ill! lil‘ll‘H I b 7 STUDIES ON DISSOCIATION OF THE BRUCELLA GROUP THESIS FOB THE DEGREE 01‘ M, S, F"r;2211~:An%}1<;)11y' Gallo 1931 \mcx ‘- ~ . I . 1H! ,. ‘ n - .,.. .. 'u v ‘5“..o'l. s' I.‘ . .- _ k.-~.'." -O . . 0 O a l 0%.? Ian. J P9.” .. .4’ ’ 6": 0‘ ,1 “l’ 4‘5} 4" . «w . 3. u. . .. .‘_.H." r ‘a\ .. I '0. b, . & "" 1:%$'1;’ . p" . ' 4% ' ’ 2.59% a “6% ‘ . fig.” k-‘\.f$§; - 'l j" o .' 4"“ I ‘ > ' W"!—i-’x \ ' J I _ . 'x . bur“ " x? u x , V .Lj‘ . -.«- (r? .‘ 4’} 3:" 3 I . s... .‘. ' . ‘ , . a. 6 > C .I. ‘ . .. ' ‘ s." ‘ »,i . "' . .‘ , . ”9 M ,g' ,u‘gf-fij‘, ' ', v . v. I "'0 kl“? ' (I I ‘ I - ' ¢ 0 ‘ n “ ' .’¢ : A " o . ‘ . B . \ ‘ .. w, it ' J M " n ~‘~ '1 “r . Lrix-fir'ril _‘ ,3 «a ' ‘l 3 " (“fig K g i Y I "‘p ~ . ', 1 A , V; a" 9‘ " " ”,2 ,3: : -‘._‘, 1)" gt .3; . ‘ 13"»?32 3119” J g"? ' ‘ W K“. ’? girl, 4 1‘. V}: E .9 .4 .545 fi ‘ 5 x ‘I’ t ' . b ‘3‘” n O I. ‘. . ‘ . ‘ ' . 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I.“ STUDIES ON DISSOCIATIOH OF THE BRUCELLA GROUP. Then“ for Degree of M. S. Frank Anthony Gallo 1931 E m r. m. m, L. 8 7m 7 a 9’3, 9 [Adah/“~56? J \.' v '2? Michigan State College of Agriculture and Applied Science Studies on Dissociation of the Brucella Group A Thesis Submitted to the Graduate Faculty For the master of Science Degree Department of Bacteriology and Hygiene by Frank Anthony Gallo East Lansing, Michigan June, 1931 \ , C? {Q i 330-1 I. II. III. Iv. v. VII. VIII. IX. X. XI. Contents Introduction Historical Resume History of Cultures Bacteriological Study of Cultures Experimental (1) methods used to bring about dissociation (2) Biochemical Studies of R strains (5) Serolorical studies of R strains (4) MorphOIOgy of R cultures (5) Reversion of R types (6) Study oi’the revert strains Discussion Summary Conclusions Acknowledgment Bibliorraphy -1- Studies on Dissociation of the Brucella Group. I - Introduction. In recent years, the phenomenon of dissociation of bacteria has engaged the interest of many workers in the field of bacteriology. Although variability of bacteria was observed as early as 1875, the forms noted were regarded as contaminants rather than atypical forms. The relationship of the atypical forms was not recognized until recently. Bacteriologists, accepting the fact that variation or dissociation does exist, are not attempt- ing to ascertain whether the phenomenon is a haphazard "hit and miss" procedure or an orderly process. Neisser and Massini (1906 - 1907) reported changes in Escherichia ggli, which were quite significant. Kolle regarded their aberrant forms as contaminants, but Kawalenko (1910) confirmed their findings using single cell cultures. Their methods of study were quickly applied to other organisms and similar results were obtained. These studies acted as a stimulus to a more thorough study of the dissociation pégnomenon. Among the many studies reported, may be cited the work of Cowan.(1922) on the streptococci, Griffith (1928) on the pneumococcus, Topley and Ayerton (1924) -2- on the Salmonella enteritidis and Arkwright (1984) on Eberthella‘typgi. The repeated confirmation of dissociation of’various organisms, by numerous workers lends convincing evidence that the phenomenon of dissociation is likely a property common to all bacterial species. 11 - Historical Resume The first studies on dissociation of the Brucella group were presented by Henry (1928 - 1929). He states that variants of the porcine strain, although culturally and morphologically similar to those of the bovine strain, appear to be widely separated serologically. Henry worked with two types of colonies, one type being the usual mmooth.§3, abortus colony described as being moist, clear, and slightly granular, and which by transmitted light shows a bluish green fluorescenoeYRQEhe second types of colony he describes as being Opaque and granular. The organisms are suspended with difficulty in salt solution. Frenzel (1951) was successful in dissociating ‘gg. abortus to the R type. He states that the R strains are less virulent than the S types. His R antisera will not agglutinate the S antigen, but agglutinates the R antigen. The R types are weak when used as antigens, producing antisera of low titer. -5- On the other hand, he finds that the S antisera will agglutinate the R antigen in low dilutions. In the light of the data which will be presented later, in this paper, the colony descriptions of Henry indicates that he was dealing with an inter- mediate or a partial R type of colony and not a true R type as he supposed. Frenzel does not give a colonial description of the colonies, but points out agglutinative differences. III ~ History of Cultures The cultures in this