(rakieten, gram-negative non-specific phagesdepartmentofbacteriology, longislandcollegeofmedicine,...

RELATIONSHIPS BETWEEN STAPHYLOCOCCI ANDBACILLI BELONGING TO THE SUBTILIS GROUPAS SHOWN BY BACTERIOPHAGE ABSORPTION'

MORRIS L. RAKIETEN AND TONY L. RAKIETENDepartment of Bacteriology, Long Island College of Medicine, Brooklyn, New York

Received for publication April 8, 1937

Experimental evidence has established the fact that with fewexceptions bacteriophages are absorbed by susceptible bacteria,either living or dead, (d'Herelle, 1920; Bail and Okuda, 1923;Prausnitz and Firle, 1924; Burnet, 1930). Exceptions have beennoted by Flu, 1923; Gohs and Jacobson, 1927; Levine and Frisch,1934; Burnet, 1934; and ourselves. The studies of Burnet (1929-34) have demonstrated that absorption of phage is largely deter-mined by the nature of the surface antigens of the bacterial cell.This concept has been strengthened by recent investigationsdealing with bacterial extracts, first reported by Levine andFrisch (1933--34). Extracts prepared from susceptible bacteriahave the power of inactivating phage, while those of lyso-resistantstrains lack this property. The majority of reports dealing withphage absorption or inactivation have resulted from evidenceobtained by a study of the enteric group of bacteria and theirphages.

Staphylococcus phages behave in a similar manner towardsusceptible cultures and extracts derived from them, (Rakieten,et al., 1936). However, toward such agents as serum and whiteblood cells staphylococcus phages in general differ from thoseattacking the Gram-negative organisms in that they are renderedinactive to a varying degree. This so-called non-specific absorb-ing power of body fluids and exudates for staphylococcus phagesmay be modified to a point of practical extinction by a method

1 Presented in part at the annual meeting of the Society of American Bacteriol-ogists, December 1936, New York City.

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286 MORRIS L. RAKIETEN AND TONY L. RAKIETEN

entailing adaptation of staphylococcus phage to serum (d'Herelleand Rakieten, 1935). Staphylococcus phages also differ fromcoli and dysentery phages in that they may be absorbed by or-ganisms other than susceptible cocci, namely bacteria belongingto the subtilis group. Such organisms as far as can be deter-mined are not affected by their contact with staphylococcusphages; the phage corpuscular count, however, is diminishedenormously. No absorbing action, on the part of these Gram-positive bacilli, was ever demonstrated against coli, dysentery,proteus, or streptococcus phages. The present study deals withthe staphylococcus-phage-absorbing ability of certain membersof the subtilis group and a discussion of the mechanism respon-sible.

In 1933 a tube of polyvalent staphylococcus phage becamecontaminated while standing on the laboratory table. Onrefiltering, the previously active phage was without lytic orplaque-producing power. The contaminant was an activelymotile, large, Gram-positive rod. In Savita broth a slight tur-bidity and a firm wrinkled pellicle develops in twenty-four hoursat 350C. The same characteristics are noted when the organismis allowed to grow at room temperature. Gelatin is liquefiedactively, and hemolysis is produced on blood agar plates. OnSavita agar the organisms produce dark brown to black colonies,and over a varying period of time the pigment diffuses into theagar, coloring it brown. The colonies are flat, dry, and wrinkled,though, very infrequently there have developed black, raised,and mucoid colonies. Associated with this temporary changethere has been a decrease in the phage-absorbing quality of theorganism. Central spores may be observed when growth occursafter twenty-four hours at incubator temperature in meat-extractpeptone broth and on Savita agar, but in Savita broth sporesappear only after several days incubation. This particular strainof B. subtilis (niger) hereafter designated as strain C3 has beenstudied with regard to its staphylococcus-phage-absorbing ability.While we have isolated 27 other strains belonging to the subtilisgroup and examined them for phage absorbing power, to a largeextent our interest has centered on culture C3. Experiments

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STAPHYLOCOCCI AND BACILLI OF SUBTILIS GROUP

with the few other subtilis strains that possess this characterlead us to believe that a mechanism similar to that of strain C3is responsible for this action.

