IRRADIATION OF PLANT VIRUSES AND OF MICRO-ORGANISMS WITH MONOCHROMATIC LIGHT

II. RESISTANCE TO ULTRAVIOLET RADIATION OF A PLANTVIRUS AS CONTRASTED WITH VEGETATIVE AND

SPORE STAGES OF CERTAIN BACTERIA,

B. M. DUGGAR AND ALEXANDER HOLLAENDER2

Laboratory of Plant Physiology, University of Wisconsin

Received for publication, June 11, 1933

The study of the effect of monochromatic radiation on thevirus of typical mosaic of tobacco and on Serratia marcescens, asdescribed in the first paper of this series (Duggar and Hollaen-der, 1934), gave some striking results. It was found that thevirus is extremely resistant to ultraviolet light at 0°C.; in fact,very much more so than S. marcescens. It was further found thatthe virus agent has its highest sensitivity at wave-length 2652A.

This paper includes work on the resistance of B. subtilis (vege-tative and spore form) and of the spores of B. megatherium f.to monochromatic ultraviolet radiation as compared with theresistance of S. marcescens and the virus of tobacco mosaic.To make possible a comparison of the results given in the firstpaper with those likely to accrue from this investigation, we usedthe same procedure except that the requirements of the speciesor stages of bacteria employed necessitated certain changes in thedetails of culture and suspension technique.The preparation of a uniform suspension, that is, a suspension

which will, upon further dilution, as in a series of poured plates, givethe expected numerical ratios, often presents a difficult problem.With the organisms used in this study more time was necessary

1 This investigation was made possible, in part, through a grant from the Re-search Committee of the Graduate School.

2 This represents work done while holding a National Research Fellowship inthe Biological Sciences.

241

B. M. DUGGAR AND ALEXANDER HOLLAENDER

in working out methods of overcoming these and other diffi-culties than in testing the organisms for their resistance to ultra-violet light. In fact, a number of organisms were investigated,but only a few of them were found suitable for our work. Foreach of these bacteria a special method of preparation was de-veloped, including special media. However, care was taken thateach suspension used by us checked in its light absorption fairlywell with the standard suspension described in the earlier paper.

PREPARATION OF MATERIALS

Bacillus sutbtilis, vegetative stage. Stock cultures were preparedas potato-glucose agar slants, incubated at about 27°C. for forty-eight hours. Five standard loops of the surface culture weredispersed in 2 drops of a synthetic medium prepared according toGottheil (1901).

KH2PO4.0.5 gramCaC03.0.1 gramMgS04. 0. 25 gramNaCi.......................................0.1gramAmmonium tartrate.10.0 gramsGlycerin.... 10.0 gramsCane sugar............ 5.0 gramsDistilledH20..1000 cc. + trace

of iron

To 9 cc. of this medium in a test tube was added 1 cc. of the sus-pension; the culture was shaken thoroughly and filtered asepti-cally through sterile cotton into another sterile test tube. Thisliquid culture was then incubated for six and one-half hours at 28°C.The organism did not grow profusely in this time interval, nordid it form any pellicle or precipitate. After the incubationperiod mentioned, the material was again shaken and filteredthrough sterile cotton, the filtrate constituting the active bac-terial suspension. The purified, pasteurized virus suspension(1:10), hereafter designated I. J. (infectious juice) was proxi-mately purified by a diatomaceous earth treatment preciselyas indicated in our previous paper. The experimental suspensionemployed was then prepared by adding 1 cc. of the active bacter-ial suspension and 11 cc. of the 1:10 virus suspension to 99 cc. of

242

IRRADIATION OF PLANT VIRUSES

physiological salt solution (hereafter designated S. S.), of one-half the strength previously used. This diluted the bacterialsuspension approximately 100 times and afforded a virus suspen-sion of 1:100 on the basis of the original I. J. Beginning withthe preparation of the stock suspensions all materials were, as inthe earlier work, plunged into an ice-water mixture, and so main-tained up to the time the material was plated out.To determine whether or not this B. subtilis suspension con-

tained any spores, 5 cc. of this material were kept in a test tubein boiling water under constant shaking for five minutes andcooled at once. When cool, 1 cc. was removed and plated out inagar, and the freedom of the preparation from spores was shownby the lethal effect of this treatment, no colonies appearing, or, inexceptional cases, single colonies. A higher survival value would,of course, indicate the presence of spores.