work were obtained from.the collection of Doctor I. F. Huddleson. The history of the cultures follows. Brucella abortus. Culture No. l was received from.the Bureau of Animal Industry prior to 1915. The source and date of isolation is unknown. Culture No. 2 was isolated from.an aborted fetus in 1915, from herd "A" at Michigan State College. Culture No. 3 was isolated from.an aborted fetus in 1915, from.herd "A“ at.Michigan State College. Culture.Ro. 4 was isolated from.the udder of cow No. 995 of the abortion experimental herd of.Michigan State College in 1915. Culture Ho. 5 was in the laboratory stock cultures -4... prior to 1915. The source and date of isolation is unknown. Brucella suis. Culture H0. 400 was obtained from Doctor Griswold of the Michigan Department of Health. It was isolated from a boar's testicle. The date of isolation is un- known. Culture Ro. 401 was obtained from Purdue University. The source of the culture is not known. It was isolated January 15, 1925. Culture No. 402 was obtained from.?urdue University. The source and date of isolation is unknown. 7 Culture H0. 404 was obtained from.Doctor Conway of the University of Missouri in 1922. It was isolated from.a premature fetal pig, from a naturally infected sow, February 1, 1922. Culture Re. 405 was obtained from.Doctor Conway having been isolated from swine in.Missouri. The date of isolation is unknown. Culture No. 408 was obtained from.Hr. Good of the University of Kentucky. It was isolated from a hog. The date of isolation is unknown. Brucella.melitensis. Culture No. 301 was obtained from.Mr. J. P. Torrey who isolated the culture from.the Michigan State College Dairy herd. The date of isolation is unknown. Culture No. 312 was isolated in 1921, from a case -5- of undulant fever in Tunis, Algeria by Doctor Burnet of the Pasteur Institute of Tunis. Culture no. 315 was isolated from an undulant fever patient, by Doctor Burnet of the Pasteur Institute of Tunis. Culture R0. 316 is of human origin and was isolated by Doctor Burnet in 192 . Culture R0. 318 was received May 10, 1921 from Doctor K. E. Meyer of the George William Hooper Foundation, for medical research, University of California. The date of isolation is unknown. IV - Bacteriological Study of Cultures. Before dissociation studies were started, each organism.was repeatedly plated out to eliminate all possible contamination. These strains were repeatedly stained by Gramfs method to further check the purity of the cultures. The pure-line strains thus obtained were then studied culturally and physiologically to further check their identity. The species of the strains selected for study were checked according to methods of identity presented by Huddleson, namely dye sensitivity and hydrogen sulphide production. Suffice it to state that Huddleson (1928 - 1929) found that the three species of Brucella can be rccOgnized as measured by the source of the organism, namely bovine, porcine and caprine strains, by the agency of dye bacteriostasis -6- and hydrogen sulphide production as presented in the tables below. 'TABLE I. GRJVTH ON DYE PLATES Thionin Basic Fuchsin Br. abortus---~-~-No growth Growth Br. suis-----~-Growth Ho growth Br. melitensis-uouGrowth Growth 5313:8131: II. PRODUCT 0H 0H HYDROGRN SULPHIDH Hydrogen sulphide production in days. 1 2 3 4 5 6 7 Br. abortus~~~ + + + + - - - Br. suis—--- + + + + o + + Br. melitensis - - - - - - - The results presented in tables 111 and Plate I show that the strains selected for study checked with the identification given them by Doctor Huddleson. The cultures were examined for their agglutin- ability by a B3. abortus immune serum obtained from an infected cow. The usual test tube method for agglutination was used. The results are presented in Table IV. It will be observed that all strains were -7- agglutinated in like titer although the maximum.titer was not determined. However, the fact that all strains reacted similarly adds further proof for the identity of the cultures. -8- TABLE III. bACTERIJSTATIC ACiIJK 0F DYLS UN ORGANISMS SELECTE FOR STUDY. Thionin Euchsin _Brucglla Abortug (Bovine Species) 1------ Ho Growth --- Growth 2—---—- No Growth -—~ Growth 3~---- No Growth ---- Growth 4---u-7No Growth -... Growth 5----—- No Growth --- Growth D--.'- Brucella Suig (Porcine Strainl 400-~-- Growth---~-- No Growth 404...... Growth------- Ho Growth 401.....- Growth - No Growth 405...... Growth------- No Growth 402----- Growth------ No Growth 408---- Growth------ No Growth Brucella Melitensis (Caprine Strain) 318--- Growth~----—~ Growth 301-.-.» Growth-~---- Growth 512¢-- Growth-----— Growth 316--- Growth-~~~-- Growth 315---~ Growth-~~~~- Growth ,.