EXPERIMENTAL

Absorption of staphylococcus phages by strain CSTo tubes of broth (approximately 10 cc.) 0.1 cc. of a twenty-

four-hour culture of strain C3 and 0.25 cc. of bacteriophage wereadded. The tubes along with proper controls were incubated at350C. At frequent intervals tubes containing culture and phage,as well as tubes containing phage alone were filtered through L5Chamberland candles, in an effort to find out at what period dur-ing the growth of the culture phage absorption occurs. Portionsof the filtrate were layered over segments of agar plates thatpreviously had been seeded with a susceptible culture of staphy-lococcus. After proper incubation the plates were counted forplaques. The protocol in table 1 summarizes the results obtainedwhen strain C3 was tested for its phage-absorbing ability when incontact with three polyvalent staphylococcus phages.

In each instance it may be observed that there is a drop incorpuscular count of staphylococcus phage after addition to atube containing strain C3. As bacterial multiplication begins,certainly within the third hour, a continued decrease in phageelements takes place until, within a relatively short period aftergrowth may be observed macroscopically, the phage titer dropsto such an extent that in the dilutions noted no plaques aredemonstrable. Under the same conditions, however, controltubes of phage alone show little if any fluctuation in titer. Re-peatedly, filtrates of phage and culture mixtures were tested forevidence of phage on several other susceptible cultures of staphy-lococci by the cross test (Asheshov, 1933), without positiveindication of lysis.

Since it appeared that a relation existed between bacterial cellnumbers and reduction in bacteriophage corpuscle count (con-trols showed that this was not associated with a significant changein pH) one should be able to demonstrate this almost immedi-ately, providing sufficient bacterial cells are added to a bacterio-

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phage. The following experiment was carried out to test thisassumption. A twenty-four-hour broth culture of strain C3 wascentrifuged at high speed for thirty minutes. The supernatantfluid (7.5 cc.) was removed and the sedimented bacteria resus-pended in the same volume of Savita broth. To this was added0.25 cc. of staphylococcus bacteriophage M13. Similar amountsof phage were added to the supernatant and a control tube ofbroth. All tubes were shaken vigorously for a few minutes andthen filtered through L5 Chamberland candles. A dilution of10- of each filtrate was then overlayed on an agar-plate culture

TABLE 1Plaque count after layering a dilution of 10-' of each filtrate on susceptible culture

Staphylococcus aureus Vnt.

IUMMDIATELY 30 1 HOUI 3 HOuRS 5 HOURS 24 HOURS

C3 plus Bp. M13.......175 201 256 112 6 0Control Bp. M13....... 500(ofi.) ofl. efl. cfl. cfl. cfl.

C3 plus Bp. P.G....... 197 142 211 17 0 0Control Bp. P.G....... 414 449 394 N.T. 401 408

C3 plus Bp. Lrk........ 206 115 111 185 80 0Control Bp. Lrk....... cfl. 458 417 489 cfl. cfl.

C3 control (growth intube-macroscopic)... _ _ _ 4 +4 +++

cfl. = plaques confluent;-= no evidence of growth; +++ = slight turbiditywith pellicle; N.T. = not tested.

of susceptible staphylococci. After an incubation period ofeighteen hours the plates were observed for evidence of plaques.The results are indicated in table 2.The results of many experiments carried out in the manner

described above with four staphylococcus phages lead us tobelieve that a relationship does exist between actual numbers ofbacterial cells of strain C3 and its capacity for absorbing staphy-lococcus phages. These results also compare favorably withthose summarized in table 1. The supernatant of a twenty-four-hour broth culture has no phage-binding power, under the condi-

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tions described. However, since the supernatant is not bacteria-free a marked absorption of the phage added to it occurs afterappropriate incubating intervals, when growth is observedmacroscopically.When undiluted filtrates of mixtures of phage and culture C3

were tested on susceptible strains of staphylococci (dilution beingresorted to for accuracy in counting) complete absorption ofphage was never observed, there remaining approximately 3 to5 per cent of the original number of phage corpuscles. Andrewes(1934) in a report dealing with the action of anti-phagic serumstated that it was possible to absorb only 95 per cent of the totalnumber of phage corpuscles. We have been able to corroboratethis interesting finding. Methods involving only the use of brothcultures would fail to show that any phage remained unneutral-ized, and it is specifically by the use of a method involving plaque

TABLE 2

PHAGE M13 IN CONTACT WITH STRAIN C3 FO3 TwO aMIUTS PLAQUE COUNT

Resuspended 24-hour culture of strain C3 plus 0.25 cc. of Bp. M13 0Supernatant of above suspension plus 0.25 cc. of Bp. M13......... 197Savita broth plus 0.25 cc. of Bp. M13............................. 181

demonstration that such unneutralized corpuscles may be de-tected. It appears that the absorption by strain C3 is compa-rable to that reported for neutralization by anti-phagic serum.

