumors or other abnormal growths. The dis-ease is initiated by Agrobacterium tumefaciens(Conn, 1942; Smith and Townsend, 1907).Although the disease is initiated by bacteria, thepresence of the inciting organism is required foronly forty-eight hours; subsequent to this periodthe fate of the newly formed tumor tissue is inde-pendent of the presence of the bacteria (Braun,1954). The manner in which tumors are initiatedis not understood. No report of a successfulattempt to obtain an active bacteria-free extract,or to demonstrate a viral agent has ever been con-firmed. This fact suggested the possibility thateither the bacteria must enter the host cells inorder to release an active tumor-inducing prin-ciple, or that the bacteria themselves enter into avery close association with the host cells and canno longer be demonstrated by standard bacterio-logical or cytological techniques.The cytological evidence pertaining to the

location of the crown gall bacteria is equivocal.Levine (1936) after reviewing the subject, con-cluded that further work was necessary to estab-lish the location of the bacteria in the host. Sincethat time the problem has not been resolved. Inview of these considerations an attempt wasmade to determine the location of the bacteriaby labeling them with radioactive phosphorusand tracing their distribution in the host tissueby means of radioautography. This report is con-cerned with the bacteriological aspects of thisproblem, i. e., the method by which viablecrown gall bacteria may be obtained which arelabeled sufficiently for autographic studies.

1 Part of a dissertation submitted in partialfulfillment of the requirements for the degree ofDoctor of Philosophy in the Department of PlantScience at Yale University. This work was carriedout at the Brookhaven National Laboratory underthe auspices of the U.S. Atomic Energy Com-mission.

2 Present address: The Rockefeller Institutefor Medical Research, New York 21, New York.

MATERIAL AND METHODS

The pathogenic Wisconsin A6, the highly viru-lent B6, and Braun's non-pathogenic IIBNV6strains were used in the labeling experiments.The composition of the medium used is given intable 1. This medium allows adequate and repro-ducible growth. Phosphorus can be made thelimiting factor, thereby insuring a maximumuptake of added P32. Furthermore, the mediumpermits turbidimetric analysis of the bacterialpopulation since it is clear and bacterial growthis associated with a minimum of clumping. In-creasing the carbon source (in this case, gluta-mate) further increases the tendency to clump.For this reason the glutamate concentration wasreduced to 0.1 per cent in some experiments. Insuch a medium, strain B6 does not clump duringgrowth, IIBNV6 clumps slightly, and A6 moreso. The clumps (except for A6) are microscopic;as the culture ages, clumping becomes more pro-nounced.The turbidimetric analyses were made with a

Coleman Model 14 Universal Spectrophotometer.Glutamate medium was used as a blank, thereadings were taken at 400 m,u. The samples,10 ml in volume, were placed in test tubes whichhad previously been checked for uniformity witha solution of dye. Graphs were made by plottingPetroff-Hausser counts against turbidity read-ings. The curves thus obtained, coupled with pHmeasurements of the cultures, served as a basisfor subsequently interpreting culture data. Underthe above experimental conditions the genera-tion time for A6 was 2.7 hr, for B6 2.3 hr, and forIIBNV6 3.6 hr. The pH shifted from approxi-mately 7.0 to 8.5.The radioactive phosphorus was obtained as

labeled phosphoric acid from the Oak RidgeNational Laboratory. The acid solution wasplaced in a quartz tube, neutralized with sodiumhydroxide, diluted with distilled water to anactivity of 1 mc/ml, and sterilized with ultra-violet light. The specific activity at the time theneutralized, sterilized p32 solution was used,

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TABLE 1Composition of medium used for labeling crown

gall bacteria*

CaSO4 .............................MgSO4*7H20...........NaCl ..............................NH4NO3 ...... ....... .....

Fe (NO 3) 3 ..........................

MnCl2 .............................ZnCl2..............................Potassium citrate..................Sodium glutamate..................NaH2PO4 ..........................K2HPO4 ...........................Distilled H20....