‘-.C“..--“CC‘-.-‘-““. PRO D UC T/OIY 07‘ H2 5 SN 00 TH S-TR’HIHS 0F GENUS BRucuLH not or GROWTH IN HOURS 48 72 96 120 144 I68 If N -_ Tn__o __i r L 1 (N BEN/é [-71.11 n n}! «Bier U) 5‘. ._i__ _—————1 l ( ~%F# : [IIIK r l r I: I (I I I I .{ Q 402. vermin” su/s’ 04 L. 1 ¢ L. a ’39: .1... -:,:__- ::-:_’ 7- ': -:- ...:‘.§'.”“ Jrgar:91.fl'.l:’3’akn::_:: ;‘:. :‘3 ../‘.:’!‘:_ 3:3", ’_.::_Z'.‘.‘;. ""3":7:315:13;Eff;::?§é;§§i€f§m:'rf‘:tf5'§§?:.:!'E“';:r’: .......,V..-........... .;. 3‘ . I “W “m‘fiux‘a _:i\'\\_'_\\;§1§\ \-\'\'_\_\f \\v \\\\\*\\‘\\“ \\ \‘x \\\\x 5% as.“ \mmmwa “35‘ \\:\ :;‘\\\\§'\g.'“ ft“: _ $3.733,“ 31.“; .\\.\\ ~~~~w \~ ' m)“ we. -. ....'..-~.,;:.:.;.z:.‘.._-.; 7L908 L h—r‘e L. LgL_gl 30! ~ ~ 6 1 “~33”: I on... '1 \ 3/; b I“ it”: 15 (D (b _( 3w ' ' 4 )— 1 I l -' :. cw .J I j ~_.__.___. __ 4 \ a U ~ #1. ++ . ++ - ++ a ++++ b ++++L ++++.++++.++++a++++b++++»0. Hbm . hi ++ . +9 #. ++ . ++++.+,++++. ++++~+++++4+++u++++.++++»1. man . . ++ .P ++ . ++ . ++++.. ++++. ++++.+++++++++.++++a++++.1b .mHm . . ++ 5 ++ . ++.r,++++ . ++++b.++++P4+++h4+++PM+++b+++wblp m m . . «w . ++ . ++ a mefl . ++++p ++++k++++b++++.++++.++++PWb . . . . a. .11 , . by! .1. . .1. (F I... III... KILL. . ++++F++++.++++.++++.++++?Ir F. GI ++ b ++ . +++.k++++ .++++ . ++++k++++.++++.++++p++++sls11 mBmPIIII. .. ++ . ++ . +++ .++++ik++++k ++++b++++k++++P+++sr4+++sls “RmPlll; b ++ .# ++ .h +++ a++++ .++++ . ++++.++++a++++.++++.++++sl. 0e . bi. ++ . ++ . +++.b++++ h++++ P +++wa++++Pw+++.++++h++++rnk HHmPlllu. .r ++ . ++ a +++ P++++ beef...b ++++.++++.++++P+++APW+++bWb. ibeldll: a. case um. . . . p - . . ++ P ++ .h +++.b4+++ .++++ p ++++.++++k++++PW+++FW++ARI. mlllll. bl. ++ . ++ . +++.bH+++ .++++L +++++W+++.++++.++++.+++anr. .v . .PI ++ 5 ++ . +++.+++++.a++++ . ++++.++++.++++rw+++.++++.l. mPlllll. - ++ . ++ . +++ .i¥++ .++++ . ++++.++++k++++r++++r++++.ir. .ILWIIIII. a ++ ++ +++ ++++ ++++ ++++ ++++ ++++ ++++ ++++klb H . . oooena.oommna.omma-~.o¢on . «and. efi-a.omua.oeua.omua.oa-~.o. seasons um. . .1 Lf - p b r b . p F a . .ROHamwoomMHQ mom QMmD mAmstwmo «Ammoamm Qua a3 HaHflHmdnHaaflowd was .bH manda -11... The sugar reactions were negative. The Brucella group, without exception, does not ferment the five sugars used. These reactions serve as a further check on the purity and identity of the cultures used. The cultures selected for study were plated out repeatedly on liver infusion agar to study the colony formation. Without exception, the colonies were round and dome-shaped with a glistening surface. Under the low power of the microscOpe, the colonies gave a slightly granular appearance. The margins of the colonies were even and regular. From every standpoint all strains were typical 3 organisms. The extreme care exercised in identifying and purifying the cultures selected for study is presented here to show that the cultures were pure-line strains of Brucella. This was done as a preliminary to the dissociation studies to rule out as effectively as possible the existence of contamination. Inasmuch as the R types of the Brucella were culturally, serologioally, physiologically and morpholOgically different than the S prototypes, it is quite essential to know that the R forms described were not contami- nants. Beef liver infusion agar medium, first described by Stafseth (1920) and recommended by Huddleson (1927) was used through this work. This medium was selected -12- for this study, as Huddleson claims that it is the best medium for growing members of the Brucella group. The medium was prepared according to Huddleson's formula except that a Buchner funnel with a cotton filter was used, in the place of Scharples separator, for the clarification of the medium. Gentian violet was incorporated in the beef liver infusion.medium, in a dilution of 1-50.000. The presence of this dye in the medium inhibits the growth of gram positive organisms. A more detailed description of the bacteriostatic action of this dye is given in an article published by Huddleson (1928). Veal infusion broth was prepared in the usual manner, and adjusted to a ph of 7 in all instances. The one per cent lithium chloride broth was prepared by adding 10 cc. of a 10 per cent solution of lithium chloride to 100 cc. of veal infusion broth. The 0.1 per cent phenol veal infusion broth was prepared in a similar manner; 2 cc. of a 5.0 per cent solution of phenol was added to 98 cc. of veal infusion broth. The medium.was then tubed and sterilized. A high titered positive g5, abortus serum was used in the preparation of the positive serum broth. Ten cc. of sterile serum was added to 90 cc. of veal infusion broth. This was then tubed aseptically, incubated at 