Filtrates of cultures C3 incubated from periods varying from24 hours to 27 days lack the ability to diminish staphylococcusphage counts. Unfiltered 20-day-old autolysates also fail toreduce the titer of staphylococcus phages. Spore suspensionshave no apparent effect on these same phages, but with thegermination of the spores and the subsequent increase in cellnumbers one observes the diminution in plaque count.The ability of strain C3 to absorb staphylococcus phages is not

lost when this culture is subjected to autoclaving at 15 pounds for15 minutes. An agar slant culture was suspended in 5 cc. ofsaline, and this suspension was autoclaved. A series of tubescontaining equal amounts of phage and progressively increasing

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amounts of autoclaved bacterial cells were set up and incubatedfor 20 hours at 350C. A dilution of 10-5 of each mixture withoutfiltering was overlayed on a susceptible culture, and after incuba-tion the plate was read for evidence and numbers of plaques.Table 3 gives the results obtained.One may show absorption of staphylococcus phage by the use

of dead bacterial cells providing attention is paid to the numberthat are utilized. The principle in the cells responsible for thisabsorption is heat stable. In a study dealing with the absorptionof staphylococcus bacteriophages by heat-killed susceptiblestrains of staphylococci and extracts derived from these strainsRakieten et al. (1936) also demonstrated that the absorbingqualities of such cultures and extracts were not impaired onautoclaving at 15 pounds for 15 minutes.

Strain C3 and twenty-one other strains belonging to the sub-tilis group have been tested for their ability to absorb severalraces of coli, dysentery, proteus and streptococcus phages. Noevidence of reduction in corpuscular count was ever obtained,even though some of these phages remained in contact with thesecultures for 72 hours. Furthermore, susceptible cultures ofEscherichia coli and Salmonella dysenteriae placed in broth contain-ing proliferating cells of any of these twenty-one strains of Bacillussubtilis are completely lysed by their respective phages, the result-ing growth being a pure culture of Gram-positive rods. One cannot however, prevent the absorption of staphylococcus phages byculture C3 by adding to this mixture an excess of coli, dysenteryor streptococcus phages.

All attempts to release and demonstrate staphylococcus phagefollowing absorption by strain C3 have failed. The followingmethods have been employed to show any evidence of phage thatmight be released.

1. Use of an indicator strain. This consists in touching, as isdone in the cross test (Asheshov, 1933), several strains of sus-ceptible staphylococci streaked on an agar plate, with a tiny loop-ful (loop 1 mm. diameter) of a mixture containing culture C3 andabsorbed phage. This method which does not employ filtration,when positive, and small quantities of phage are present, is indi-

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cated by a clear zone of lysis on the susceptible strains aroundthe growth of the superimposed contaminating culture. Thismethod is often utilized to determine whether colonies of second-ary cultures are free of bacteriophage. No evidence of bacterio-phage was obtained when cultures of C3 which had absorbedstaphylococcus phages were tested by this method.

2. The addition of a susceptible culture of staphylococci to a cul-ture of CS following absorption of a bacteriophage. All attempts toshow that the added culture of staphylococci had been in contactwith phage and therefore resistant, or partially so, were negative.

3. A culture of strain C3 which had absorbed bacteriophagewas spun in the centrifuge at 3000 r.p.m. for three hours; the

TABLE 3Absorption of bacteriophage M1: by autoclaved culture of strain CS

AMOUNT OF AUTOCLAVECULTURE AMOUNT OF Bp. M13 ADDED PLAU COUNTSUSEP

ADDED TO CC. OF BROTE

CC. CC.

0.1 0.25 1940.2 0.25 1960.5 0.25 1820.7 0.25 1611.0 0.25 312.0 0.25 00 0.25 Over 500

supernatant and the sediment were then tested for any trace ofphage by a plaque method. No positive evidence of any disunion of phage was ever obtained.