0.1 g0.2 g0.2 g2.7 g5.0 mg0.1 mg0.5 mg10.0 g2.0 g1.5 mg4.4 mg1000 ml

* The phosphorus concentration may be varied.The final concentration in the medium above isapproximately 1 ,ug/ml, sufficient for growthalthough it is still a limiting factor. pH 6.8.

usually approximated 20 ,uc/,ug. The solutionwas added to the medium so as to produce thedesired radioactivity in a final volume of 10 ml(in 25-ml Erlenmeyer flasks). Usually the mediawere inoculated just prior to the addition of theP32; occasionally the bacteria were allowed toadapt themselves for several hours to a mediumbefore the p32 was added. The size of the inocu-lum was calculated from turbidity readings.

Although sterile controls were not run with allexperiments, no contamination was ever observed.The bacteria were allowed to grow for some

time in the radioactive medium and theil sam-pled. The amount of P32 assimilated by the bac-teria was determined by withdrawing 5.0 ml ofthe culture and analyzing it by means of the pro-cedure described in figure 1. The centrifugationswere carried out at 2500 g for 30 min. The 0.4 mlradioactive samples were pipetted onto alu-minum planchets, dried, and counted in a con-ventional-type counter with a self-quenchingGeiger-Muller tube of known efficiency. A Na-tional Bureau of Standards Radium D and Esource was used as a standard. Corrections weremade for background and decay, and all figureswere converted to disintegrations per min per mlof original culture. Almost all of the nonbacterialp32 was removed by the first washing. When thetwice-washed bacteria were resuspended andrecentrifuged, less than 5 per cent of the activityremained in the supernatant. Most of this super-natant activity represented bacteria which hadbeen stirred up while sampling, or had not sedi-mented. Further washings did not appreciablydecrease the nonsedimenting p32. In samplesconcerned only with the loss of p32 from labeledbacteria into nonradioactive media, the PMvalues were compared with the PT values.

5.0 ml culture

4.6 mlcentrifuged

Hsedimented bacteria,resuspended in 5.0 mlmedium, recentrifuged

sedimented bacteriaresuspended in 5.0 mlmedium, recentrifuged

sedimented bacteriaresuspended in1.0-10.0 ml of medium

used as inoculum

0.4 mlcounted -+ total P32 (PT)

supernatant

4.2 mldiscarded

0.4 mlcounted -+ p32 left inmedium (PM)

supernatantusually discarded, occasionally -

0.4 mlcounted - p32 of firstwash (Pwi)

supernatant

4.6 mldiscarded

0.4 mlcounted-+ p32 of secondwash (Pw2)

0.4 mlcounted p. p32 retainedby bacteria (PB)

Figure 1. Procedure for determining uptake of p32 by crown gall bacteria.

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Autographs of bacterial smears were made bystreaking onto Kodak dental X-ray film a drop ofa suspension of the washed, labeled bacteria.The films were desiccated with barium perchlo-rate at 10 C and exposed for various lengths oftime. Following exposure, the films were treatedwith Kodak D-19 developer, and Kodak dentalX-ray fixer, and rinsed several times with dis-tilled water. The films were left in cold (8 C)water overnight and then soaked in 50 per centglycerin for 24 hr. Strips of film containing theautographs were cut out, mounted on a micro-scope slide in 50 per cent glycerin and desiccated.

EXPERIMENTAL RESULTS

The uptake of p32 by the bacteria. Preliminaryexperiments concerned with the labeling of bac-teria established that the percentage of P32absorbed from the medium varied directly withthe amount of bacterial growth, but inverselywith the total phosphorus content of the medium.A criterion more useful than "percentage uptake"is that of "specific labeling," i. e., the actual p32content per bacterium. The specific labelingachieved varies directly with the specific activityof the labeling medium if the size of the inoculumdoes not exceed one hundredth of the totalgrowth at the time of harvesting. To insure thatno excess of phosphorus would be introducedvia the inoculum, the bacteria were grownthrough two transfers in a glutamate mediumcontaining 1.0 Mlg P/ml prior to inoculation.The labeling medium consisted of 10 ml of theglutamate medium containing 100 uc P32/mland from 10 to 20 ug P/ml including that con-

i:...:.:F::. 3||:~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~.....

