THE BACTERIOPHAGY OF A SPORE-FORMINGORGANISM

PHILIP B. COWLESDepartment of Immunology, Yale University School of Medicine, New Haven,

Connecticut

Received for publication January 29, 1930

Since the discovery of the first bacteriophage by d'Herelle in1916 there have been isolated many races virulent for a widevariety of bacteria. It is an interesting fact, however, that of theorganisms for which specific races of bacteriophage havebeenfound almost all are non-sporulating. d'Herelle (1926) himselfmentions the discovery of a bacteriophage for B. subtilis, butreports no study of it. Koser (1926-27) isolated one for a thermo-philic spore-forming rod, but was interested in it chiefly from thetemperature standpoint. More recently Adant (1928) hasdescribed a bacteriophage active against several strains of B..subtilis.

In view of this scarcity of information about bacteriophagyamong spore-forming organisms, and since a study of this problemmight conceivably throw new light on the lytic process and,perhaps, upon sporulation as well, it seemed of interest to attemptsome investigations in this field. This paper is concerned with adescription of two lytic principles active against B. megatherium, acommon aerobic spore-former.

B. megatherium was selected for study because, in contrast tomost similar organisms, it grows with diffuse turbidity in brothand develops on agar with a relatively homogeneous growth whichis easily emulsified. Moreover, it is one of the largest of thebacteria, and as such lends itself especially readily to microscopicobservation, even with so low a magnification as that afforded bythe 16 mm. objective. A stock laboratory strain (no. 1) wasused in the isolation of the bacteriophages and in all routine

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work, but six other strains were found to be susceptible. Theseincluded another laboratory strain (no. 2), a culture labeledN.Y.U., the American Type Culture Collection strains nos. 72 and270, as well as two strains (nos. 20 and 20A) obtained from Dr.W. W. Ford. All produced, on agar and potato, the creamywhite or yellowish growth, later changing to brown, which ischaracteristic of the species. The Type Culture Collectionstrains nos. 71 and 943, listed as B. megatherium, were not sen-sitive to the bacteriophage, and on agar did not develop as thisorganism usually does.

In all routine work young organisms from an eighteen- totwenty-four-hour agar slant were used. The culture mediaregularly employed consisted of alkaline extract broth, pH 7.8,and nutrient extract agar of pH 7.0. Filtrations were madethrough L5 Chamberland candles, and all cultures and culture-phage mixtures were incubated at 30°C.

ISOLATION OF LYTIC PRINCIPLES

Sewage was used as a possible source of lytic principles. Thiswas first filtered through paper to remove the coarser particles,and then 90 cc. of this filtrate were added to 10 cc. of broth madeup in ten-fold strength. The reaction of the mixture was testedand, when necessary, sufficient N/10 sodium hydroxide wasadded to bring the pH to about 7.6, after which the material waspassed through a candle. Ten cubic centimeter amounts of thisclear filtrate broth were inoculated with enough young mega-therium organisms to produce distinct turbidity, and the culturewas incubated. Control broth cultures of the same turbiditywere prepared each time, in order that any differences in growthmight be observed. After twenty-four hours the sewage-culturemixture was filtered, and the filtrate inoculated and incubated.This process was repeated dafily, with the addition of sterilebroth when needed to replace the loss in filtration. The firstthree passages showed no difference in turbidity from that of thecontrols, nor did 0.1 cc. amounts of the filtrate spread overheavily seeded agar plates give visible plaque formation. Thefourth transfer, however, seemed slightly less cloudy than the

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control, and on plates the filtrate from this transfer showed manyareas somewhat more translucent than the body of the culture.Successive passages resulted in the rapid development of lyticpower, and the tenth filtrate produced complete clearing of brothsuspensions in a dilution of 10-6, as well as,-in proper dilution,-clearly defined plaques on agar.By the use of the same method, there was obtained from another

sample of sewage, several months later, a second lytic filtrate.It may be mentioned that twenty successive passages in which