4. Attempts to produce anti-staphylococcus bacteriophagic serumby injecting rabbits with cultures of strain CS and absorbed phage.It was thought that if, in such a mixture, any bacteriophage mightbe liberated in-vivo, an antibody response to such release phagemight be obtained. No significant increase over the naturallyoccurring anti-staphylococcus bacteriophagic principle in theserums (average 1:60) was obtained by this method.

5. Subjecting to lysis a culture of strain CS which had absorbedstaphylococcus bacteriophage. An extremely active and specificbacteriophage for strain C3 was isolated from sewage in 1934.

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An entire broth culture of this organism which had absorbedphage was lysed, and the filtrate, lysate, or both were testedagainst several susceptible cultures of staphylococci. All at-tempts by any method at our disposal failed to reveal that anystaphylococcus phage had been obtained from the dissolved cul-ture. On two separate occasions it appeared that indications ofphage incompatability were discovered. In tubes in whichequal quantities of staphylococcus and subtilis bacteriophageswere placed we observed a complete loss of the staphylococcusphage, whereas the controls containing only the same quantitiesof each phage were unchanged. Asheshov (1933) has also de-scribed instances of phage incompatability.During the past three years 27 strains of Gram-positive spore-

forming bacilli (aerobic), belonging to the subtilis group havebeen isolated from agar plate contaminations. Only one of thesecontaminants, a strain of Bacillus mycoides has in any way beenable to absorb staphylococcus phages. We have also studied 31strains of organisms belonging to the subtilis group, which wereobtained from Dr. L. F. Rettger's department at the Yale Uni-versity School of Medicine. From this group, three cultures ofB. subtilis and one strain of Bacillus cereus, partially diminishthe plaque count of staphylococcus phages, and two strains, nos.8, and 79 (B. subtilis) are as capable of absorbing staphylococcusphages as strain C3. The remaining 25 strains lack any demon-strable staphylococcus phage-absorbing power. Altogether 58strains, noted above, and four others including Pseudomonasaeruginosa, and Bacillus pseudotetanicus have been tested for anycapacity to inactivate coli, dysentery, proteus and streptococcusphages. No diminution in plaque count of these phages was everobserved.

It appeared therefore, that we were dealing with anotherexample of so-called non-specific bacteriophagic (staphylococcus)absorption. It is well known that staphylococcus phages are,or appear to be, rendered inactive by a variety of non-specificsubstances. Shortly after the appearance of the reports byLevine and Frisch (1933-34) and Burnet (1934) dealing withphage inactivation by extracts of susceptible cultures of bacteria,

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STAPHYLOCOCCI AND DACILLI OF SUfTILIS GROUP 293

we prepared extracts of culture C3 and two other staphylococcusphage-absorbing strains of B. subtilis by the method of Burnet,in order to determine whether such extracts would act towardsphage in the same manner as those prepared from strains ofsusceptible staphylococci (Rakieten, et al., 1936). Extractsprepared in this fashion lack any phage-inactivating power,either towards staphylococcus phages or toward phage forstrain C3.Our experience with phage has led to the view that only rarely

are so-called non-specific factors responsible for phage absorption.In his early studies with phage d'Herelle maintained that therewas a definite relation between susceptible bacterial protoplasmand phage absorption. The recent studies with bacterial ex-tracts of susceptible cultures, noted above confirm this viewpoint.It seemed logical therefore to assume that subtilis cultures thathave the ability to absorb staphylococcus phages have in theirantigenic constitution a factor similar to that of susceptiblestaphylococci. The demonstration of such a principle, by sero-logical means might serve to explain the reason for phage absorp-tion. This antigenic component, acting in much the same man-ner as susceptible staphylococcus protein with regard to phageabsorption, ought to be detectable in properly prepared anti-serum. Antigenic relationships of widely removed species ofbacteria have been demonstrated by Lancefield (1925), Julianelle(1926), and Sugg and Neill (1929).Young white rabbits were immunized with living cultures of

subtilis strains C3 and no. 8 (three with C3; two with no. 8).Prior to the initial injection, these five rabbits were bled from theheart and their serums tested for the presence of natural occurringagglutinins for eight strains of B. subtilis, in which were includedstrains C3 and no. 8. No agglutinins were detected in titersfrom 1: 10 to 1: 320. However, all five serums had some naturallyoccurring agglutinins for our susceptible strains of staphylococciin dilutions ranging from 1: 10 to 1: 40. After a six weeks courseof immunization the animals were bled and agglutinations weredone on the homologous strains as well as on several other strainsof B. subtilis and staphylococci. The results are recorded intable 4.