*W * 8:

*~~~ ."}LSm.o

TABLE 2

Calculations of phosphorus content per bacteriumand specific labeling*

p32 content of medium. 28 ,c/mlP content of medium. 1.4,g/mlSpecific activity ....... 20 pc/,ugBacterial p32 (PB) .... 7.3 uc/mlBacteria per ml ....... 1.5 X 108 bacteria/mlSpecific labeling ....... 4.9 X 10-8 pc/bacteriumP content per bac-terium .............. 2.5 X 10- p,g/bacterium* Strain A6.

tributed by the stable phosphorus added at OakRidge. Thus, the specific activity of the mediumvaried roughly from 5 to 10 Ac/MAg, i. e., from18 to 36 p32 atoms per 1 X 106 p31 atoms.The specific labeling of the bacteria can be

calculated by dividing the amount of bacterialp32 per ml of culture (PB) by the number of bac-teria per ml. The data of table 2 show the maxi-mum labeling achieved in these experiments.The specific activity of the medium in which thebacteria were grown was the highest obtainablewithout using carrier-free p32. It can be calculatedthat under these conditions one disintegrationoccurs within a bacterium about every 10 mn.The bacteria used in the autographic studiescontained one-half to one-sixth of this amountof radioactivity. Figure 2 shows an autograph offour labeled crown gall bacteria in a smear madeon X-ray film. Each bacterium caused an averageof about 160 grains to be developed. The fourspots depicted are considered to represent four

..*

*0 AS

.t,

Figure 2. Autograph of four labeled crown gall bacteria in a smear made on X-ray film. The specificlabeling of the bacteria at the beginning of the 11-day exposure period was 1.3 X 10-8,c per bacterium.

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individual bacteria because: (1) the number ofbacteria counted on the smear approximatedthe number added; (2) most of the spots in theemulsion were of the size illustrated, and thissize was the smallest encountered; (3) the sizeof these smallest spots was relatively homogene-ous, i. e., the grain count did not vary by more

than a factor or two. The distribution of labeledbacteria in host tissue will be reported elsewhere(Stonier, 1956).The observed value of 2.5 x 10- lAg phos-

phorus per bacterium is typical for calculationsmade on 12 A6 cultures. This value representsa rough approximation since the estimation ofthe number of bacteria per ml is not accurate tomore than about 30 per cent, and since themedium may have contained more phosphorus(as impurities contributed by the salts used)than indicated. However, this value is in agree-ment with the reports of Mesrobeanu (1936)and Geiger and Anderson (1939). The formerreports 1 per cent, the latter 2 per cent, ofthe dry bacterial weight as being phosphorus.This would imply that 0.25 to 0.5 per cent of thewet weight is phosphorus. The bacterial cell inlog phase has a volume of 7.85 X 10-'3 cc (seeappendix), its specific gravity is about 1.1(Porter, 1946); therefore it weighs 8.65 X 107

,g. On this basis, 2.5 X 109 jAg phosphorusrepresent 0.3 per cent of the wet weight.

The release of p32 by the bacteria in vitro. Severalexperiments were performed to determinewhether or not the bacteria released p32.Labeled and washed bacteria were inoculatedinto a variety of nonradioactive media. Meas-urements of the P32 content of the culture as a

whole, and of the supernatant medium followingcentrifugation, were made at various timesfollowing inoculation. In most cases more thanhalf of the bacterial p32 could be found in thesupernatant after 5 days. In distilled waterkept at a temperature of 45 C for 18 hr,three-fourths of the p32 was released. Much ofthe p32 was released prior to log phase, and some

of this phosphorus was reabsorbed when the bac-teria were grown in media containing a lowlevel of phosphorus. The amount of P32 foundin the supernatant solution depended on (1)the physiological state of the bacteria in theinoculum, (2) the time of sampling after inocu-lation, and (3) the composition of the non-

radioactive medium. The physiological state

of the bacteria appeared to be determined bythe nature of labeling medium and by the ageof the culture from which the inoculum wasobtained. This phenomenon is under furtherstudy.

