they lingen (1923), '29a, '30), wyckoff (1932),...

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

I. THE VIRUS OF TYPICAL TOBACCO MOSAIC AND SERRATIAMARCESCENS AS INFLUENCED BY ULTRAVIOLET AND

VISIBLE LIGHT

B. M. DUGGAR AND ALEXANDER HOLLAENDER2

Laboratory of Plant Physiology, University of Wisconsin, Madison

Received for publication June 11, 1933

The effects of visible light and, in recent years more especially,the bactericidal effects of ultraviolet light have received in-creasing attention. Most of those engaged upon such problemsprior to very recent years have been content with results essen-tially qualitative in nature. Nevertheless, progress was madeand it was to be expected that with the development of vastlyimproved physical instruments available to the biologist, andwith analogous advances in biological technique, definitely quan-titative studies would be made. This advance is now in progressand during the last five years, especially, contributions of muchfundamental importance have appeared. Such studies are ofinterest not merely because they afford definite data regardingthe lethal effect, or lack of lethal effect, of monochromatic lightof various wave-lengths upon bacteria, but also because theyindicate the rate of killing, or diminishing rate of survival withtime and with increasing intensity of illumination. Prominentamong those who have contributed these recent advances maybe mentioned Bayne-Jones and Van der Lingen (1923), Coblentzand Fulton (1924), Gates (1929, '29a, '30), Wyckoff (1932), andEhrismann and Noethling (1932). Virus and bacteriophage have

1 This work was made possible, in part, through a grant from the ResearchCommittee, Graduate School, University of Wisconsin.

2 In part this represents work done while holding a National Research Fellow-ship in the Biological Sciences.

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B. M. DUGGAR AND ALEXANDER HOLLAENDER

received far less attention. Nevertheless, progress has beenmade, notably through the contributions of McKinley, Fisher,and Holden (1926), Fisher and :McKinley (1927), Olitsky andGates (1927), Rivers and Gates (1928), and Baker and Nana-vutty (1929). Obviously more quantitative data on the effectof radiation on viruses are most desirable, and in many cases atleast are experimentally obtainable. The ease of handling plantviruses at once suggests the desirability of working upon someone or more of these forms. This study was undertaken withprimary interest for the moment centered upon the virus. Ser-ratia niarcescens was selected as a species of bacteria suitable forcomparative study.The purpose of this paper is accordingly to report the technique

employed and also the results obtained with the influence ofapproximately pure monochromatic light, primarily ultraviolet,on a plant virus, the agency of typical mosaic of tobacco, in com-parison with the vegetative phase of a species of bacteria, car-ried out under conditions which are believed to be as nearly quan-titative as is practicable with standard procedures and availableapparatus.Although the amount of work done with the bacteria has been

extensive, such studies have been primarily for comparison andcontrol. Were it not necessary to make this comparison as nearlyabsolute as possible, it would be entirely feasible to approach bynew and improved technique a still more closely quantitativedetermination of the influences of radiation of different wavelengths on the bacteria. Such a study is in progress.A fundamental difficulty which underlies the utilization of any

virus as an object to be irradiated is found in the fact that virusesdo not lend themselves as yet to methods of intensive purifica-tion without great possibility of loss of activity. I1 a final analy-sis of the influence of light of any wave-length it would be desir-able, of course, to eliminate from the virus suspension all otherparticles or substances which might absorb light. MIoreover,there is no way in which the extent of this constant error of in-efficient absorption can be accurately determined. Unfortu-nately, in the past investigators have made the exposures of viruses

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IRRADIATION OF PLANT VIRUSES

primarily in the virus-containing fluids, and frequently have madecomparisons between these results and the results obtained withbacteria or other organisms exposed under entirely different con-ditions-usually on agar plates. The agar plate method is ob-viously unsatisfactory for virus work, and it possesses disad-vantages as applied to bacteria. In the studies here reported adevelopment of the suspension technique has been founid pecul-iarly applicable for both materials, as indicated later.