37°C. for a period of 24 hours, to eliminate contaminated tubes. The R immune serum -13- broth was prepared in a similar manner. The test tubes used in serial transfer of the cultures were of standard height, having an internal diameter of one am. This size tube was selected because it was found that the Brucella organisms can not be transferred serially by transferring a 100pful of innoculum into 5 to 10 cc. of nutrient broth. Serial transfers can be obtained by transferring into 2 cc. or less of the broth. With this small amount of culture medium, the smaller diameter tubes were ’ more satisfactory as evaporation was lessened by the smaller surface of the broth. V - Experimental I - Methods used to Bring About Active Kicrobio Bissociation. Experhment No. 1 All strains of the Brucella group were seeded in 10 per cent positive serum broth, transferred serially and plated out every 48 hours. A series of these cultures were aged and plated out at weekly intervals. A11 plates were carefully examined for R typegcolonies that showed rough colonial character- istics were fished and transferred on liver agar slants. The data are given in table V. In 18 serial transfers of five strains of Q3. abortus no R forms were observed. Opaque smooth forms were observed in the eleventh transfer in all five strains studied. These persisted throughout the experiment with a complete disappearance of the 8 forms. The cultures that were transferred weekly showed similar results. In the case ofg§£,{§gig strain 408 produced R forms after the fifth transfer. The other five strains produced opaque-smooth ferms after the eleventh transfer, but no R forms were observed. The data presented in table VI shows the results of aging on dissociation of the species of Brucella. Opaque smooth forms were observed inwgg. abortus at the fifth and sixth weekly transfer. Till then these cultures retained their original smooth characters. Strain 408 oflgg..§gig produced R type after the third weekly transfer; the remaining cultures retain- ed their original smooth character until the last transfer, when they reverted to the smooth opaque forms. Strains 301 and 518 of pa, melitensis produced R forms after the fourth and fifth weekly transfer. The remaining cultures reverted to the Opaque smooth, at the fourth transfer, going back to the S form at the fifth and sixth transfer. museum on u I .3300 we can» ammo.“ ... m hsoaoo 90 09.3 oodmmonnpooeu I we hdoaoo no 0&3 ...Boofiu I m 1- JI W n H q a u a m“ a n u n u a n u o h a — mHmH ..d. .. mm. _ rm _ m _ mom _ MT. 41$. . n . a . IEIFEEPE. mat? 4 u a n p n p m . m u m - D a _ u _ p o o - QHN mo. mm _ m _ m _ mm _ mm a ma . n . m . m . . n . 1 . . . u . . mHm iéiilmmrws.m.m.m.m.fi ... leurm Ell u in 11 dw . m .mflmfimpflfloa Hm. - o r if F —M+.m .W -m Limn .m PM .m L WU» mtmr Mr mpwrmtm.mL mL mow b .m0. mo _ mo L mo P I _ m0 L mOIIL m0 L 11m . m L m L m p m t m L I TI r m — m r 0.0% F Limb? m0 L I . m0 P m0 L mo . mo . mo . m _ m - m — m L m P m — m — m P m t m L «sown P E0 . I P I . mo - mo L MD L m0 . m L m P m p m L m L ume w. rim: L m LT m t mow F LmO. I - mo - I . m0 _ m0 . m0 . m0 . m . m — m L m L m P m L m L m L .m t m p 80¢ - th-I LIMDme- mormo meLmOL b. MIL! m} mrthLm-mi-mtmL’Ihp 00¢ - . nadm .hm. . a L L A... L L f m L ..m k L fl b “m b - F m. P IIrILWLrIIILIIILIIlrIIIrLWLrII.. m . m .fi Imam 0.0m mamN n.0N m.¢m m.mm 0-ON m mH m-QH mu¢H\m-NH m-OH mum mam 0.6 mum m.0n fi. mfiphopw L! L L L L r L b L L - — L n r n + b F llllIII—u a Magda—HOHDHQHC HO Dada . Dmofldpfldo - k L L ( ._ . $4.3..m Snmmm mafiumwm 530 mam OH EH 02H...om¢ Wm n..).§»..r«.. AMI. 38%: SSE anemones... 3...... as magmas. S magmas ..osdfioomma .> mamas mu DISSOCIATION CHARGES BY’MEEFERB OF BRUCELLA INDUCED g.”ffl_ BY GROWING IN 10 PER CENTA SERUM snows. ' 1 . I 'Oultures ' ' ' r r T I I I Fr smooth type of colony opaque-smooth type of colony rough type of colony no growth ltuahm I II I Of the Br. melitensis strains, culture 301 produced R forms after the eleventh transfer. Culture 318 produced R forms after the fifteenth transfer. The remaining three strains produced Opaque-smooth types which persisted throughout the period of transferring. EXperiment No. 2 Tubes containing a one per cent solution of lithium chloride in veal infusion broth were inoculated with the cultures of Brucella. These cultures were transferred and plated out at weekly intervals for a period of men weeks. All plates were examined for R types. The data are given in table VII. From the data presented in table VII, it will be noted that no R forms of‘gg. abortus were obtained after ten weekly transfers. Opaque-smooth forms were observed in all cases