The toxic effect of P32. An investigation wasundertaken to determine the damaging effectof p32 on the bacteria. It had been observed thatbacterial growth was slower in P32-containingmedia than in the controls and, similarly,labeled bacteria when inoculated into non-radioactive media appeared to grow more slowlythan unlabeled control bacteria. Such an ob-served toxic effect might arise from any one ofthree causes: (1) it may be due to some toxicimpurity, such as a heavy metal; (2) it may bea radiation effect, the ionizations producingdirectly, or indirectly, physiological or geneticdamage; or (3) damage may occur when ap32atom incorporated into a biologically activemolecule undergoes transmutation to an S2atom, thereby disorganizing the molecule.To investigate the nature of the toxicity the

following experiments were conducted:(1) Chemical toxicity: Bacteria were labeled

under similar conditions on two separate occa-sions, approximately 7 weeks apart. In bothcases the same batch of p32 was used at an activityof 100 ,uc per 10 ml of medium. All experimentaldetails were duplicated as closely as possible.Eleven times as much of the p32 solution wasused in the second run to achieve 100 ,uc (thehalf-life of p32 is 14.3 days). Growth in these twocases was comparable and perhaps somewhatbetter in the second case. Chemical toxicity wastherefore ruled out as a cause of significantgrowth inhibition.

(2) Radiation damage. Data from variousexperiments indicated that this factor did notplay a major role. For example, two culturesyielded 1000 and 1100 million bacteria per mlat the time of harvesting, although one culturecontained 100 ,uc per ml, the other 6 uc. Growthin neither case appeared appreciably depressedwhen compared to nonradioactive controls. How-ever, in both instances the specific activity of themedium was low (1000 ,g/mc). In this case a.fifteenfold increase in the radiation dose did notappear to have any appreciable consequence.

(3) Transmutation effect. The type of experi-ment illustrated in figure 3 indicated that mostof the p32 toxicity observed reflects damage

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0.10

0.01

1 2 3 4 5 6 7Days

Figure S. The effect of the specific activity ofthe medium upon growth of crown gall bacteriain P32-containing media.

caused by transmutations. Four sets of cultures(strain B6, in 10 ml glutamate medium) weresupplied with varying amounts of stable andradioactive phosphorus. Cultures of group A, B,C, D received 0 (control), 100, 50, and 50 jAcP32/ml, respectively. The stable phosphorus(P31) content of cultures A, B, C, D was adjustedto 2.5, 50, 25, and 2.5 ,ug P31/ml. This resulted ina p32 p31 atomic ratio of 7 per million in culturesB,C, and 70 per million in culture D. The radia-tion received by the bacteria growing in culturesB, C, and D may be calculated to be 160, 80, and100 rep per hour per bacterium (see appendix).Group C exhibited aimost normal growth;group B showed a normal log phase but a some-what increased lag period. Group D culturesmanifested not only an increased lag period,but the log phase was highly abnormal, probablyreflecting the fact that as the p32 p31 ratio in-creased within the bacteria, the growth rate ofthe culture as a whole was correspondingly de-pressed. The difference between cultures B andC may be attributed to radiation. The differencebetween D and C cannot be accounted for interms of increased p32 since both received thesame amount. The difference in growth betweenD and B cannot reflect a difference in the radia-

tion dosage received by the two sets of cultures,since D received a lower dose. However, thebacteria in culture D suffered 10 times as manyatomic transmutations as did the bacteria ineither culture B or C. The most cogent con-clusion, therefore, is that the growth inhibitionobserved can be attributed to the transmutationsoccurring within the bacteria.