B. megatherium was grown in its own broth culture filtrates failedto show the development of any lytic principle whatsoever. Mostof the criticisms directed against experiments which have ap-parently shown the "spontaneous generation" of bacteriophagehave stressed the fact that the original culture perhaps containeda minimum amount of the latent principle. By using sporesheated at 90°C. for fifteen minutes, as was done in this case, anybacteriophage present was inactivated, and this criticism couldbe avoided.The two lytic filtrates seemed to be essentially alike. They

were active in the same dilutions, produced similar plaques onagar, and were heat labile. Fifteen successive passages of filtrates,which had been heated at 80°C. for ten minutes, failed to showany regeneration of lytic power, and other tests showed that atemperature of 75°C. was sufficient to inactivate them. Thissensitivity to heat is wholly in accord with what is known aboutthe behavior of other bacteriophages, and, together with otherpoints of similarity, is strongly suggestive that these lytic prin-ciples are of the same nature as those for non-spore-formingbacteria.No secondary growth has as yet been observed in filtrates

prepared in the usual way and kept at room temperature forthree months. In broth cultures which have undergone ap-parently complete lysis, however, secondary growth may occur,sometimes within several days, and sometimes not until weekshave passed. This growth is usually scanty in amount andflocculent in character, made up of what appear to be typicalorganisms.

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The fact that the bacteriophage is destroyed by heat makes itpossible at any time to eliminate it from a culture, providedspores are present, as is usually the case. Abundant growthresults when a clear, lysed culture is exposed to a temperature of80°C. for ten minutes.

Spores do not seem to be actively attacked by the bacteriophagebut will not germinate in its presence. Microscopic observationof spore-phage mixtures on agar shows no change in twenty-four

FIG. 1. j3 PLAQUES, 2 DAYS, SHOWING PLAQUES WITH ONLY A SUGGESTIONOF ZONE FORMATION. 3 X

hours, and inoculations of spores from such mixture into brothfail to develop on account of the bacteriostatic action of thebacteriophage carried over with them. Spores seeded on thesurface of an agar plate which had previously been flooded withbacteriophage and dried, failed to grow, but when inoculationswere made from such a plate into broth, and then heated, abun-dant growth occurred. If duplicate bacteriophage titrations areprepared, containing as indicator in one case a visible number of

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young megatherium organisms, and in the other so few sporesthat no turbidity is produced, and then incubated, they willpresent the same appearance. In the first case the turbidity willhave disappeared in the first six tubes (10-6), for instance,although persisting in the last tubes, while in the second case thefirst six tubes will remain clear, as the bacteriophage has inhibitedthe development of the spores, and growth will have occurred inthe others. This aspect of the problem will receive further study.

_E, -- --; o .... >. ..e-* ... ,.ri

d X '@ 'R'R ,.w j

,.g eFs '' w ;t

*fi&.' 2'S

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FIG. 2. a PLAQUES, 1 DAY, SHOWING DEFINITE DIFFUSION ZONES. 3X

It was stated above that the plaques formed by both lyticfiltrates are similar. This is true, but in each case these plaquescan be divided into two main types which will be referred to forconvenience as the a and i8 forms. The clear plaque proper withboth types is 0.5 mm. or less in diameter, and does not increasein size upon prolonged incubation. The a principle, however, ischaracterized by the ability to diffuse from the plaque throughthe surrounding culture with the production of a translucentzone, which, upon incubation for a week or two may attain a

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width of 10 mm. or more. During the first few hours of incuba-tion the zone formed may be so clear as to suggest that it is identi-cal with the plaque, but microscopic observation reveals thepresence of numerous organisms, increasing in number as thedistance from the true plaque increases. The outer parts of thisdiffusion zone, at any stage of its development, are composed ofmasses of spores with occasional vegetative rods, and shadowforms.The s principle, as a rule, does not show the presence of a

diffusion zone, and microscopic observation of a ( plaque reveals amore sharply defined boundary than is seen with the a plaque.

FIG. 3. a PLAQUES, 5 DAYS, SHOWING EXTENSIVE DIFFuSION OFBACTERIOPHAGE 3 X

Both principles can be recovered from their respective plaquesor from any portion of the zone, when this is present, and, as faras can be told, both principles continue to breed true.

It should be borne in mind that all of these descriptions referto observations made on only one strain of B. megatherium grow-ing on the usual nutrient extract agar. Numerous observationshave indicated that the strain of culture used and especially theamount of nutritive material available have pronounced effectsupon the process of bacteriophagy. Details regarding theintimate behavior of the two types of bacteriophage under varyingconditions and with diverse strains of B. megatherium will bepresented in a later paper.