PREPARATION OF THE BIOLOGICAL MATERIALS

In our work we have purified the virus of typical tobacco mo-saic up to a certain limit by using a diatomaceous earth adsorb-ent and precipitant. This material eliminates all chlorophylland much of the proteinaceous materials, likewise much of thecomplex carbohydrates of the leaf juices, yet leaves the plantextract essentially in a "natural" condition with respect to so-lutes. The virus suspension obtained still contains a smallamount of some soluble pigmented substances, possibly flavonesand some tannoid complexes.' The difficulty of making an ac-curate comparison between the tolerance of the virus and of thebacteria, determined separately, was recognized, since the in-effective light absorption of the two suspensions would introduceerrors not readily computed.The difficulty referred to above was eventually very simply

solved by diffusing the bacteria in the virus suspension and thusexposing both the bacteria and the virus agency under identicalconditions. The combination of virus and bacteria in the sus-pension, under the conditions maintained, affects neither materialdeleteriously, so far as can be measured. Such technique would

3Fresh, succulent, diseased tobacco shoots are finely ground in a food chopper;the juice is then squeezed through gauze, diluted by the addition of nine parts ofwater to one of juice, giving a 1:10 suspension of the virus. To 100 cc. of thisstandard 1:10 suspension is added 10 grams of super celite (a diatomaceous earth)and after standing (with occasional agitation), the material is centrifuged atapproximately 4000 r.p.m. for five to ten minutes, and the supernatant, clear,slightly colored suspension is used as a source of virus. Subsequently, as notedlater, this suspension is again diluted to a concentration, relative to the naturaljuice of 10-2, here designated as 1:100 suspension.

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at least furnish comparable results. This was not accomplished,however, without considerable experimental work and the in-troduction of several other modifications of the usual procedure.In the first place, when the 1:10 virus-containing plant juice isprepared as described, some of the bacteria accidentally presenton the leaves may be found in the juice, nor does the "precipita-tion" by means of diatomaceous earth remove all of these. Itwas then found perfectly practicable to pasteurize the 1:10 virussuspension by exposure in a water bath, with stirring, at a tem-perature of 65 to 67°C. for fifteen minutes. Such spores of bac-teria as may remain in living condition are not a factor of impor-tance in view both of the dilution to 1: 100 of the juice for exposureto radiation, and of a further 100-fold dilution when tlle bacterialplates are poured, as subsequently indicated.

Moreover, very early during our preliminary work it was clearthat the bacteria used would not form a perfect suspension eitherin distilled water or in the diluted virus suspension. Clumpingoccurred and this gave rise, of course, to erratic results when thebacteria were poured on the dilution plate. Eventually thedifficulty of clumping was erntirely prevented by the utilizationof a physiological salt solution, that is, a balanced salt solutioncontaining the chlorides of calcium, sodium, and potassium.4It was found less desirable to use sodium chloride alone.

Accordingly our procedure was to prepare a virus suspensionin the usual way; likewise to arrange by a definite culture systemfor a fresh culture of the bacteria which, under the conditions ofgrowth and time interval, would furnish a rather definite numberof organisms per cubic centimeter. From an agar slant cultureof the bacteria, grown for twenty-four hours at approximately280C., a suspension of standard opacity was prepared. Tubes ofstandard beef bouillon were inoculated from this suspension, eachreceiving 1 cc., and the broth cultures were incubated for eight

4 This salt solution was of the following composition:

NaCl...6 gramsKCi.. 0.4 gramCaCl2.. 0.4 gramWater.. 1,000 cc.

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hours at 28°C. These tubes were used immediately as stocksources of bacteria, or, if any short delay occurred, they wereplunged into crushed ice. As a matter of practice we arrangedthat the stock solutions of the virus suspension would be one-tenth the concentration of the virus in the plant juices andthe stock bacterial suspension would contain approximately100,000,000 bacteria per cubic centimeter. These two stocksuspensions were then added to the physiological salt solution insuch quantity that the final suspension of virus was 1:100 andthe final suspension of bacteria of the order of 1,000,000 percubic centimeter.From the preceding statements it will be seen that the suspen-

sion for exposure actually contained in 100 cc. of the salt solutionapproximately 1 cc. of bouillon containing the bacteria and whatamounts to 1 cc. of the virus suspension calculated on the basisof the full-strength juice. The ineffective absorption of the finalsuspension is, of course, to be ascribed chiefly to the addition ofthe virus fluid, though in part also to the bouillon added. Thisexposure suspension was then kept in an ice mixture until used.

APPARATUS

For a quantitative study of the effect of radiation it is desirableto work with light as nearly monochromatic as possible and anaccurate determination must be made of the amounts of energyused. It is with monochromatic light alone that it is possibleto determine effects such as the localization of the influence oflight in the different parts of the spectrum; while exact energydeterminations are required for a quantitative interpretation ofthe results.