If it is true that the concentration of internalp32 is a critical factor responsible for the growthinhibition observed, then one can predict thatonce labeled bacteria start to grow in nonradio-active media, their growth should be normal.In another experiment it was found that dilutingthe internal p32 by a factor of four resulted in amarked alleviation of the observed growthinhibition. Therefore, it is probable that if alabeled bacterium is viable, a few generationsproduced in an unlabeled medium will com-pletely overcome the toxic effect of p32 bydilution.To test this hypothesis the following experi-

ment was performed. Two turbidimetric flaskscontaining 30 ml of medium were inoculated withstrain A6. One flask received approximately2 X 108 labeled bacteria (specific labeling 5 X10- Auc/bacterium), the other received approx-

A 6

B

4"I-

4cr

0Z

loB

0 4 8 12 16 20 24 28 32 36 40 44 48

HOURS AFTER INOCULATION -

Figure 4. The effect of labeling bacteria withp32 on bacterial growth (see text).

o BACTERIA NOT LABELED. BACTERIA LABELED WITH p32

IO7[

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imately 3 X 108 unlabeled bacteria. Growth ofthe two cultures (as measured by turbidity) isgiven in figure 4.

Figure 4 shows that the growth of the labeledbacteria in the post-lag phase is identical to thatof the unlabeled control, except that it trails by7 hours. It is probable that a large part of theincreased lag period may be ascribed to the lossof bacteria during washing. Precisely what por-tion of the increased lag period reflects genetic orphysiological damage, or more probably, a par-tial destruction of the labeled population was notascertained. If these in vitro studies are applica-ble to events occurring in the plant, then thelabeled bacterium is either viable and in thecourse of a few generations behaves perfectlynormally, or it cannot overcome the toxic effectof the p32 and probably lyses. That the labeledbacteria which show the capacity to grow arenormal is borne out by the fact that no depressionof crown gall production as a result of labelingthe bacteria was ever observed. In three exper-iments the labeled bacteria were plated out onnutrient agar and a total of 178 isolated colonieswere inoculated into tomato plants; all inocu-lations resulted in tumors.

DISCUSSION

Many papers have reported on the labeling ofbacteria with radioisotopes, but most of themhave been concerned with bacterial metabolismor bacterial virology. Relatively few papers havedealt with the use of labeled bacteria in studyinghost-parasite relationships. Only one paper hasbeen published making use of such a techniquein plant pathology; approximately a score areconcerned with human disease.Warren (1951) labeled Bacterium stewartii

with p32 and introduced the bacteria at the baseof the stem of corn plants. He then followed themovement of the bacteria in the host tissue witha laboratory monitor, or by means of radioau-tographs of the whole plant or plant parts. Noattempt was made to differentiate between P3-labeled bacteria and p32 which might have beenreleased by the bacteria. However, bacterialisolations were made to check the presence ofbacteria as indicated by the radioactivity.The earliest papers dealing with the use of the

labeling technique are those of J. 0. Ely (1941,1942) in which he describes the method used forlabeling 16 species of bacteria and for injecting

them into 5 different regions of rats. The distri-bution of the bacteria was contrasted with thedistribution of inorganic sodium radiophosphate.Much of the medical work since that time hascentered around labeled tuberculosis bacilli(Hevesy et al., 1948; Strom and Rudback, 1949;Pearson et al., 1949; Hammer et al., 1950;Tubiana et al., 1953). Pearson et al. (1949)labeled a variety of fungi and Mycobacteriumtuberculosis with I'3l and injected them into dogs.Relative rates of destruction were demonstratedby the decrease in radioactivity of the reticulo-endothelial system and by an increase in radio-activity of the thyroid (where inorganic iodineaccumulates). The relative indestructibility ofthe pathogens as compared with the non-pathogens was illustrated by the retention of thetracer in the lesions of blastomycosis and tuber-culosis. These workers also attempted to obtainautographs of individual tubercle bacilli. Pho-tographs of two of these autographs (Hammeret al., 1950) are not convincing since the auto-graphs consist of only a few, dispersed granuleswhich match up poorly with the stained bacteriapresented in adjacent photographs. Withoutreference to the stained bacteria, it is impossibleto estimate the number of autographed bacteria.More recently Tubiana et al. (1953) labeled M.tuberculosis with p32 and stated that they wereable to obtain autographs upon several daysexposure. Goldberg and Leif (1950) appliedlabeled suspensions of Pasteurella pestis as aer-osols to mice and compared the P3 content of therespiratory tree with that of the gastro-intestinaltract 30 min after infection. Buckland et al. (1950)attempted to label Bacillus subtilis spores withP32 for autography but were unable to achievethe level of activity per spore that was required.Two years later Harper and Morton (1952)described this work in more detail and pointedup one of the major problems in this technique,viz., the higher the degree of labeling, the greaterthe damage to the bacteria as a result of theincreasing amounts of radioactivity.There are several pitfalls connected with