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294 MORRIS L. RAXI1TEN AND TONY t. AAII~tNX

Both antiserums agglutinate homologous antigen to a fairlyhigh titer. Anti-C3 serum in addition also agglutinates severalother members belonging to the subtilis group (B. subtilis Mar-burg, no. 81, air no. 1, and B. mycoides Rhgld). However, whilethese strains appear to have agglutinins similar to strain C3, asdemonstrable above, the very active phage against strain C3 has

TABLE 4

STAI 1:50 1:100 1:200 1:400 1:800 1:1600 1:J2000

Agglutinative power of rabbit serum (pooled) anti-B. subtili8 8

B. subtilis No. 8................. 4 4 4 4 4 4 2B. subtili8 C3.................... 4 4 4 2 1 0 0B. subtili8 Li6................... 0 0 0 0 0 0 0Staph. alb. V.................... 4 4 4 4 1OOStaph. aur. Vnt .0

Agglutinative power of rabbit serum (pooled) anti-B. subtilis C3

B. subtilis C3..................... 4 4 4 4 4 4 4B. subtilis 81.........& .......... 4 4 4 4 4 4 0B. subtilis 8..................... 0 0 0 0 0 0 0B. subtilis air No.1............. 4 4 3 0 0 0 0B. subtilis Marburg.............. 4 2 1 0 0 0 0B. subtilis Jordan.0 0 0 0 0 0 0B. subtilis Novy................. 0 0 0 0 0 0 0B. subtilis N. Y. No. 82.......... 0 0 0 0 0 0 0B. subtiis C9..................... 0 0 0 0 0 0 0B. mycoidesRhgld.2 2 3 0 0 0 0Staph. alb.V.................... 4 4 4 3 1 0 0Staph. aur. Grazl................ 2 1 0 0 0 0 0Staph. alb. Grazl................ 4 4 4 0 0 0 0Staph. aur. Vnt.................. 4 3 3 1 0 0 0Staph. Gec. (resistant) ........... 0 0 0 0 0 0 0Staph. Grnspan (resistant........ 0 0 0 0 0 0 0

4 = complete agglutination; 0 no observable agglutination.

no action whatsoever on these same strains. Nor is the anti-C3 phage absorbed to any degree when placed in a medium inwhich any of these serologically related strains are proliferating.The serum containing anti-no. 8 antibody is able, to a fair degree,to agglutinate subtilis C3, but a reciprocal action is not demon-strable by the anti-C3 serum toward subtilis no. 8.

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Both anti-subtilis serums also possess agglutinins increasedto a significant degree over those found naturally for phage-sus-ceptible strains of staphylococci. These staphylococcus cultures,as well as extracts prepared from them, will absorb the samestaphylococcus phages that are absorbed by subtilis cultures C3and no. 8. Neither of the two phage-resistant strains of staphy-lococci are agglutinated by these anti-subtilis serums. While itmay be assumed that during the course of immunization withsubtilis cultures the naturally occurring staphylococcus agglutininsare also stimulated, this criticism is not entirely warranted be-cause of the fact that the phage-resistant strains of staphylococciare not affected; especially since sera prepared against some of

TABLE 5

SERUM DILUTIONS

1:100 1:200 1:400 1 1:800

(a) Anti-subtilis serum (C3, diluted 1:50) absorbed with Staph. Vnt.