It appeared early in our investigation that the energy neces-

sary to inactivate the virus agent with the suspension method ofexposure decided upon is unusually large, very much larger, infact, than the energies necessary to kill microorganisms, alsolarger than the energies usually available in work with trulymonochromatic light.The use of filters which are now available for the differential

selection or elimination of certain parts of the spectrum would at

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once suggest itself. However, while the loss of energy wAith theuse of such filters may be only moderate, they show certain otherdisadvantages which render them unsuitable for our work. Thebest filters available will tranismit relatively broad regions of thespectrum and the band transmitted will not show a definite cut-off. If practicable, it would have been desirable to work with adouble monochromator by means of which very pure monochro-matic light might be obtained. This possibility was precludedby the fact that the energies needed in this experiment were verymuch larger than could be obtained with a double monochroma-tor, so that we had to compromise and be satisfied with the useof a single monochromator with very high transmitting power.There was available for part of our work, fortunately, a mono-chromator with very large fused quartz optics and a source oflight considerably more intense than the commercial mercuryvapor lamp.

Source of radiation. The greater part of this investigationwas carried out with a water-cooled, capillary, mercury-vaporlamp of the Daniels-Heidt (1932) type, burning on 150 to 400volts and using 5 to 2 amperes. The high intensity of the radia-tion (about 20 times greater per unit area of the arc than that ofcommercial lamps) made this lamp veiy suitable for our work.

Monochromators. MAost of the work here reported was donewith a large quartz monochromator consisting of a 60' fusedquartz prism, with faces 12 by 14 cm., and a pair of plano-con-vex quartz lenses each with a diameter of 15.25 cm. and a focallength of 35 cm. (Heidt and Daniels, 1932). This instrumentwas used only down to X2650A, on account of the absorption ofthe fused quartz at shorter wave lengths. In connection withthe instrument just described, there was available a large sur-face thermopile5 of 48 copper-coinstantan junctions in connectionwith a Leeds and Northrup galvranoineter, H. S. type, the sen-sitivity of this set-up being about 170 ergs per second per centi-meter deflection.

It is expected that a complete description of this thermopile, as well as of thethermopile and monoclromator described in the following p)aragraph, will begiven by Daniels and associates in an article to appear in the Journal of theAmerican Chemical Society.

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The entire work in the visible part of the spectrum was done ona monochromator of the constant deviation type, consisting of a60 hollow glass prism (sides 12 cm. square) filled with ethylcinnamate, and a pair of biconvex glass lenses each with a di-ameter of 12 cm. and focal length of 18 cm. This instrument wasequipped with a large surface thermopile of 13 copper-constantanjunctions, connected with a Leeds and Northrup H. S. typegalvanometer, sensitivity 165 ergs per second per centimeter de-flection. This, as well as the previously described monochroma-tor, was used only with the capillary mercury vapor lamp.Both of the instruments described in the paragraphs on source

of radiation and monochromators were kindly put at our disposalby Professor Farrington Daniels of the Department of PhysicalChemistry.A Bausch and Lomb constant-deviation type, quartz mono-

chromator in connections with a vacuum thermopile and a Kippand Zoonen galvanometer was used in some of the work. Theseinstruments were kindly put at our disposal by Professor C. E.Mendenhall of the Department of Physics.The work on the 2537A line and the checking work on the other

lines of the spectrum were done with an apparatus built around aBausch and Lomb monochromator loaned through the Radia-tion Committee of the Division of Biology and Agriculture,National Research Council. A description of this experimentalset-up will be published shortly.

In the following list are given in Angstrom units the wavelengths employed; in certain cases it was necessary to groupcertain lines together, and in such cases an approximate desig-nation is given, the actual lines being given in parentheses:2537 (2535, 2537), 2652 (2652, 2653), 2804, 2952 (2925, 2967,3021, 3023), 3130 (3125, 3131, 3132), 3342, 3650 (3650, 3654,3662, 3663), 4047 (4047, 4078, 4108), 4358 (4339, 4347, 4358),5461, 5790 (5770, 5791), 6120 (6072, 6123).The purity of the lines was tested in many cases with a quartz

spectrograph. The spectrum showed that there was never morethan 10 per cent, and in some cases not even 2 per cent, of lightof other wave-lengths present.