interpreting data obtained from experimentsmaking use of labeled bacteria. Several of thepapers cited above make no attempt to differ-entiate between p32 as labeled bacteria and p32released by the bacteria either as a metabolicproduct or as a result of destruction. In A.tumefaciens the label may be readily released in

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vitro, and other workers have reported the releaseof isotopes by various other labeled bacteria. Thismatter will be discussed more fully in a subse-quent publication.

Labeling results presented solely in terms ofper cent uptake by the bacteria, are of onlylimited value. To ascertain the labeling achieved,and to be able to reproduce it, the specific activ-ity of the medium must be known. For example,Bon6t-Maury and Deysine (1953) report anuptake of p32 by Escherichia coli of 0.2 per cent at100lc/ml, and of 6 per cent at 1 mc/ml ofsynthetic medium. The fact that the bacteriaassimilated 0.2 Ac in one case and 60 ,uc in anotherprobably reflected differences in the specificactivity of the medium (providing bacterialgrowth was comparable).The specific activity of the medium also is of

prime importance in respect to toxic phenomena.Several authors have emphasized the killingeffect of the ionizing radiation emitted by PI*atoms. At high levels such ionization has a lethaleffect on resting cells. Schmidt (1948) reports that58 per cent of E. coli cells were nonviable afterbeing suspended for 48 hr in 0.075 M phosphatebuffer containing 100 M&c p32 per ml. However,this figure is probably not applicable to growingcultures inasmuch as in the case of X-rays theeffect on growing cultures is very much less thanon resting cultures. Rubin (1954), also workingwith E. coli, reports that a continuous flux of upto 10,000 r per hour does not alter the rate ofincrease of the cell mass nor the maximum tur-bidity, only the lag phase increased. He also findsthat similar results may be obtained in thepresence of 4000 tc P32 per ml (roughly 6000 repper hour). In the experiments described above,the bacteria were grown in cultures containingno more than 100 ,uc/ml at a calculated dose ofabout 150 rep per hour. The alteration of thebacterial growth curve obtained in cultures ofgroup D (figure 3) cannot be explained in termsof ionizing radiations. The conclusion that thetoxic effect exerted by a radioisotope results fromtransmutations occurring within the organism,rather than from a radiation effect, has beenimplied by the physiological or genetic effectsobserved in other organisms: e.g., Powers (1947)on Paramecium; Hungate and Mannell (1952) inNeurospora; Friedrich-Freska and Kaudewitz(1953) on Amoeba; Michaelis and Kaplan (1953)in Epilobium; King and Wilson (1954) in

Drosophila. Rubin (1949), working on the dam-aging effect of P32 on E. coli, arrived at a con-clusion essentially identical to the one presentedabove. Unfortunately, no data are given. Hersheyet al. (1951) have provided evidence for thetransmutation effect in T4 bacteriophage. Onlythese last authors have presented actual cal-culations to indicate that it is primarily trans-mutation that must be involved in the observedkilling effect. Other authors have, for the sake ofsimplifying the calculations, either assumed thatmost of the radiation comes from the medium, orthat the distribution of PE in larger animals ishomogeneous. Neither assumption is valid. Theradiation dose to an organ or cell organelle ac-tively metabolizing p32 will automatically begreater than the average for the whole organism.The calculations presented in the appendixindicate that an object as small as a bacteriumreceives an increased radiation dose of 20 per centwhen the specific activity of the medium is in-creased tenfold. If the diameter were 30 times aslarge (Neurospora spore), 30 times as much of theemitted energy would be absorbed and the in-creased radiation dose becomes appreciable. Thefraction absorbed becomes even greater when oneis dealing with a softer ,B particle as in S35; and therelative radiation dose received doubles when theorganism is grown only at the surface of a radio-active medium. Therefore, any conclusions basedon an increased effect dependent upon an increasein specific activity of the medium, cannot beconsidered valid until the precise increase inradiation dose caused by the increased accumula-tion of the isotope has been taken into considera-tion.