after the third transfer. Strain 408 of’§£.'ggig produced R forms after the fifth trans- fer. Opaque-smooth forms were observed in the remain- ing five strains after the fourth transfer. In the case of figs melitensis, strain 518 produced R types after the seventh transfer. Opaque-smooth forms were observed in the remaining four strains after the fourth transfer. In all instances these Opaque-smooth forms were quite stable and did not return to their original 8 forms. EXperiment No. 5 All cultures were transferred on liver infusion agar slants, and aged for a period of six weeks. They were transferred on plain veal broth and plated out each week for a period of eight weeks. The data are given in table VIII. hfloaoo «o 3.3 AME?” .. 2300 no on? swoon 08s .3023 n hdoaoo mo 8.3 35303585 .- hfloaoo Ho 3.3 spoons .. . 0. mo. mo . . f L . m . m. — 8— WOLIB b p p — 8 n m. P m Two. mo? .Erm L LmOLmO. {Faker .8. m. Lm. mo 8 8 mo L L .m Em 5.0m 0.0a 0.3” 0.0 0.0m 0.0m 0.33.03. r L L r L L f . L L L . L Q9335? .mmbmm QQHmOAmo .505qu 850 mam fine a Gasofla Hm gonna MDomo amobmfl Ma mo mmmmag Mm @3030 ZOHadHBmMHQ ”My Wanda a. hsoaoo moo :0 a son a m hmoaoo we can» recess a m L; iwh L 1 M L MW L _ L m L M Ll m. r m L L L P m L m L I L m L Danni! Lb m L L L Lr Wm L m L L r Mr Li m, P W L m P L. L g P NH L F L m L slh!‘ L m L m L rm L L LII-lb L m L m L {P I! L at P m L L . duamnwm Raffles . L .Hflflmudfimg can)... L L F L .. L mmuw L _ . L mm. L t . L L L . H L P L ,_ L L L L L L L . wagon L L Ida-m cha- L L r F . nuances .nm. woman mac. . own. an 343m on. Emmamawma .59: cadm mind. mags to M464 Hm \ ; 58: .328 asses .. magmas... L... 3936 saanoomfin ....flua same... ll'lI-\ Three out of the five strains cflggp sinners studied procuccd R four . The R forms were observed as early as the third transfer and as late as the seventh transfer. In ten weekly transfers five out of the six strains of £3..gggg studied iroduccd R farms. Dissociaticn of this species was noted as early as the second transfer and as late as the sixth transfer. 1 Strains 301, 316, $18 of the Lr. zclitcr3 is cultures produced R forms. ftcr the first as mdi fl“ "th weekl;r trans- fers. the rmmaining cultures that did net dissociate retained their smoothness. Experiment R0. 4 The cultures were inoculated in tuhes of veal broth containing 0.1 per cent phenol. They were trans- ferred serially and plated at dd hour intervals. A11 pla tcs were c'reizllv examined for R types 'ho da :a are fiven in table II. From the results indicated in ts ble IR, it will be observed that all of the strains studied, retained their orig nal smoothness after fourteen serial transfers. Strain 438 of‘gg,‘§3;§ produced R types after the fifth transfer. It is interesting to note that strain 438 produced R types in each of the euperiuente that were performed to bring about dissociatien. .Lis organism was apparently quite unstable. 58.30 093 Anion .. m usages mo .93 50er I m ..lnlbllolbllolLllmP tfipolrlhlt tbmrtEP .lnm. .[TLIbIL-Inwt _ elrlPlrlhlLIhL . E . . . shoe assure . Iuosopunoa .um. - . . . . . . o . a a . . . . L . _. . 33.0.. ..w.._m..n .... ..t ..., ... ..u.....h .m ..,--..m.. “sheenfli. . . . . . . . . . . . . . . . Duchess chm” - b HP b ? b p P P b b h . ti Fr Ur . g. b P H oaOHuDAom Nansen Add“; Aozqflm Hand mum doc 4 EH 9:. duo ha ammo was. .5..ch axonmfl ”an. .343 mmmawad Nrm macahwo 3,199ng mMHQ oHH Manama our? ’5‘“. Growth of Cultures in 10 per cent cog. The inoculated plates, in ell esporiments performs ed in this work, were placed in large glass jars, secl- ed and grown in on M‘losnhore conteinin3 10 per cent carbon dioxide. "he plates were then incubated at 37°C V. Huddleson (1921) found that all the species of the Erucelln group grew best in en etnosphcre containing 10 per cent carbon di side. There are e number of ineitents used for microbic dissociation. In this work chemical agents, positive immune serum.end o3in3 were tee moons employed to bring about dissociation. All of the shove mentioned agents have at some time been used successfully as dissociat- in3 inci tents. The process of e31n3 the cultures on liver ogcr slants, followed by weekly transfer on pic in veol broth, gave the greatest amount of dissociation. She other etents used brought about dissocietion in only a few of tiie strains that were under observation. Biochemical studies of R strains. All of theR strains were repeatedly plated on plain liver agar, and the th10&1 R types were fished end trensfrrrcd on liver agar. The action of those selected R trpes were studied on thionin and fuel win. "he dye pletes were prepared as recommended by Huddleson (1-28). "he plot es were seeded with heavy suspension of a 48-72 hour e3or slant growth. Qhe suspension was obtained by washing the growth from an agar slant culture with a smell amount of sterile broth. The seeded plates were then incubated at 87°C. for 48 hours. The data are given in tabloid. Eron.the data presented in table.i, it will be noted that all R types 05.:rueella irrespective of their species grow equally tell on both the thionin end fuchsin eger'medium. It will be recalled that the smooth types OanEh shortns are inhibited in thionin medim, while the mooth types of $53. 