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Exposure vessels. (a) The virus-bacterial suspension alreadydescribed was consistently irradiated, for the greater part of thisinvestigation, in a standard fused quartz cell (fig. 1). The di-mensions of this exposure vessel were as follows: height 11.5cm., width 1.5 cm. The faces of the cell were parallel, and thesewere ground and polished up to a height of 5.5 cm. The throat

FIG. 1. QUARTZ EXPOSURE-CELL, WITH STIRRER AND LIQUID SEAL

of the cell was cylindrical and provided with a ground mouthinto which fitted a ground stopper through which was introduceda quartz stirrer reaching well into the liquid to be exposed. Theend of the stirrer was so constructed that when turning it wouldnot only mix the liquid by rotation but would also lift the mate-rial and thus guarantee perfect circulation. Care was taken thatthe stirrer should make at least 300 revolutions per minute, andno exposure-interval was started until the material had been in

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IRRADIATION OF PLANT VIRUSES 227

motion for two to three minutes. Moreover, the location of thestem of the stirrer was so arranged that it was not in the path ofthe beam of incident light.6 This cell was used for ajl work downto X2652A. The absorption of the cell down to this line wasnegligible. The work at x2537A and the checking of the effectsat other lines were done in a cell of novel design, with crystallinequartz sides, which will be described in a later paper.

Experimental procedure. The cell and stirrer were cleanedwith the concentrated sulphuric acid-dichromate cleaning solution

r;-EXIT SLIT OF MONOCHPOMR SC H E WV-TANK FILLED WiTH ICE WATER EXPOSURE CELL -THEIOPLEF- THERMOPLE TANK FILLED WiTH COTTON ARRANGEMENTH- EXPOSURE CELL (,ARTZ)X-LEADS TO GALVANOMETERT-CONDENSING LENSC -THERMOflLE CELLV- THIERMOPILEW- OUARTZ "INDOON

FIG. 2. EXPOSURE-TANK, CELL, AND THERMOPILE ARRANGEMENT

and they were then washed at least 10 times with distilled waterand at least twice with sterile distilled water. The cell was filledwith distilled water and placed at H in front of the thermopile inthe tank containing ice water, the entire set-up being obviousfrom the illustration (see fig. 2). The calculation proceeded asfollows: First, there were measured the values of the energytransmitted by the cell filled with water; secondly, values were

6 Mr. W. H. Bauer of the Department of Physical Chemistry did the quartzblowing required, assisted in some of the apparatus set-ups, and in some of theexposures of material.


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obtained (1) by measuring the energy transmitted by the cellfilled with the standard suspension, (2) subtracting such valuesfrom corresponding values of the water-filled cell, (3) dividing thisresult by the number of cubic centimeters of suspension in the cell,and (4) multiplying this quotient by the magnitude (in seconds)of the exposure-period.

Controls, employed with every experiment, were handled in amanner identical with those of the exposed, except for the com-plete protection from irradiation. The absorption of the differ-ent batches of exposure-materials as prepared for the severalexperimental series varies somewhat. These variations are as-cribable to the virus suspension, that is, to the influence of theenvironmental conditions under which the diseased plants aregrown on the color of the plant juice. Since the virus materialbefore exposure had been diluted to 1:100, small changes werenot very obvious in the absorption spectrum. If the purifiedand undiluted virus suspension exhibited too much color, it wasusually discarded and a new lot of fresh material prepared. Evensmall changes in the absorption spectrum of the material to beinvestigated for the influence of monochromatic light are not ideal,even though the material may appear clear and colorless to theuntrained eye. So long as there are no better methods for purify-ing the virus suspension we had to be satisfied.

In order to make the results as comparable as possible, eachset of experiments was run with one batch of exposure-suspen-sion, kept in crushed ice during the entire interval. The absorp-tion of the material (stored in an ice water bath) did not changein sixty hours sufficiently to appear in the absorption readings.For best results the entire spectrum was investigated from onebatch of material, this requiring a continuous sequence of ex-posures for a period as long as sixty-five hours.

TREATMENT OF SUSPENSIONS AFTER EXPOSURE AND DETERMINA-

TION OF LETHAL EFFECTS

After exposure, the irradiated suspensions and the controlmaterial were pipetted into sterile test tubes and the pluggedtubes held in crushed ice. From each tube of the exposed sus-

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pension (representing one interval of exposure) and of the control,unirradiated suspension, there were prepared two series of inocula,the one to be used in making a dilution series of poured platesfor bacterial counts and the other for inoculation of tobacco plantsas a means of determining the extent, if any, of virus inactivation.Poured plates and counting the bacteria. The "original" of the

exposure-dilution and of the unirradiated control, each contain-ing around 1,000,000 bacteria per cubic centimeter, of each ex-posure interval, was designated No. 1. From each No. 1, assoon as removed from the ice water bath, a series of dilutions wasaccurately and aseptically prepared in the physiological salt solu-tion previously described, and corresponding agar plates werepoured.