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MICROSCOPIC OBSERVATION OF LYSIS

As has been mentioned,> the large size of B. megatheriumfacilitates microscopic study and, hence, permits the lytic processto be followed more readily than is possible with the smallerbacteria which have heretofore been used.In preliminary experiments the ordinary hanging drop was

tried, but the active motility of the organism rendered thismethod unsatisfactory. As a substitute the following technicwas employed. A drop of melted agar is spread over the surfaceof a sterile cover-slip and allowed to harden. A small amount ofbroth containing both megatherium cells and bacteriophage isnext placed on the agar and dried. The cover-slip is theninverted on a concave slide and sealed with vaseline, after whichthe slide is placed on the warm stage under the 4 mm. objectiveof a microscope.When a suitable field, containing perhaps a dozen or more cells,

is found, it is charted on paper for easy reference and thenobserved at intervals until growth is apparent. This occurs, as arule, in from one to three hours, and at about the same timelysismay be seen. Generally lysis is not evident before actual celldivision has been noticed, although at times a single organismmay vanish before there has been any apparent growth. Thedissolution of the bacteria is not instantaneous. Instead, theorganism seems to become more and more transparent, it cannotbe focused sharply, and, finally, in from five to ten seconds afterthe first change has been noticed, it fades away entirely. Thereis no apparent gradual decrease in size such as exists when asubstance dissolves, and there is no visible residue, althoughoccasionally there may be seen, occupying the site of the cell, afaint shadow which usually vanishes within a few minutes.It is worthy of note that in numerous observations of the lysis ofB. megatherium there has never been seen any swelling or rupturesuch as have been reported with other bacteria.The following observations, typical of many others, may be

briefly described. A single organism was watched while it grewinto a chain of three. An end cell of this chain underwent lysis,

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but the two remaining cells continued to divide, growing over thespot previously occupied by the cell which had vanished. Whenthis chain had grown to comprise five cells it broke apart and atlength another end cell disappeared, twenty-five minutes afterthe first had gone. The end cells need not be the first affectedhowever. A chain of four or more may undergo lysis as a whole,or the middle one of a series may succumb. Sometimes one cellof a pair will disappear, while the other will resist the lytic actionfor many minutes. Frequently a typical raft of many cells willdevelop, only to undergo lysis eventually, both from within andwithout.A preparation in which bacteriophage was acting on organisms

from a four-day agar culture contained many cells which failedto grow. Some of these seemed to be susceptible to the lyticagent, but required periods of several minutes to an hour betweenthe initial change and final dissolution. Heat killed organismswere observed for twenty-four hours without the appearance ofany lysis.

All of these facts are quite in accord with the usual statementsthat the most actively growing organisms are the most susceptibleto the bacteriophage, while old cells are less so, and dead cells,except perhaps in the presence of living ones, are not affectedat all.

SUMMARY

The isolation of two bacteriophages specific for B. megatherium,a common aerobic spore-forming orgam, is described. Variousstrains of this organism, seven in number, obtained from differentsources, were susceptible to the lytic action.Each bacteriophage contained two principles, one of which was

characterized by the ability to diffuse from the initial plaqueand cause changes in the surrounding culture.

Spores seem to be resistant to the action of the bacteriophage,but are inhibited from developing by its presence.The lytic principles were destroyed by exposure to a tempera-

ture of 750C. for ten minutes, and could in this way be eliminatedfrom cultures containing organisms in the spore stage.

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BACTERIOPHAGY OF SPORE-FORMING ORGANISM 23

The lytic process as seen under the microscope is described.B. megatherium does not, apparently, swell or burst in lysis butrather fades from view.

REFERENCES

ADANT, M. 1928 Bacteriophages actifs pour des Bacilles du groupe subtilis.Compt. rend Soc. de biol., 99, 1246.

D'HERELLE, F. 1926 The Bacteriophage and Its Behavior. Tr. by Geo. H.Smith. Baltimore.

KOsER, S. A. 1926-1927 Action of the bacteriophage on a thermophilic bacillus.Proc. Soc. Exper. Biol. and Med., 24, 109.

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