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The bacteriophage Wilson, Grace A. 1923-12-31

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THE BACTERIOPHAGE  by Grace A. Wilson. 1923.  THE BACTERIOPHAGE *y  Grace A. Wilson.  A Thesis submitted for the Degree of MASTER OF ARTS i n the Department of BACTERIOLOGY  THE UNIVERSITY OF BRITISH COLUMBIA MAY, 1 9 2 3 .  The Bacteriophage. In the last few years attention has been focused on the works of d'Herelle, T^ort, Bordet, Gratia and others on the so-called "Bacteriophage," a phenomenon which consists of the bn-.king down and solution of bacteria by a filtrable a^»nt which may be a^ociated with pure cultures. This b<*cte iol/tic action was e*rly spok«n of by Hanken (1) while working with the waters of India.  However, the first real investigation of it  was made in I915 *>y Twort at the Brown Institute. Twort, (2) after experimenting with varied materials obtained results with vaccinia.  He innooulated agar  slants with the vaccinia before the glycerine had completely sterilized it and found that the micrococci showed at times a translucent or transparent change which started as clear spots at the mnr^ins.  He found that  sosis of these oolonies could not be sub-cultured, also that if s colony of the white micrococcus that had started to become transparent was plated out, then the micrococcus grew and a pure streas culture *"rom certain of these colonies could be obtained.  But if plate  cultires, (made by inno-ulating the water of condensation of a series of tubes and floating this over the surface of the medium) were left, the colonies turned transparent this action starting from the edge.  Other experiments  float'4....  -3.  shoved that if * nonsil colony »f the mieroooocus ^ M touched v i t h aorae of thf* glaasv -solony, the norar-*! colony would become "'watery,"  I t « f founl that till* trans -  parent material vaa s t i l l active i3 hi ;n d i l u t i o n s <*ad a l l attempts to sub-culture tne f i l t r a t e proved negative for i t wouli no'., jrow by i t e i l f on any m-iium. Ey further expeiira<mt, Tvort found that t h i s f i l t e r pa^ein;? matori-.l 1.  Resisted heat to 60  for one hour.  2.  I t could be separated from tn» organism from wuich  i t was derived by f i l t r a t i o n . 3.  (BeriCfeldj.  i t aad ao action on dead or^ni'saa end nae most  s a t i r e agsinst young actively groeing organisms. *.  It increase-l in m e n t i t y vnnn allovel to set on a  culture. •T.  It acted to a 1e*s iegree on elo*ely r-l^ted or-  ganisms and did not act upon unrelated forras sesh ae ths colon -trchoii group. 6.  It oould be transmitted iri4?finitely from one culture  to another. T-*ort also obtained similar result1* vita a micrococcus, and a mambsr of the -Jolon-tyTj-ioid .{roup of baoilli obtained from th  suoous  sembrane of -* iog  suffering from distemper; aleo with a bacillus from the intestinal tract. Ha cl »ias in the case of vaccinia, the transparent mateiibl contains na e.iayiae.  If it ie p»*rt  Cont'd,...  -3-  of the micrococcus it may be either a sta^e in its life history which will not grow on artificial media but stimulates fresh cultures of the micrococcus to pass into the same stagg, or an enzyme secreted by the micrococcus which leads to its own destruction and the production of more enzyme.  He does not however, insist that his  experiments have definitely disproved tne possibility of its bein^ an ultra microscopic virus, Bordet and Ciuca (3). *ho hav«* al«»o dona a consider^ble amount or fork on th« bacteriophage, entirely disagree with -' ^r^lle in thinking that lysis is due to a living filterable virus.  They believe tnat  the microbes, when exposed to external influences, such as a leucocytic exudate, undergo a modification by which they art then capable of producing an autolytic principle. This property is transmitted to the following generations by the germs wnich were sufficiently resistant and thus could multiply, Bordet was the first to declare tnat a lytic principle could be obtained without starting from a stool filtrate but could be isolated by filtering an exudate.  This method however is not conside ed satis-  fad opy by the majority of those who heve tried its duplication,  H" and  his co-workers were aleo the first  to produce an antilytic serum by innoculetinT rabbits with  Cont'd....  -4-  lncreaslng amounts of dissolved culture filtrate.  