In order to insure the highest accuracy in the bacterial countsthe greatest care must be taken in mixing or shaking, adequateshaking being given in every case prior to removing a pipettesample either for further dilution or for transfer to the correspond-ing Petri dish. Ordinarily we used dilutions Nos. 3, 4, 5, and 6.Transfers of 1 cc. each were accordingly made to a duplicateseries of Petri dishes, and the agar poured, as usual, at 43 to 45°C.Standard potato glucose agar was used, at pH 6.5 to 7, althoughthe organism grows well on various nutrient agars.

Serratia marcescens grows rapidly at 24 to 28°C., with readilyidentifiable colonies, and with little spreading of the surface col-onies if the agar is moderately hard, so that the counts weremade ordinarily after forty-eight to twenty-four hours, the entireplate being carefully counted.

Since a control series was arranged for each of the longer ex-posures and one control for each of two of the shorter exposures,the "lethal effect was directly determined as the percentage re-duction in the number of colonies in the exposed compared withthe control. It appears that this dilution plate count method isas quantitative as it seems practicable to apply in bacteriologicalwork. Nevertheless, exact knowledge of the behavior of theexperimental organism is a necessary preliminary to careful work.

Inoculation with the virus. Since the only available method ofdetermining inactivation of this virus is by the decrease in the

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incidence of disease induced when the virus is inoculated into asusceptible host, it was necessary to use such a host, yieldingcharacteristic symptoms. An established strain (WisconsinHavana 142) of cultivated tobacco was selected and growni inpots in the greenhouse for this purpose. For each exposure andsimilarly for each control a lot of not less than twenty plants wasemployed, ten plants of each lot being inoculated at the exposure-dilution (1:100) of the virus and ten plants at greater dilution(1:1000). The latter dilution usually affords, with a standardneedle-prick method of inoculation 100 or 90 per cent of disease.At a dilution of 1:100 it is seldom that inoculation fails to yield100 per cent infection, although during extreme weather infectionmay be erratic, and under unfavorable growth conditions thesymptoms may be temporarily masked.

It is obvious that since the number of plants practically avail-able for inoculation must be limited, the degree of accuracy tobe expected in the virus work cannot approach that attainablewith the bacteria used for comparison. There is also the inocu-lation factor to be considered. We have invariably repeated anytests made during midsummer, or those otherwise demandingconfirmation. Nevertheless, it is to be recognized that the valuesfound for the virus agent are reproducible only within the limitsof the error of inoculation.

RESULTS AND DISCUSSION

To illustrate the results obtained in respect to the lethal actionof the irradiation, a table for one set of experiments, X2652A, isgiven (table 1). This table exhibits under the heading "incidentenergy" the amount of energy which strikes the exposed material;and under "absorbed" is given the energy which is lost by thebeam in passing through the exposure-cell and before reaching thethermopile. These values are corrected for the absorption andreflection of the windows, the latter being the largest source oferror when not taken into account. In addition, there are twofactors which modify very slightly the values given, and thesewill not be evaluated in this paper. The factors referred to are(1) the light scattered by the material exposed, and (2) the ex-

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hibition by the exposure-material of a faint bluish white fluores-cence. In further explanation of the first factor it may be saidthat the virus particles (or the material carrying the virus), thecells of the bacteria, and such other particles as are furnished bythe very dilute bouillon and virus fluids are all jointly concerned.The factor of fluorescence is an extremely small one and is usually

TABLE 1Radiation of virus-bacterial suspension, at X2652 A, employing a monochromato

with fused quartz optics and a capillary mercury vapor lamp as the source ofradiation

ENERGY LETHAL EFFECTSTIME OFEXPOSURE Bacteria Virus inactivated

Incident Absorbed killed1:100* 1:lOOOt

8econds X 103 ergs X 103 per ergs X 103 per per cent per cent per centCC. CC.