Bacillus subtilis, spore stage. Stock cultures were prepared asfor the vegetative stage, but these were incubated for ten days,the last three days being at 37°C. About 4 loops of this culturewere then distributed in 2 drops of bouillon and pasteurized inboiling water for two minutes under constant shaking, thus kill-ing the vegetative stage of the organism. Then 9 cc. of standardbouillon (8 grams bacto nutrient beef, in 1000 cc. water) wereadded, and the suspension shaken, and finally filtered asepticallythrough cotton.As before, the final suspension contained 1 cc. of the bouillon

culture + 99 cc. S. S. + 11 cc. I. J. 1:10, held in melting ice, asin the case of all prepared suspensions.

Bacillus megatherium sp., spore stage. The stock cultures, pre-pared as in the preceding case, were held four months (first four,weeks at 37°C.). Five loops of surface growth were added to 9cc. S. S., shaken for five minutes, then pasteurized at 80°C. forten minutes under constant shaking, and filtered through cotton.Of this suspension 1 cc. was used in preparing the exposure sus-pension as above.

Serratia marcescens, vegetative stage (no spore stage occurring).Stock cultures were prepared on agar slants, as for the preceding,but incubation was for twenty-four hours only. Two loops of

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B. M. DUGGAR AND ALEXANDER HOLLAENDER

this fresh surface growth were diffused in 9 cc. bouillon; then 1cc. of this diffusion was added to a tube containing 9 cc. bouillon,and this last culture incubated at 28°C. for eight hours, duringwhich interval no visible pigment develops. The exposure sus-pension, comparable to both of those preceding, was prepared with1 cc. of this culture.

It should be noted again that the material consisting of 1 cc.bacterial suspension, 99 cc. S. S. and 11 cc. of virus suspension1:10, was essentially optically clear, and had a very faint flavoustinge. The absorption of this material has been discussed ade-quately in our earlier paper. The number of bacteria variedbetween the limits 100,000 to 600,000; and a virus dilution of1:100 gives under favorable conditions a disease incidence of 100per cent.

In the case of all the organisms the controls indicate that thenumber of bacteria in the unexposed suspension remains rela-tively constant during the intervals of the several runs made.

APPARATUS AND PROCEDURE

In the first paper of this series there is given a description of theapparatus which makes possible the irradiation of biological sus-pensions with large and measured amounts of monochromaticlight to X2652A. This apparatus was particularly serviceablefor work centering upon the virus, relatively large amounts ofenergy being required. For the present study, however, withthe work centering uponl different forms of bacteria the require-ments were somewhat different. Quantitative determinationsof the effects of monochromatic light on bacteria require thehandling of small and correctly measured quantities of radiation,for which a new apparatus has been constructed, fulfilling theconditions imposed.3 The monochromator used was not as light-strong as the instrument used in the earlier work, but (having

I This apparatus has been built around a 13ausch and Lomb quartz monochro-mator of the constant deviation type, loaned through the Radiation Committee,Division of Biology and Agriculture, National Research Council. A detaileddescription of this apparatus and of thc method of irradiation of biological suspen-sions will be givecn in a seplarate paper. For this reason only a brief description ofthe experimental set-up will be here included.

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IRRADIATION OF PLANT VIRUSES 245

crystalline quartz optics) it permitted work with radiation ofshorter wave-length, especially with x2537A.As a light source the capillary, quartz, mercury-vapor lamp was

used, as in the earlier work, but in the construction of this lampseveral changes were introduced (Hollaender and Stauffer, 1933).The thermopile exposure-cell arrangement used in these stud-

ies is shown in figure 1. The arrangement is simpler than that

- To Gulvanom,elerCa

-To Pump

FIG. 1. THERMOPILE, AND EXPOSURE-CELL ARRANGEMENT

A, exit slit of monochromator; B, quartz window of constant temperaturetank; C, exposure-cell with stirrer (a, liquid seal); D, thermopile.