1:33 are in- hibited on fuchein.mcdium. The B type of culture apparently deveIOps a resistance or tolerance to the bacteriostetic ection of thienin end fuchsin. This esperinentel work with the R strains has been repeated severel times to confirm the original reaction of these strains on the dyes end to strengthen the belief that these cultures that showed this ‘ particular character were not the results of contami- nation. Che results in each instance were identi~elly the some. Fermentatively the R and S cultures do not differ. Che R strains were tr nsferred several times on sugars and each instance the results were identical. Testing R Strains of the Brucella Group for Hydrogen Sulphide Production. this method first described by Ruddleson (1928) consists in the determinntion of hydrogen sulphide ~25- TABLE 11, 21111 mama’s 0:;- ms 21021111; 05 R 19021.13 (“22‘ 1:11: LEI‘JCEELLIA man? :1»: :12: mags 211103131 MED L‘UUJSIII. I Tf T“ T :W ; 231193111 : zuohs 1n ' 'Br ab ort ’ ' ' .—.6 W I r‘ ‘ 1? I ' O O I I 1 Grow 5 ' Growth T r grow: ; ' Grovfi: ' Grout Grg: ; 5h .26- production over a delimite period of 6. She Lermcl smooth cultures o£.Lrueells produce var; ing escorts of hydr03en sulphide. ‘15. 6L is produces s considerable amount of hydrogen sulghide gee over a period of four days..§g, stortus produces a considerable amount of hydrogen sulphide see over a period of two days, while 'Lg, melitensig does not produce hydr030n Eglphide gee. Shin, therefore, divides t? e sgpeeies of this group into three classes, namely those that produce hydr03en sulpiide (21.. sLi s) for a period of four days, those that produ1c hydrOjen s 1;‘ hide (Ir. shortns) for a period of two 6‘" end lestly the treiua cf‘gg. melitensis the t do n t produce hydrogen sulphide. Strips of lead acetate p's per are placed inside the tubes beside the cotton plug, the paper extending slightly below the plug. Che gar slants ere heavily seeded previous with cultures to be studied. Ihe paper is removed at 24 hour intervals and a fresh piece inserted in its piece. This is repeated daily for seven dejs. ixperinent fie. 5 "he R strains were examined in a similar mankier to determine their ability for hydrogen sulphide production. The date are presented in plate II. The R strains ofLQQ. shortus did not produce hydr03en sulphide as did the homologous smooth strains. the R strains of 1r. stis did not produce as much h"dzo:ezi sylphide as did the homelesous smooth strains. The R strains of Brucellstmelitensis behaved the some as .37. their 8 prototyges. lizper went E10. 6. SerQIOficsl dtudies on R Strains of the rrueells Grcup. ”ebbits were innunized using smooth and rou h antigens of Lrucells. Lech of the sni1sls received a series of five injections, which were 'iven at weekly intervals. Anti-ens of all the rough and smooth streins were finds, the or;snises being sus;ended in e 3.5 per cent phenolized physiele-icel salt solution. S1voth antiserum and R antisera were then obtained from the i;nunized animals. Cress-ujjlutinetien studies were then made on all of these various antigens using the different antisera. The cuts are firesented in tables PRODUCTION or ”as .23 y 79' TYPES or Cir/us BRUcILL a ”GE OF GROWTH IN HOURS 24 g 75"“ 7g_ I o 159 1L5— I I I I 1/ . I I ' I I I h L.J LA I I I I I I , L I - .. {/42 I ‘ L._J L. I I I 9’40 H d I I Var ‘ 5 pm I I F L_- I ‘ I I I I ..3/6 I I I Jo/‘ -28- onhu a cannon» .nm acumen o c e :.an, . 09h» m case .um coupon 00¢ ,onhp m namnouaaua .um a cannon oou . ++ . +++ b +++ P.+++ P.++++. ++++p++++b++++Pw¢++.++++p I . mm . .. 5‘. p I p I p I n I p I P I p I p I P I P I b I p I ., .. . I p I p I p I P I I I I I P I P I h I P I p . 4 o . mfi . . . O . o I p I u I p I P I b I h I P I I I p I b I P . ++++ . ++++ P ++++ b ++++b ++++. ++++P4+++P++++p++++p+++¢p I b N . ++++ b ++++ P.++++ . ++++b ¢+++p ++++b++++p++++p+I++P§+¢+. I . _ . ghmmuvfld. . .Hudzu .hm. . .moofi. . . P I . . I n I I I . I p I b I I I I I b I P I p I p .1 v ++++ ++¢+II.I¢4+ I+++ ++++ ++++ ++4+ I . . _ .2 ++++ ++++ ++++ ++++ ++++ +++ ++ I ++++ . “w . ONHQIH.oanIH. u I .zixI .