0.06 12.82 9.6 700.16 36.12 27.2 930.20 46.38 34.78 99.70.23 54.93 41.2 99.980.26 64.59 48.4 1000.30 53.8 40.4 100 0 00.60 112.4 84.3 100 0 01.2 225.32 181.9 100 0 01.8 367.12 289 100 0 02.4 530.62 412 100 0 01.2 734 551 100 0 602.4 1,468 1,102 100 20 604.8 2,936 2,204 100 30 907.2 4,404 3,306 100 60 10010.8 6,606 4,959 100 100 100

* This being the exposure dilution, the same dilution was also used for inocula-tion.

t Exposure-dilution was diluted to 1:1000 for inoculation.

ignored. Furthermore, while we consider that our results ap-proach absolute values, they are primarily intended to be quan-titatively comparative. In particular relation to this last state-ment, it should be noted that even though we worked with aconsiderable number of wave-lengths, actually the effective spec-tral range, or range of characteristically strong lethal effect (with-in the limits investigated by us), is confined to a very narrow region

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232 B. M. DUGGAR AND ALEXANDER HOLLAENDER

of the spectrum; and since the difference in the amount of scat-tering relative to wave-length is small, it can be wholly ignoredin this comparative study.

Regarding the effect of wave-length on the virus, our resultsindicate that lethal action (inactivation) is confined to wave-lengths shorter than about 3100A. At approxinmately this wave-length about 100 times as much energy was used as at X2652A.At such high values, there was probably some scattered light.It will be noticed that the energy values representing 100 per

-c

0

Ergs X jQ6FIG. 3. PERCENTAGE INACTIVATION OF TOBACCO VIRUS, EXPOSED AT 1: 100

DILUTION, DILUTION OF 1:1000 AIADE AFTER EXPOSURE AT X 2652 A

cent killing of the bacteria are far below the values having anymeasurable influence on the virus.With the bacteria lethal effects are also primarily confined to

wave-lengths shorter than about 3100A, but we are not yet pre-pared to say that there are no effects at longer wave-lengths. Infact, with energy 50,000 times what was needed to give killingeffects at X2652A we were able to show, with Serratia miiarcescens,some killing effect in certain wave-lengths of the visible spectrum,in spite of the fact that we took all practicable precautions toexclude ultraviolet light, using a monochromator with glass optics,a liquid (carbon bisulphide) prism, and in addition several filters


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of window glass. It is proposed to investigate further this par-ticular relation, that is, wave-lengths in the range of the visible.As presented in figure 3 the graphs show, in a typical manner,

the lethal effect of the irradiation of the virus at X2652A. At-tention is drawn particularly to the virus behavior at concentra-tion 1:100. Representing this concentration two curves aregiven for percentage killing effects, the one curve displaying inci-dent energy, and the other the energy absorbed. At virus con-centration 1:100 the results, as would be expected, are less sub-ject to error through the inoculation procedure. There are alsopresented in the same figure corresponding curves for the virusdilution 1:1000, and here, as previously indicated, the reliabilityof the results is somewhat lessened. Moreover, variability inthe results is always slightly intensified with increasing lethaleffect of the wave-length, so that at X2652A we obtain our greatestdivergencies.

In the case of the virus the middle part of the curves invariablyoffers the greater satisfaction. This, however, would be truewhether dealing with the lethal effects of irradiation, of tempera-ture, or of toxic agents. On the one hand, a slight effect meansone or two healthy plants in a lot of ten or twenty; whereas apronounced inactivation of the virus means one to two diseasedplants in a similar lot. Intermediate effects obviously furnish amore reasonable number of plants in each class, and accordinglybetter values are obtained.The same type of curve displayed in figure 3 has been obtained

for the various wave-lengths investigated with which lethal effectswere obtained. These curves are not presented in detail, butsmoothed curves for the virus at 1:100 and 1:1000 are given infigure 4, exhibiting the incident energy employed. In order topresent clearly the results, points taken from the curves in figure4 above are used in figure 5 to portray for the virus the behaviortowards wave-lengths in relation to 25, 50, and 75 per cent inac-tivation. In the same figure are given the killing effects withSerratia marcescens of the various wave-lengths-based on thedeterminations in the presence of the virus-and using points

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corresponding to 70 per cent killing. It will be seen that maxi-mum inactivation occurs at X2652 A.

Attention is drawn to the dotted lines for X2537 A, which wereobtained under somewhat different conditions, and accordinglyare represented here only tentatively. The relation to this wave-length will be treated more in detail in a subsequent paper.