described in our previous paper, and it proved more efficient inthe use of small amounts of energy. The thermopile was of thevacuum type, consisting of three bismuth-tellurium junctions,having a surface of 2 by 5 mm. It was connected to a type HSLeeds and Northrup galvanometer, and was read on a scale 2meters from the galvanometer, 0.08 ergs/sec. /nun. deflection, asstandardized against a Bureau of Standards lamp. The thermo-

B. M. DUGGAR AND ALEXANDER HOLLAENDER

pile was constructed by MIr. J. P. Foerst of the Department ofPhysics. Since a thermopile of this degree of sensitivity is highlysusceptible to vibrations, etc., special care was taken to preventsupporting any of the moving parts (stirrers, etc.) from the ex-perimental work table, but rather directly from the ceiling orfloor of the laboratory.

RADIATION CELL

AND STIRRER'^6gmm

S7mn< 14m

CBFIG. 2. EXPOSURE CELL

A, sectional view of cell proper; B, representing longitudinal section of entirecell; C, longitudinal section of stirrer and attachment.

A new type of cell was constructed which rendered possible theexposure of smaller amounts of biological materials, and this cellwas likewise better suited for quantitative investigation, especi-ally at shorter wave-lengths. It consisted of an inverted T tubewith the horizontal tube cut close to the vertical on each side,the short cylinder thus resulting at the bottom being closed oneach side with a crystalline quartz slip to serve as windows. Thiscell was equipped with a stirrer and a liquid seal (fig. 2).

246

I-- 23mnL--- P

IRRADIATION OF PLANT VIRUSES

It is perhaps important to emphasize one or two features inthe operation of the stirrer. Exposure of the material was neverstarted until the stirrer had rotated for at least three minutes.Moreover, since the stirrer made several hundred rotations perminute, all the material in the cell had abundant opportunity tocome frequently into the path of the light, as referred to later.The extent of the area of the beam of light and the influence ofstirring will, however, be considered more in detail in the pro-posed article on apparatus.The following lines of the mercury spectrum in the ultraviolet

were used: 2537, 2652, 2804, 2952, 3130, 3342, and 3652A, someof these actually representing groups of lines not readily sepa-rated, as previously explained (Duggar and Hollaender, 1933).The irradiation and the testing of the effects (i.e., the dilution

plate series and the virus inoculation) were carried out as pre-viously indicated. The arrangement of the exposure cell-thefact that not all the material was exposed to the radiation at everyinstant (fig. 1), but rather was brought into the beam of light at arate of about 600 times per minute-brings up the question ofthe effect of short-interval interrupted exposures. This has beentested separately both with virus and with bacteria. The effectsof the total energies in frequently interrupted exposures corre-sponded to those of the same energy at a single (continuous)exposure within the limits here considered. This special ar-rangement, whereby the irradiated material was practically sur-rounded by material not in the beam of light, simplifies theconsideration of scattered light, the latter being largely reab-sorbed so that it need not be treated separately.

DISCUSSION OF RESULTS

The present discussion, with an abundance of new data on otherbacteria, may be regarded as supplementary to our previous ac-count of the influence of monochromatic light on the virus oftobacco mosaic and on S. marcescens.Our detailed results have been analysed and the salient fea-

tures presented in smoothed graphs (figs. 3 to 7). The effect ofthe three longest wave-lengths (all in the near ultraviolet) have

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B. M. DUGGAR AND ALEXANDER HOLLAENDER

been ignored in the graphs for the following reasons: Only veryslight and not at all uniform results are recognizable at thesewave-lengths. If only 1 per cent, or even somewhat less, of thelarge energies to which these organisms were exposed at thosewave-lengths were of the very effective short wave-lengths, itwould be more than sufficient in some cases to induce the effectsobtained. However, we feel that this point merits a more care-

0 80 t60 240 320

80 t t

BC

40~~~~~~~~~~~COO~~~~~~~~~~~~~~~~~~~~~~~~1

20____i0 2 2 40 ) 8 IXl;0iz

X 103 Ergs/cm.1/seC.FIG. 3. BACILLUS SUBTILI,S, VEGETATIVE STAGE ONLY (SEE TEXT FOR PREPARA-

TION), SHOWING THE EFFECTS OF MONOCHROMATIC ULTRAVIOLETRADIATION BETWEEN WAVE_LENGTHS 2537A AND 2950OA

ful investigation, which wre hope we shall be able to make, at alater time, with a double monochromator.