If the conclusion that assimilated Pn may exerta damaging effect by virtue of its transmutationis correct, several papers dealing with "radiation"effects of radioisotopes need reinterpretation,(Mackie, Blume and Hagen, 1952; Schlegel, Goldand Rawlins, 1953.)

ACKNOWLEDGMENTS

The author is greatly indebted to Dr. V. T.Bowen, under whose direction this work wascarried out; to Dr. R. C. King for constructivecriticism of this manuscript; and to Drs. R. C.King, F. Forro and W. Rubinson for discussionsof the calculations.

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SUMMARY

A method has been described for labeling crowngall bacteria for radioautography with P32. Theaddition of 100 uc of p32 per ml of syntheticculture medium was found to be most satisfac-tory, providing the specific activity of the mediumwas adjusted to be in the 5 to 10 ,uc/,ug P range.At this level the specific labeling achieved wasroughly 1 to 2 X 10 ,uc per bacterium; i.e., eachbacterium contained approximately 1000 P12atoms.

Lowering the specific activity of the mediumresults in a lowered specific labeling of the bacte-ria. With increasing specific activity, the toxiceffect of PI2 becomes correspondingly more pro-nounced. At 5 to 10 ,uc/,ug P growth inhibitioncan be observed; however, neitherthe morphologynor the pathogenicity of the bacteria appears tobe significantly affected. It could be shown thatthe toxic effect of P32 is primarily caused by P"2atoms assimilated by the bacteria, and probablyresults from a transmutation rather than a radia-tion effect. Once labeled bacteria start to grow ina nonradioactive medium, their growth is indis-tinguishable from unlabeled controls.The phosphorus content of the bacteria calcu-

lated on the basis of the phosphorus uptake fromthe medium, was estimated at 2.5 X 10 ;tg perbacterium for strain A6. This amounts to 0.3 percent phosphorus by wet weight.

Labeled crown gall bacteria released largeamounts of P32 to the medium when they weretransferred to nonradioactive media.

APPENDIX

The radiation received by an organism growingin a radioactive medium may be resolved intotwo components: the external radiation, R.,which it receives from the medium, and the in-ternal radiation, Ri, received from P32 atomsincorporated into the cell itself. The total radia-tion, Ri, consists therefore of R. + R,.

(1) Calculations of Re: The energy released perml of medium equals the number of disintegra-tions per minute per ml multiplied by the averageenergy per disintegration. It is assumed that inan infinitely large, homogeneous volume of radio-active solution, the energy absorbed by any smallpart of that solution is equal to the energyreleased, since as much energy as is lost to thesurrounding medium will be absorbed from thesurrounding medium. The average path of the P3

B particle in water is 0.25 cm (Glendenin andCoryell, 1946). The culture contained 10 cm' ofliquid in the shape of a flat cylinder (r = 2.3 cm;h = 0.6 cm). Since the volume of the culture islarge in respect to the average path of the j3particle, the finite dimensions of the culture donot appreciably alter any calculations based onthe assumption that the volume is infinite. Usingequation (7) of Richards and Rubin (1950), itcan be shown that of the order of 17 per cent ofthe energy is lost from the culture. This does nottake into account the energy returned to themedium as a result of backscattering. The cal-culations below assume that the bacteria havethe composition of pure water, whereas theirspecific gravity is probably 1.1 (Porter, 1946,p. 18). This increased density will increase theradiation dose by 10 per cent, offsetting the 17per cent loss from the medium. For this reasonthese two factors will be omitted from subsequentconsiderations.