Absorbed serum with Staph. Vnt............ 0 0 0 0Absorbed serum with B. subtilis C3.... 4 4 4 4

(b) Anti-subtilis serum (C3, diluted 1:50) absorbed with B. subtilis C3

Absorbed serum with Sta$h. Vnt............ 0 0 0 0Absorbed serum with B. subtilis C3

.. 0 0 0 0

4 = complete agglutination; 0 = no agglutination.

our phage-susceptible strains of staphylococci do agglutinate theseresistant strains. Also, sera prepared against the resistant strainswill also agglutinate phage-susceptible cultures. In this instanceagglutinogenic similarity is not entirely associated with phagesusceptibility as Burnet (1933) points out. On the basis of theabove results there appears to be some justification for concludingthat there is a correlation between the staphylococcus phage-binding power of certain members of the subtilis group of bacilliand the ability of these same strains to produce agglutinins forphage-susceptible strains of staphylococci. It appears that inthe antigenic components of these strains there is a staphy-lococcus-like stimulant, and that this heterogenetic antigen be-

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haves under certain conditions like staphylococcus substance sothat it may be detected by the absorption of staphylococcusphages.That the staphylococcus agglutinins in the serums are the

minor ones may be proven by an agglutinin absorption method.The results set forth in table 5 indicate what takes place when ananti-subtilis serum is absorbed with Staphylococcus aureus Vntand B. subtilis C3.A relationship between strain C3 and susceptible cultures of

staphylococci may also be demonstrated by placing the phageactive against this strain of B. subtilis in a medium containingan actively growing culture of susceptible staphylococci. Underthese conditions a considerable amount of this subtilis phage isabsorbed in much the same manner as the staphylococcus phagesare absorbed by an actively growing culture of strain C3. Noevidence of subtilis phage absorption has as yet been obtainedwhen lyso-resistant strains of staphylococci were utilized.

DISCUSSION

In general, bacteriophages regardless of type tend to becomeabsorbed by susceptible protoplasm (bacterial), either living,heat-killed, or in the form of a watery extract. Staphylococcusphages, in particular, while they also conform to the generalprinciples stated above, in addition may also be absorbed by suchcellular elements as yeast and white blood cells (d'Herelle andRakieten, 1935). That white blood cells may also take up otherviruses has been recorded by Douglass and Smith, (1930), Daub-ney (1928) and Schein (1917). Coli bacteriophages as well asother phages acting especially on the enteric group of bacteriaare scarcely affected when allowed to remain in contact with leu-cocytes. Lysis of susceptible E. coli readily occurs by bacterio-phages in concentrations of pus that have a definite inhibitoryeffect on staphylococcus phages.

Bacteriophages attacking staphylococci are also absorbed andinactivated irreversibly by certain bacteria not ordinarily re-garded as related to staphylococci. These organisms, describedin the present report, are members of the subtilis group. It is

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believed that some portion of their antigenic constitution re-sembles that of susceptible staphylococci, and that it is this factorwhich is responsible for their staphylococcus-phage-absorbingquality. This factor also resembles the factor responsible forphage absorption by susceptible staphylococci in its heat sta-bility. Finally this factor is able to stimulate formation of anantibody that reacts with susceptible staphylococci, and may beidentified by serology.

It is becoming more and more evident that bacteriophages areuseful agents in the detection of antigenic differences in closelyallied strains of bacteria (Leveine and Frisch, 1934, Burnet, 1934,Craigie, 1936, Almon and Stovall, 1936, and Sertic, 1936). Thepresent report indicates the usefulness of bacteriophage in de-tecting antigenic similarities in strains widely removed from eachother.Union between phages and susceptible bacterial protoplasm,

irrespective of the state in which it is presented (living, heat-killed, or in the form of an extract) takes place very rapidly atincubator temperature, the largest proportion of phage beingtaken up in less than thirty minutes. Once the phage has beenabsorbed it is difficult, under ordinary conditions, to effect anyrelease. In all instances where staphylococcus phages have beenabsorbed by strains of B. subtilis all of the several methodsutilized to free absorbed phage failed to show that any lytic agenthad become disunited.Burnet in a series of reports (1929-33) dealing with phages

attacking bacteria of the enteric group has shown that a relationexists between the heat-stable agglutinogenic constitution ofthese bacteria and their susceptibility to phage. With regardto the phage isolated for B. subtilis C3 this relationship does nothold, since organisms that have agglutinogens in common withstrain C3 are not attacked by this phage, nor is this phage ab-sorbed by placing it in contact with any of these related cultures.However, it is not our purpose to conclude on the basis of theresults with one phage that a relation in the subtilis group doesnot exist that is comparable to that in the Salmonella group asdescribed by Burnet.