Contrasting the radiation results as between bacteria and virus,the energies required at the effective wave-lengths to produceequivalent lethal effects when the agents are irradiated coin-

1 73 q I- 1 4S 7 J9-0

I ~~/0 /01

FIG. 4. I-NACTIVATION OF TOBACCO VIRIJS AT ALL EFFECTIVE WAVrE-LENGTHS

cidently in the same suspension are 200 times as much for thevirus as for the bacteria.For a quantitative interpretation of the results from the stand-

point of the work with the virus, it is desirable to compare theabsorption coefficienits at the several wave-lengths with the cor-responding lethal effects of these wave-lengths. The curvesrepr-esenting these values showv a close agreemen-t down to X2652A. At X2537 Al the lethal effect again decreases, while theabsorption coefficient increases very rapidly, this increase beingprobably related to the coloring matters present, as previ-ously indicated. Accordinigly, this does not initroduce as large anerror as might at first appear. Iincidentally it will be observed

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that the values reported relative to wave-length are consistentfor the bacteria, for example, with values which have been re-ported where the bacteria alone were irradiated, as by Gates(1929), and by Ehrismann and Noethling (1932).Results of very different types have been obtained by various

investigators working primarily with certain animal viruses andthe bacteriophage. Many of the investigations here referred tohave been essentially general and qualitative. The apparatus

3100

<zqoo -

'32700-

m7rcescens -(a) V- r us--b)(c)(d)X 10, E i-gs X 10',

FIG. 5. TWENTY-FIVE, 50, AND 75 PER CENT INACTIVATION OF ToBACCO VIRUSAND 70 PER CENT KILLING EFFECT OF SERRATIA MARCESCENS

Note different energy scales

and techniques employed were not in those cases intended todetermine the influence of monochromatic light, nor were meas-urements of intensities given. In certain instances killing effectswith viruses were reported under conditions approximating thoseinducing bactericidal action. With respect to plant viruses,Mulvania (1926) and Smith (1926), working with leaf extractscontaining the virus of tobacco mosaic, found inactivation of thevirus when exposed to the full spectrum of the mercury arc.Using a purer preparation, Arthur and Newell (1928) found in-


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activation of the same virus at wave-lengths shorter than X3000A. For the most part, however, the results do not warrantdefinite comparisons.From results obtained by Olitsky and Gates (1927) it is ap-

parent that under the influence of monochromatic radiation theenergy values required to kill Staphylococcus aureus and to inac-tivate the virus of vesicular stomatitis are of the same order,and that X2675 A is most effective for both virus and bacteria.

80

i 15 2500 21,00 2700 2800 2900 3000\Wave Lenqtin A

FIG. 6. RELATIVE BIOLOGICA4L EFFEC1TS WITH RE.SPECT TO WA\ E LENGTH, IN THE1ULTRAVIOLET B3ELOW A 3100 A, SELECTED FROM VARIOUJS AUTHORS

(See text)

The materials were separately irradiated on the surface of agarplates. Similarly, Rivers and Gates (1928), using the same agarsurface technique and monochromatic light, report that the curvesof inactivation of vaccine virus approximate those exhibitingkilling effect with Staph. aureus, the maximum killing effectbeing at X2675 A. Baker and Peacock (1926) and Baker andNanavutty (1929)Temployed a suspension method, diffusing bothagents in the same saline, exposing in an open dish to the fullspectrum of the mercury arc, and stirring the suspension merelyby means of a jet of air. Their results indicate that the suscep-

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tibility of the bacteriophage is of the same order as that of B.coli, while the resistance of the virus of Rous's chicken sarcoma isapproximately five times as great as that of the bacteriophage.

In view of the fact that, considering relative energy values,the curves for bactericidal action and virus inactivation, relativeto wave-length, are of the same general form, it is of interest tocompare these curves with curves representing the results of otherinvestigators using different biological materials. Accordingly,there are displayed in figure 6 graphs illustrating (in comparisonwith our data for virus and bacteria) protein coagulation, fromSonne (1928); lethal effect on paramecium, Weinstein (1930),erythema production, Hausser (1928) and Coblentz, Stair, andHogue (1931), and hemolysis, Sonne (1928). It will be seenthat the curves representing bactericidal action, virus inactiva-tion, lethal effect on paramecium, and protein coagulation (seealso Rivers and Gates, 1928) are of the same general type. Thisstriking similarity will be discussed more at length in later publi-cations.

SUMMARY

The physical installation used has included a quartz mono-chromator, an intense source of radiation (Daniels-Heidt capillarymercury vapor lamp), a quartz exposure-cell (mechanicallystirred), an exposure tank provided with a quartz window, and asensitive thermopile. Twelve spectral lines (or groups of lines)were investigated in the range X2537-6120 A. The temperatureof the exposure was maintained at 1 to 2°C. by means of meltingice.The biological materials consisted of a fresh suspension of semi-

purified tobacco virus and of cells of S. marcescens taken duringthe logarithmic growth phase from a bouillon culture. For thedetermination both of lethal effects on bacteria and of inactiva-tion of the virus, materials were combined in the same suspension,so that comparative values might be obtained. Exposed ma-

terials were accompanied by controls similarly treated, except asto protection from radiation.