‘ . :... I... I .1 .o I . I;. . . klI - b P I P b b P p b P u . . EEMmununa. . , q. . ”c: I , .,naunopauqa..un. . ‘ I . . » . - , m @Hmu. . P . / f CKmdeHWHS m wmemD EWHSQNJZE 434AQD..J ”5”»... .5 ma. 41KHT Z a? WHEHHJSHDQGGfi M 3&8 ) _ U I u o .IIA Y. 4 . :n . 2 ,\ ... . /. . . ., . C. O . . sum :5 ocuhmn co... 3:53.32. 35 no.7;- com Em. E . 4 . . . . I . I . I . I . mom“ . .IIIIIIIIIIIII‘IIJIII‘IIJIII-Illqll'lll-IIIIII-IIIIWIJ I d I a I I I J Nil- .IIIIIIIIIQIIIIIII-IIIIIIIIIIIQIIIIIIIIIBI-IIIIIII I 4 I 4 I II a ..HWWIIII. .lli‘iwflI‘tfl ‘I‘I¢§d *‘+‘d I d 4.1T- .jldilbfiufi 35:33.44. I I $J . bikini}... . . II:::I:I3I1‘ - a 4+ +§ . 00A. . III #4: . - gfififl . . . .9... u o o . I wr ”H50 Cum.- . b I bod... OWfiPIHW‘V: . I I . I . I . I . I . I . I . I . I . I . 2.3 . u I H IIIvh I h I I“ I II I H I J I J I I I J I J $1. a I In I . «I I II I III I I I I I In I dI I Jl I d I J IT- . . II M to I IIII I +I+ 4 ......I 4 #1:. +++mfi+m++I+II+4£ 4 I I f. . II II IIII +4.2. I I++¢I II ++¢+4 III? JIII. IJIIJMI...‘ .13 I I I IxTII. ‘ II I‘ b1“ 4 III ‘ III III III I‘ HIIIHI‘ . ., 1 II -I II II II- I w... .... ......nfl... ...._...._.Ihwi_.h.. ._ Era. o__m_ A. p_ ofiom. . «2.332.. ...m. . b I . I I. I. I.I.I. I.I.I.I.I. ”.826... a I I» I n I In I III I JI I I: I H - J I J I J I J I¥a . a I II” I q I Ihl I H I h I “I I J I d I J I J I d To . . +4 I. +++I IWI +¢++ w ++++ Wot; MIIII 4 «It‘d I+¢44J++4¢ I??? I J i... H §¢ I §§+¢ 1 0.9.9... I IIO... JOIOO J+§+§ d I‘.I§§¢J §§§+J :IIJ OOO‘N I JI *mlllu J a J J d J 0.. . . .&.€,namfih3%hmnéu ngngwrahn avafiap . . P . . (...:o:3.... .23., 4 . ....ho. 30...... . . .. _ w. ,c . Iflnduuona ohm. . I E omd‘émm wwHwoammEd a ...4. .3 aim. macaw?" Hgmoh 0..ng maamu 1444403.... a 4.4.... ...A... mwumgnm mu a“: M». NO NHHAHaAMHHDflmw< OHIHNH am...“ J. .I- MK... Rough etreine of :3. £112 and 13;. mlitcneie show cross-agglutination, while the rough strains of £59 cherte. e111 egflutinete L313 with its own specific R antiserum. there is no egjlutiLesiee of the R antigens by the smooth eerum, nor of the S antigens by the K enti- eere. It will be cheervcd from date proeentcd in tables i and i1 that the R antigens when inoculated into retbite reeult in the formation of antibodies that will agglutinete the R but not the smooth antigen of the corresponding hemologcue strain. inter-egjlutinebility I. of the R ant gen occurs between the R‘gg. enie en& E 3.11;. melitcneie, using; th correepenaing; 3-“: 11012101030118 antisera. The range abortue antigens are agglutinated by their own specific entiserem. these ett gene will not be agglutinated by the antisera of either R'gg. ‘ggigrnor R.§£p meliteneie. Reugh ebortue antiserum will not efglutinete the R antigens offl. 3333 end E23 melitcneia. Qhe smooth entiecrum.doue not e3;lutin:te any of he rough antigens, irreepeetive of the antiserum used. lorphology of R Cultures. The R strains studied were characteristically different morph0103icelly from their 3 prototypes. they differ both in size and shape. Che R ferns were without exception considerably larger then the 3 forms, sometimes being from 3 to 5 times larger. The orgegiema are long rode, very granular in structure and markedly c.21- plconorphic. Photomicrogreghe of R strains are presented on page 53. 1:3 all instances stair 313 : reactions with Grum'a stain were eigilar to the original S fonts. Colonic 1 Aepoc to of { Cultures. The colonial orpcarance of the R tyge colon: of Erucelle is very ei‘xiilnr in type to the R ferns obtain- ed in 331 1039113 3roup. Che colorioe are c311.ctericed by a very irregular contour, 1w1 ca es boin: extremely Jejjed, resembling the usual soil Spore-former colony. Rho surface of the colonies are wrinkled. The colony is flat Jit}l e dul.l eng2e crunce. The cOIOLiec Have a brownish tinge, which beco3ee more mg3kcd vith ego. Photorro p118 of smooth and rouyh colonies are presented on page 35. within the last ten years, many workers have ct some time or other cone in contact with 0.3331833 that mavo ruthe:r peculiar end interest 133 rca(r.icne. Arkwright (1:21) noted a variation in bacterial aggluti- nation by the use of oalta and specific serum. Griffith (1928) working “ith the gnoumocooci noticed the marked influence of L ;~1une eerum on the biolo;ical progertieu of the organism. Hhite (1“25) made eorolooice 1 studies of the Salmonella group with the hepes of classifying them and he too otee ~vea the peculiar re- action of these strains to BPCu ific iLmure scrum. This and the work of many others is too significant to be ignored, and to be looked upon as contamination. Photcmicrogruph of R Typos. 31511110 10 Lrnaella 3h0?3u3 culture 10. 