Bacillus sutbtilis, vegetative stage. The relatively rapid rise ofthe curves for this organism (fig. 3) would indicate that a largenumber of bacteria are in an identical stage of sensitivity anddevelopment. Attention should be drawn to the different scale

248

IRRADIATION OF PLANT VIRUSES

used in representing the effects with X2952A, showing that thesensitivity of the organism decreases fairly rapidly toward x3000A.The maximum of sensitivity is at X2652A (see also Gates, 1929).It is, furthermore, remarkable that this organism proves to bemore sensitive to x2805A than to X2537A. The absorption of thematerial is, however, higher at X2537A than at x2805A. Thelarger part of this absorption is attributable to small amounts of

0-_

0

c3'g-

v ocu QU 6D0 X0 /00 120

X 103 Ergs/Cm.2/sec.FIG. 4. BACILLUS SUBTILIS, SPORE STAGE (SEE TEXT-FOR PREPARATION), WITH

GRAPHS REPRESENTING 3 WAVE-LENGTHS

other materials (flavones, soluble proteins, etc.) present in thevirus suspension, and not to the bacteria present.

Bacillus subtilis, spore stage. The curves presented for thisorganism (fig. 4) have, in the higher survivor ratios in general, thesame appearance as the curves for the vegetative stage. How-ever, the exceedingly slow rise of the lower survivor ratios isremarkable. This would indicate a large variation in sensitivity

'C~~~~~~~~~'

40~~~~~~~~~~~

ZG

249

B. M. DUGGAR AND ALEXANDER HOLLAENDER

and probably the presence of a small number of more resistantspores. Previous investigators have found the spores to be about2 to 7 times as resistant as the vegetative stage. Since theyused methods which would not show the difference between highand low survivor ratios, their results may well be based, actually,on the presence of a few very resistant spores. The liquid sus-

3000

2q0c~~~~~~~~~~~~'tiV ,8

qitt~~~~~~~~~~C9Qn~~~~ _____________~~~~~~1

2700 _ I

2500, Qna /7-n Inn ni----u 4v 1# v bu aOUW{u} *v

X 103 Ergs/cm .2/sec.

FIG. 5. BACILLUS SUBTILIS, SPORE AND VEGETATIVE STAGES AT 2 AND AT 85 PERCENT SURVIVOR RATIO, SHOWING, ESPECIALLY, GREATER DIFFEIRENCES,

IN CONTRASTING THE STAGES, AT THE LOWER VALUES

pension and dilution count method avoids this error and givesprecise information. Nevertheless, even with the suspension-dilution method, a very few organisms may survive through beingheld temporarily out of the beam of light, as for instance, in con-

tact with the glass vessels, especially at the seams, but this is an

extremely small factor by comparison with the survivor ratiosin general. We feel that an attempt to get absolutely 100 per

cent killing would give a very distorted picture.

blJH

250

a)

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IRRADIATION OF PLANT VIRUSES

It is apparent from figure 5 that the curves for the spore and thevegetative stage are generally conformable for any survivor ratiocomputed. More particularly, it is noteworthy that the 85 percent survivor curves are close together, especially in the region ofshorter wave-lengths; whereas the 8 per cent survivor curves aremore widely separated. This difference may conceivably be re-lated to different types of cell walls, or membranes, in the twostages. At X2652A the ratio of the vegetative to the spore stage,considering a survivor value of 8 per cent, is approximately 4:5in respect to energy level employed.

Apparently there is very little relation between heat resistanceand light resistance. B. subtilis spores will stand boiling forfifteen minutes, the vegetative stage will scarcely withstandfifteen minutes at 65°. This point is brought out more emphati-cally in the graphs illustrating the behavior of the organism nextdiscussed.