Dilutions of the bacteria were prepared in physiological salt

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solution. Poured plates (agar) were made and the counts gave aquantitative comparison of the irradiated cultures with the un-irradiated controls. The percentage inactivation of the viruswas determined by inoculation of tobacco plants and by compar-ing the incidence of disease induced-exposed versus unirra-diated.

Inactivation of the virus is confined to wave-lengths shorterthan about X3100A. The energy required to produce any per-ceptible effect at approximately x3100A is more than 100 times asmuch as is necessary at X2652A. The energy values represent-ing 100 per cent killing of the bacteria are far below the valueshaving any measurable effect on the virus. For both of thesebiological materials, using the range of wave-lengths stated, thegreatest influence is at X2652A. The resistance ratio of virusto bacteria, as represented by these results, is about 200:1.Relative to wave-length, graphs of certain "biological effects,"collected from various authors, show striking resemblances.

REFERENCES

ARTHUR, J. M., AND NEWELL, J. M.: Amer. Jour. Bot., 1928, 15, 623.BAKER, S. L., AND NANAVUTTY, S. H.: Brit. Jour. Exp. Path., 1929, 10, 45.BAKER, S. L., AND PEACOCK, P. R.: Brit. Jour. Exp. Path., 1926, 7, 310.BAYNE-JONES, S., AND VAN DER LINGEN, J. S.: Bull. Johns Hopkins Hosp., 1923,

34, 11.BIEMOND, A. G.: Zeit. f. Hygiene, 1924, 103, 681.COBLENTZ, W. W., AND FULTON, H. R.: Scientific Papers, Bureau of Standards,

1924, No. 495, 641.COBLENTZ, W. W., STAIR, R., AND HOGUE, J. M.: Bureau of Standards, Jour.

Res., 1931, 7, 723.DANIELS, F., AND HEIDT, L. J.: Jour. Amer. Chem. Soc., 1932, 54, 2381.DUGGAR, B. M., AND HOLLAENDER, A.: Science, 1932, 75, 567.EHRISMANN, O., AND NOETHLING, W.: Zeit. f. Hygiene und Infektionskr., 1932,

113, 597.FINSEN, N. R., AND DREYER, G.: Mitt. aus Finsens Mled. Lysinstitut., 1903, 3,

72.FISHER, R., AND MCKINLEY, E. B.: Jour. Inf. Dis., 1927, 40, 399.GATES, F. L.: Jour. Gen. Physiol., 1929, 13, 231.GATES, F. L.: Jour. Gen. Physiol., 1929, 13, 249.GATES, F. L.: Jour. Gen. Physiol., 1930, 14, 31.GILDENMEISTER, E.: Centralbl. Bakt., Abt. I, 1922, 89, 181.GILDENMEISTER, E., AND HERZBERG, K.: Centralbl. Bakt., Abt I, 1923, 91, 228.HAUSSER, K. W.: Strahlentherapie, 1928, 28, 25.HEIDT, L. J., AND DANIELS, F.: Jour. Amer. Chem. Soc., 1932, 54, 2384.

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MCKINLEY. E. B., FISHER, R., AND HOLDEN, M.: Proc. Exp. Biol. and Med.,1926, 23, 408.

MIZUNO, K.: Japanese Jour. Med. Sci. (Part VI, Bacteriology and Parasitology),1929, 1, 53.

MULVANIA, M.: Phytopath., 1926, 16, 853.OLITZKY, P. K., AND GATES, F. L.: Proc. Exp. Biol. and Med., 1927, 24, 431.RIVERS, T. M., AND GATES, F. L.: Jour. Exp. Med., 1928, 47, 45.SMITH, F. F.: Ann. Missouri Bot. Gard., 1926, 13, 425.SONNE, C.: Strahlentherapie, 1928, 28, 45.WEINSTEIN, I.: Jour. Opt. Sci., 1930, 20, 433.WYCKOFF, R. W. G.: Jour. Gen. Physiol., 1932, 15, 351.ZOELLER, C.: Compt. Rend. Soc. Biol., Paris, 1923, 89, 860.


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they lingen (1923), '29a, '30), wyckoff (1932),...

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