(a) per ml of medium, R = aE

where R = the radiation energy absorbed inMev/min/ml

a - the no. of disintegrations(dis)min/ml

E = the average energy in Mev/dis

/

in culture C of figure 3,a - 50 X 2.2 X 106 dis/min/ml (1 ,uc

2.2 X 106 dis/min)E = 0.7 Mev/dis (Hollander, Perlman, and

Seaborg, 1953)

Then R =0.7 Mev/dis x 50 X 2.2x 106dis/min/ml

7.8 X 107 Mev/min/ml

(b) per bacterium, R. = vR

where R. - the energy absorbed by a bacteriumfrom the medium in Mev/min/ml

v = the volume of an average bacterialcell in ml <

Under the experimental conditions, a bacterialcell in log phase can be characterized as an ellips-oid of revolution with a diameter of 1 ,u along theminor axis, and 1.5 IA long. The volume of such acell is therefore 0.785 .3.Then R. = 7.85 X 1-13 ml/bact x 7.8 x 107

Mev/min/ml= 6.1 X 10-' Mev/min/bact

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(2) Calculations of R,: In culture C the spe-cific activity of the medium was 0.5 ,ug/,uc. Thebacteria contain approximately 2.5 X 10 ,jug ofphosphorus per bacterium. The bacteria afterseveral divisions in the radioactive medium maybe considered to achieve a specific activity ofbacterial phosphorus approaching the specificactivity of the medium. Thus in log phase, thebacteria may be considered to contain 5 X 10-9,ucP"/bacterium. This amount of radioactivityrepresents:5 X 10-'jc/bact X 60 X 3.7 X 10' dis/,uc/min

= 1.1 X 10-' dis/min/bactIn terms of energy released this equals:

0.7 Mev/dis X 1.1 x 10-' dis/min/bact=7.8 X 10-3 Mev/min/bact

However, the energy that is actually absorbed bythe bacterium is only a small fraction of theenergy emitted. Asuming that the P'2 within thebacterium is homogeneously distributed, theaverage path (p) of an electron within an ellips-oid is equal to 0.75 of the radius of a sphere withthe same volume (Lind, 1928). Since

%6rr' = 7.85 X 10-13 cm'

then r = 0.57 X 10-- cm

and p = 0.43 X 10 cm

This figure is valid if it is assumed that the elec-trons travel in a straight path. This assumptionis warranted because the average straight part ofits path is 0.095 cm. For essentially similar cal-culations for this part of the problem, see King(1952).

Since the average path of the P' ,B particle inwater is 0.25 cm, the energy absorbed by thebacterium is only a relatively small part of theenergy emanating from the bacterium; specifi-cally, the fraction

0.43 X 10-4 1.7 X 10-0.25

so that

Ri = 1.7 x 10- X 7.8 X 10-3 Mev/min/bact= 1.3 x 10- Mev/min/bact

(3) Calculations of Rg: If it is assumed that theradiation received by the bacterial cell from themedium stays constant, then during the life cycle

of culture C, Rs changes from

Rs = 6.1 X 10-' + 0 Mev/min/bact

to

Rt = 6.1 X 10-' + 1.3 X 10-6 Mev/min/bact= 6.2 X 10'- Mev/min/bact

Similarly, for culture B,

Rs = 1.2 X 10' + 1.3 X 10-6 Mev/min/bact= 1.2 X 10- Mev/min/bact

and for culture D

R= 6.1 x 10-' + 1.3 x 10-' Mev/min/bact= 7.4 X 10-' Mev/min/bact

Since most reports concerned with radiationdamage to bacteria express the dose in terms ofroentgen or roentgen equivalent physical per hr(rep/hr), these values may be converted by usingthe equation:

1 rep 5.8 X 107 Mev/gm= 5.8 X 107 X 7.85 X 10-13 Mev/bact

4.6 X 10-' Mev/bact

to calculate that the bacteria in cultures B, C, andD received 156, 81, and 97 rep/hr, respectively.

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