This  H r t a wss able to neutralise completely tne aotion of their bsoteiioph<ige. Since then Kuttner (4) has obtained this lytio prinolple from normal tissue ertracte of guinea pig* and from normal rabbit sera.  3he does not agree vith  d'Herelle but favors the idea that the bacteriophage is a secretion of the bacteria of the nature of an autolysin.  This autolysin usually liberated in old  bacterial cultures, as a result of cell disintegration aots as a oatalyet which destroys the equilibrium occurring in actively sowing cells between the constructive and destructive forces in favor of tne latter. Solution of the bacteri .1 cell consequently results and thus more of the autolysin is liberated. D'Herelle'e work h«.s undoubtedly aroused the greatest interest in the Bacteri ojj'i^gs*  He has recently  published a book on this subject, the result o^ four years of oareful study.  He ma*e daily examinations a*  the stools o* a pati«nt suffering from a severe dysentery (Shiga).  He innoculated broth with feces, incub.ted  it overnight, filtered through a Beracfeld filter and a^ded about twelve drops of tlie fOtrste  o youn* active  broth cultures of 3niga b-cillus, usin^j one tube innoculated with Shiga only as a control.  During the  heighth of the disease such cultures yielded normal  Cont'd... growths.  -5jjo^ever, when the patient presented symptoms  of improvement, the "bouillon innoculated with culture and filtrate appeared sterile,  vhen he a^ded a drop of this  dissolved culture to another youn? broth culture it also was dissolved.  After repeating tais process several times  it w.s found t vat the broth cultures were dissolved even more guiokly than at first.  IVom this he concluded  that the B^ct -^riopha^s was increasing in potency mid that it was capable of cultivation in series. I^'ierelle also examined th* absve cultures aft«r Incubation perioii o * o^e, t *o wnd t1! re'3 ho.rs by innoculatin^ tna n*»teri 1 on to arar sl^nt^.  ,  Jf  examined imicUately after -planting on a^ar, th? agar was covere'2 by a normal filn jf 3 ii -;a Bacilli, but with t'^o areas about 2 mm. in iianeter entirely free of growth.  In the one hour tube six of these clsar  areas were found.  In the two hour tub? there were about  one hundred and in four hours no growth of Ihiga bacillus oould be s »en. rifter many experineats of t.iis type hi  c .is  to the conclusion that tnis dissolving principle may be present in trie intestinal contents of all livin-? beings. It is usually parasitic for Bact. coli but in the course of intestinal disease it becomes H parasite on the invading o r ^ n i ™ by the process of adaptation.  D'Herelle  Coat ' < ? . . . .  -6-  holds s t e a d f a s t l y t o t'i« idea t h a t he i * de<ilin:; with an ultr^raicroecopic f i l t r « < b l o v i r u s Vnich i s p ^ r a ^ i t i o on b a c t e r i a and »« proof h« s t a t e s the ^ollo-dn» f a c t s : I . "The d i s s o l u t i o n of b a c t e r i a by t h e B a c t e r i a r h <gic p r i n c i p l e W«es pl^oe in e a r i e s , "  He found t h l t to be  t r u e s f t ° r mors th<an 3 COO rasa^/ree.  If i t  -* re an  ^nfsyrae a c t i o n i t would <*oon cease to show because of the g r e a t e r and g r e a t e r ^ i l u t i ,n of the ensyaa s o l u t i o n in the course of the successive p r e s a g e s . II.  M  The l y t i c enzymes come from m a t e r i a l  corpuscles  •which 7?ill Base through f i l t e r s ; these corpuscles m u l t i p l y in the course of the b a c t e r i o l y s i s . " III.  "All bacteriophn ;,ic u l t r a m i e r o s c o p i c  corpuscles  grown a t the expense of nay b a c t e r i a l s p e c i e s constitute one an* the same s n f i ^ e n . "  By axoorlment he fouT 1 t h a t  the serum of an anira^l nrepere* bv a c u l t u r e of any b a c t e r i a l ?p<*ci«s dissolved by b^.cterionh'-ge eont< in» an amboceptor wMch fix*e i t s e l f on sny oth«r b ^ c t e r i o phaged c u l t u r e .  Henc* the =anbocapt;>r i s s n e c i a l l v  an t i - b a c t e r i o p h a g e md not ant i-oa it^ ial." IV.  "The l y t i c a c t i v i t y of the c o r p u s c l e s v a r i e s . "  This v a r i a t i o n in a c t i v i t y i s due to a d i f f e r e n c e in the raul t i r j i c a t i o n of the corpuscles i n n o c u l a t e d .  To explain  t h i s , the a c t i v e p a r t i c l e must be a microbe, as a microbe a c t s by i t s v i r u l e n c e and t o x i c i t y while an enzyme a c t a only by i t s q u a n t i t y . .  -7V, " The virulence of, a lytic sgent m-^y be increased by successive x:'39,i'?e9-0 VI, "Bacteria -when attacked by bacteriophage defend themselves and are capable under certain condition of acquiring an immunity against the osrasite.  This could  happen only if the \ict«5 ;ionh ' ge ^ere narasitee of a living organism." VII, "Tne resistance of bact^riop lage to the motion of physical and cn^mical reagents is that of a living being and not that of an ^nzyme.  Bacterioph ge is killed  by ei/ght days contact with -;lycerin, this being the liquid used to preserve indefinitely nn enzym^ in soluble form.*  V I I I , " I t i s p o s s i b l e t o e x t r a c t the l y t i c enzymes free from the l i v i n g b a c t e r i o p h a j e IX. %.  raicro-organianis."  "Bacteriophage i s capable of a d a p t a t i o n . " "The p r o p e r t i e s of bacteriophage are v a r i a b l e , t h i s  being a l s o a c h a r a c t e r i s t i c of l i v i n g b e i n g s . Although d ' H e r e l l e does not b e l i e v e t h a t the l y t i c m a t e r i e l i s o l a t e d by hii~'---,lf end the t r a n i r - t rent m a t e r i e l of Twort to be the same, raoet of th« ^or'cere new consider t h a t the a*-.-ne phenomena are involved i n both c a s e s . Op to t h i s time tnere nas been comparatively little  *ork done on the s t o o l s of typhoid c a r r i e r s .  Therefore i t waa decided to study i n what proportion  Cont'd.»..  -8-  of carrier o»ses a bacteria image is found.  It was  thought possible that the isolation of an anti-typhoid bacteriophage from a suspected carrier might serve as another me«ns for their positive identification. A stool was obtained from an individual suspected of being a typhoid-carrier on account of the presence of a rsositive &idal test.  Tnis a%n had  typhoid fever four or five years nreviou3ly and two years ago had given a negative fidal.  Tile present  positive result was (fhThinned on three examinations at weekly intervals. B. coli but no 13. typhosus W R S isolated from the stool. About 5 3SR* of the fec^s w=*s carefully suspended in 50 co, of bouillon and incub?*ted at 3? hours.  f  or fourteen  It was then filtered through an ordinary filter  and paper mash and finally through a Handler filter previously sterilised.  The day before the test W H S  made agar slants were innocuJatod with B. dysenteziae Shiga, £. dip. "Flexner, B. typhosus, B. P-ira typhosus * 13 para typhosus B. and the aomologous strain of B. coli From all of these young cultures seven groups of four tubes of peptone broth each were innooulated.  To the  first tube was added one drop of the filtrate, to the second ten drops and to the third t-*o cubic centimeters. One tube was iinoculated with the organism to serve as a control.  All were Incubated at 37  0.  Aft^r twelve,  Cont'd,...  -9-  eighteen and twenty-four nours they were examined. All tubes showed a normal growth.  In order to be sure  that there was ao bacteriophage present, active against these organisms, a platinum loop full of each tube was spread over the surface of slanted agar.  After incuba-  tion these tubes presented « normal growth and no lytic colonies occurred.  It voul1 appear from this isolated  trial that in this •«»rrier*«" stool, at the time of ex%miriation, there was no bacteriophage active for ta« stock strains of B. Shiga, B. Flexner, B. typhosus, B. P&ra typhosus A., B. Para typhosus B. or B. Goli. Other experiments with a normal stool were carried out in the same manner as the previous attempt. The results however, were in this ca^e aleo negative. The number of typhoid carrier cases in routine examinations has been disappoi tingly limited to the single case quoted above.  This work is to be continued on  future cases as thev appear, and results appended l--,ter.  »  » t t i » i  B»TT*r?l)e - L ' b c t i o n b a c t e r i c i d e ties e-iinc de 1% Jumna e t du Gange •'ur l e v i l r i o n iu cholera Ann. i e 1 ' I n s t . P a s t e u r l 6 ; 6 , 1 0 / 5 / 1 1 .  T*ort "Tie Bftct ^riophage"  B r i t i s h Nodical Journal  August 19, I 9 2 3 . Lordet &. Ciuca,  "lae Baoteriopii-ige", B r i t i s h  Hodical J o u r n a l , August 19, 1923.  S u t t n e r - "Baoteriophv^e Phenomena." Journal of Bsoteriolosyr Wo. 1, Vol. 7 I I I , J^n-; ry, 1 ?3,  P ' H e r e l l e - "The Bacteriophage."  jfn^lieh  tr%nshtion, 1922. B r l t i e h Ke-iic&l J o u r n a l , August 19/1923.  »»»»!»!  


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