Bacillus megatherium sp., spore stage. By comparison with thedata presented in the previous paragraph for the spores of B.subtilis it will be noticed that the level of energies required toproduce similar effects with B. megatherium is distinctly higher(fig. 6). The large amounts of energy necessary may be relatedin this case also to the special characteristics of the cell walls.Otherwise, these curves show the same general features as thoseillustrating the relations of spores of B. subtilis. It is interest-ing, however, that B. megatherium, according to these data,exhibits a maximum sensitivity at X2804A, rather than at X2652A.The form of these curves, as plotted, is very suggestive of thetype of curve indicating the effect of temperature on germinationof spores of Botrytis cinerea (Henderson Smith, 1923) and othertemperature effects. In this connection, attention may be drawnto the fact that the upper and lower stretches of these curves arebased on fewer data and are, accordingly, not so exact as the mid-regions. Referring again to the relation between heat resistanceand light resistance, it may be observed that the spores of B.megatherium will not withstand an exposure of more than tenminutes at 80°C.; yet these spores are more resistant to the effec-tive rays than are those of B. subtilis, previously referred to, theheat resistance of which is considerably greater.

JOURNAL OF BACTERIOLOGY, V'OL. XXVII, NO. 3

251

B. M. DUGGAR AND ALEXANDER HOLLAENDER

Resistance of thle virus agent. In the previous paper the resultsobtained with the virus and with S. marcescens have been dis-cussed. We shall now compare in one illustration (fig. 7) thevirus with the two stages, so far as they occur, of the severalspecies of bacteria thus far considered, the graphs being made on

the basis of a survivor ratio of 50 per cent. That the virus wouldprove so much more resistant to radiation of these wave-lengths

00 0

80~~~~~~~~~~~

60~~~~~~0

40h

C0

0 40 SO /20 ll0 zoo 240

X 103 Ergs/cm.2/seC.FIG. 6. BACILLUS MEGATHERIUM, SPORE STAGE ONLY

than spore stages of bacteria was not anticipated. However,these values again emphasize the lack of concordance with tem-perature effects. The virus is inactivated by an exposure of tenminutes at 90°C., and is therefore intermediate in heat resistancebetween B. megatherium and B. subtilis, spore stages.

It is well in this place to call attention again to the fact thatthe bacteria in the investigation reported here have been ra-diated in the virus suspension for the special purpose of securing

252

IRRADIATION OF PLANT VIRUSES

comparative data. For this reason it is not permissible to com-pare the absolute energies necessary to kill the bacteria in ourcase with the values obtained by other investigators. Practicallyall the quantitative work reported by others recently on bacteriahas been done on an agar surface. Since we feel that the liquidsuspension method has certain distinct advantages, we will at-

Curves A, B, C, D, X 103 Ergs, curve E, X 106 Ergs/cm.2/sec.

FIG. 7. RELATION BETWEEN 13ACTERIA (VEGETATIVE AND SPORE STAGES) AND

VIRUS AT 50 PER CENT SURVIVOR RATIO, SHOWING CLOSE RESEMBLANCE IN

SENSITIVITY RELATIVE TO WAVE-LENGTH, BUT ENTIRELY DIFFERENTENERGY MAGNITUDES

tempt in another paper to approach a more precise quantitativeinvestigation with this method, that is, one unaffected by thematerial necessarily added when the bacteria are used in com-

parison with virus.While an absolute comparison of the results of our investiga-

tion with that of other investigators is not yet permissible, it is

bo

c36L)

253

B. M. DUGGAR AND ALEXANDER HOLLAENDER

interesting to compare the relative sensitivity to wave-lengths;further, to compare the sensitivity of vegetative and spore stages.A very large number of papers have appeared on the sensitivity

of bacteria to light. It is expected that a full review will appearin a forthcoming publication. However, several papers report-ing work with approximately monochromatic light will be dis-cussed.