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Our failure to demonstrate that extracts of culture C3 are ableto inactivate staphylococcus phages may be due to the fact thatthe methods used to prepare such extracts were faulty. Extractsso prepared had no phage-inhibiting action toward the homolo-gous subtilis phage.From a practical standpoint the results described may be of

some value in explaining the failure to find staphylococcus phagesin sources that are rich in coli and dysentery bacteriophages.

CONCLUSIONS

1. Staphylococcus bacteriophages are absorbed by living orheat-killed cultures of bacteria belonging to the subtilis group.

2. The bacilli are not influenced by their contact with staphy-lococcus phages.

3. Absorbed bacteriophage can not be detected, even afterlysis of the absorbing strain.

4. Coli, dysentery, proteus, and streptococcus phages are notaffected by contact with these same strains.

5. There appears to be an antigenic relationship between strainsof Bacillus subtilis that are capable of absorbing staphylococcusphages, and phage-susceptible strains of staphylococci.

6. Phage-susceptible strains of staphylococci also absorb asubtilis phage.

7. Bacteriophage may be a very valuable aid is studying theantigenic composition of bacteria.

REFERENCES

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ASHESHOV, I. N., ASHESHOV, I., SARANJAM, KHAN, LAHIRI, M. N., AND CHAT-TERJI, S. K. 1933 Studies on cholera bacteriophage. Ind. Jour.Med. Res., 20, 1101-1188.

BAIL, O., AND OKUDA, S. 1923 Der Abbau Lebender Bakterien durch Bakterio-phagen. Arch. f. Hyg., 92, 251-291.

BURNET, F. M. 1929a A method for the study of bacteriophage multiplicationin broth. Brit. Jour. Exp. Path., 10, 109-115.

BURNET, F. M. 1929b "Smooth rough" variation in bacteria in its relation tobacteriophage. Jour. Path. and Bact., 32, 15-42.

BURNET, F. M. 1929c Further observations on the nature of bacterial resist-ance to bacteriophage. Jour. Path. and Bact., 32, 349-354.

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STAPHYLOCOCCI AND BACILLI OF SUBTILIS GROUP 299

BURNET, F. M. 1930 Bacteriophage activity and antigenic structure of bac-teria. Jour. Path. and Bact., 33, 647-664.

BURNET, F. M. 1932 Lysogenicity as a normal function of certain Salmonellastrains. Jour. Path. and Bact., 35, 851-863.

BURNET, F. M. 1933a The classification of dysentery-coli bacteriophages.I. The differentiation by Bail's methods of phages lysing a typicalB. coli strain. Jour. Path. and Bact., 36, 299-306.

BURNET, F. M. 1933b The classification of dysentery-coli bacteriophages. II.The serological classification of coli-dysentery phages. Jour. Path.and Bact., 36, 307-318.

BURNET, F. M. 1933c The classification of dysentery-coli bacteriophages.III. A correlation of the serological classification with certain bio-chemical tests. Jour. Path. and Bact., 37, 179-184.

BURNET, F. M. 1934a The phage-inactivating agent of bacterial extracts.Jour. Path. and Bact., 38, 285-299.

BURNET, F. M. 1934b The chemical nature of the phage-inactivating agentin bacterial extracts. Jour. Path. and Bact., 38, 301-311.

CRAIGIE, J., AND BRANDON, K. F. 1936 The identification of V and W formsof B. typhosus and the occurrence of V forms in cases of typhoid andin carriers. Jour. Path. and Bact., 43, 249-260.

DAUBNEY, R. 1928 Observations on rinderpest. Jour. Comp. Path. andTherap., 41, 228-263.

DOUGLAS, S. R., AND SMITH, W. 1930 Study of vaccinal immunity in rabbits bymeans of in vitro methods. Brit. Jour. Exp. Path., 11, 96-111.

FLU, P. C. 1923 Die Natur des Bakteriophagen und die Bildung von Bakterio-phagen in alten Bouillonkulturen pathogener Mikroorganismen. Cen-tral. f. Bakt., I. Orig., 90, 362-374.

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