In the work of Bayne-Jones and Van der Lingen (1923), Staph-ylococcuts aureus was spread on an agar surface and irradiatedbehind a spectrograph. Lethal action began around X3500A,and continued with increasing effectiveness to the limits of trans-mission of the quartz instrument. Coblentz and Fulton (1924)irradiated B. coli spread on an agar surface with measuredamounts of energy. Screens were used to separate fairly definiteparts of the spectrum. The major part of the spectrum mosteffective in bactericidal action was found to be below X3100l.These investigators believed that they found a weak bactericidaleffect up to X3350A. While there are slight bactericidal effectsexhibited by our results up to X3650A, we feel tlhat the smallamounts of scattered light present may be responsible for these.In a series of investigations with S. aureus and B. coli, Gates(1929, 1929a, 1930) also employed the agar plate method, butused a monochromator. He found the maximum of sensitivityat X2650A.Ehrismann and Noethling (1932) used a double monochroma-

tor and irradiated cultures on an agar surface with measuredamounts of ultraviolet light. They found the highest sensitivityat x2650A for Micrococcus candidus, Bacillus pyocyaneus, Staph-ylococcus pyogenes-aureus, Saccharomyces cerevisiae and VibrioFinkler; at x2805A for B. prodigiosuts.

Pothoff (1921), working with B. anthracis, B. subtilis, and B.mesentericus, exposing these organisms in distilled water in water-cooled tubes, found the relation of the resistance of the spore andvegetative stages of the first organism as 6:1, the second organ-ism 4: 1, and the last 1.25: 1. He likewise reviews the work doneearlier in this field, so that consideration of such papers may herebe omitted.

254

IRRADIATION OF PLANT VIRUSES

The results of certain other investigations have been contradic-tory, and mention need not be made of those in which the stand-ard of comparison would be unsatisfactory.

SUMMARY

1. The present investigations have been made with a physicalinstallation modified from that of our previous studies, and es-pecially with a new type of exposure cell. A suspension techniqueand dilution culture procedure adjusted to the requirements ofthe organisms selected have been employed in a study of resist-ance of B. subtilis (vegetative and spore forms) and of B. mega-therium (spore form) as compared with the resistance of S.marcescens and the virus of tobacco mosaic subjected to mono-chromatic ultraviolet radiation.

2. The results are given in the form of survivor curves for thedifferent wave-lengths and for different intensities of the wave-lengths employed. The curves for spore and vegetative stagesare generally conformable. The level of energies required to givea particular survivor value with spores of B. megatherium at anylethal wave-length used is higher than that required for a similareffect upon spores of B. subtilis.

3. While vegetative stages (B. subtilis and S. marcescens) aremore sensitive to ultraviolet light, there is, in general, little rela-tion between heat resistance and light resistance.

4. The resistance of the virus irradiated coincidentally andin the same suspension with the bacteria is so much greater thanthe resistance of spore stages as to be of a different order of mag-nitude.

REFERENCES

BAYNE-JONES, S., AND VAN DER LINGEN, J. S.: Johns Hopkins Hosp. Bull., 1923,No. 34, 11.

COBLENTZ, W. W., AND FULTON, H. R.: Scientific Papers, Bureau of Standards,1924, No. 19, 641.

DUGGAR, B. M.: Proc. Soc. Exp. Biol. and Med., 1933, 30, 1104.DuGGAR, B. M., AND HOLLAENDER, ALEXANDER. Science, n.s., 1932, 75, 567.DUGGAR, B. M., AND HOLLAENDER, ALEXANDER: Jour. Bact., 1933, 27, 219.EIIRISMANN, 0., AND NOETHILING, W.: Zeit. f. Hyg. u. Infektionskrankh., 1932,

113, 597.

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256 B. M. DUGGAR AND ALEXANDER HOLLAENDER

GATES, F. L.: Jour. Gen. Physiol., 1929, 13, 228; 1929, 13, 249; 1930, 14, 32.GOTTHEIL, O.: Centralbl. Bakt., Abt. II, 1901, 7, 627.HOLLAENDER, A., AND STAUFFER, J. F.: Science, n.s., 1933, 78, 62.POTHOFF, PAUL: Desinfektion, n.F., 1921, 6, 10.SMIT1H, J. HENDERSON: Anni. Appl. 13iol. and Med., 1923, 10, 335.