43 magnificmtion 756 Figure 2. Brucella‘mclitcnsis culture Ho. 9 SR {lain-‘11 i‘ieafion 97 a Figure 5. Brucella suia culture No. 40¢R Magnificatisn 726 -33- { . J \ ‘\\~" ,‘z -/ .. I ‘1 T‘ \.‘ 1 | . ‘ ” 77/ 49:? ' a, “fig! _ / . _ t)? ’ [ Figure I. - ’1' \I ~--; ( / . ‘a—Il «7.31/4 < / . > 1" "l \V, f \ ' / Figure 2.. Photograph. of Rough and Smooth Colonies. Figure 4. Brucella euie culture 4023 Mcgnificati on $6? :; I? Figure 5. Brucella.melitensie culture 301R magnification 920“ éfi Figure 6. Brucella auie culture 402R Magnification 460 4}} Figure 7, Brucella abortue 4R magnification 460 5-; “I.— _A Figure 4. Figure 5. .136- The changes induced when e culture shifts from an 3 type to en E type are true variations. The changes induced are the results of shifts in cell character rather then the develoyuent eifn zed strains. The microbie inoitents merely cause the R ehcfieoter of the cell to develOp to a greater extent than the 8 character with the result that a change in colon; is obtained. The rate of the change may be quite greéuul Int generally it is of the letter type. Reversion of R Types. The R strains of trucelle were inoculated in 10 per cent R entiee em broth. e series of these strains were aged and transferred weekly. emother series was transferred serially every 48 hours. 311 strains were plated on plain liver infusion eger and gentien violet agar medium.es describeu by Hudelesou (1928). She date are presented in teblea.XIII. 317, AV and 3V1. beoaoo no o9? eevaounaoosu .- no 332. me 093 specs. .. a finance we sang n38 ... x -37- u . .4 - ...L. . . . ... . b- .m- n... m... 3.. n.> ..m e.n.fl.; 6.5m. . . a. F N- b L b; ii! IL 41 u . .. .. r... ...... r. ...: ... k . \ ...... ...:Q . .. «.354 55.3 E... a. as 333.3 3?. @9533V .503 “sexual"... m also . .....Z......,._...i_l_.¥_ Vita; I\ L _ v . .a 4 K J» lethr f -58- 333 we on? 03305908.. ... no 3300 no Oahu. flees. ... n 9300 no 09? smack I m a I it: I ff? _. o 65.3 ~3qu3 E5 so 393.4 new...“ 9... 3.. 3.3....” .523 32.13%. also mum 0H ...: 939.. ha nausea auburn. m $32523“ .55.. 33% as... .3300 «o 093 33.30.5033 I no .3333 Ho .83 goes. I a 9333 no can» seven I a ”I, ..3. 19A... . . U m. o p D e .1 OHM u o .P . tr: . . P . . p o . o.,. . . . nA-u . o ...le . u . . ”I: . . . . . .. . . . rd n.mH m. ..H .n.o .nub n.o non @- n.§ . ..N .nm N. .euh.ne a. . . 3. Pl E if _ 480.03 .. .2 3.33» 3,333.33 233333333333 333.93 339333: 3.3.33.3 3333.333 330 3.3.: $8 .3 3333.338 3-..... ....3 3313...? 3 433330 333.3 3 my -333... 3:33 20 3.33 33393.33 m3-» 3 e 3. 53333.33 .83 3339.33. -40- TABUS 32.71. RIL‘HZRSIQN Jr.“ R TYPES HELUCLD LY GHQ r3110 $11,141.; C211 10 PER 0115'} R 11.31.31.803. BR 3'3" TILLSL 1161116 01111171111138 .LRE PLATED A'L' ..LJZLY Iii 1L V1113 OI} PULL“ LIViiR AGAR COIETAIB- 11:0 1131:2131: 3110111111 (1 - 50.000) 1 O 0 :W 'Br ab-rtu. 115% '3‘2'3 9'3 -.'5 23'; 29' 1_. ' AK . I I I '8 z, I 0 $1.; ' ._.'R v ..4' 0 (my; ' .0 v I 0 '= m .-tcn ' ' .ol~ ' ‘ ' : o ‘ . - ' H 1 :' R - rough typo of colony 8 :- smooth typo of colony 00 - smooth-opaque typo of colony From the data presented in table 18, it will be observed that the reversion from the R to the 8 type was obtained in two instances. Br. abortus (culture No. 5R) and Br. suis (culture No. 408R) reverted to their homologous types. The R colony did not shift to the S form after twelve serial transfers. Table 14 presents data, showing the effect of aging the cultures. It will be noted, that the same strains referred to above re- verted to their S forms after the second weekly trans- fer. The remaining strains retained their rough colon- ial appearances. It is interesting to note that these strains were plated on plain liver infusion agar. This type of medium favors the R forms, resulting in the appearance of B type colonies on the plate. The data presented in table 15 demonstrated the reversion of the R types back to their homolOgous S forms. A shifting of the R colony to the S type was observed with all of the R strains of Br. abortus and Br, suis reverted to their homologous S forms. The R types of fig. melitensis retained the rough colonial characteristics. In this 085769., the cultures were plated out on liver infusion agar medium containing gentian violet in a dilution of l-50,000. This type of medium favors the shifting of the R colony to the 3 forms, for a greater number of strains reverted to their homologous types when this medium was used. Reversion of R types -42- by animal inoculationnsith a suspension of 3 killed orgmnisrls and living; R orgasm ens. Gr ffith (1928) was able to:revert B types of pheunococci to their homologous e typrs by injecting the R organism together with the killed 3 organism subcutaneously into white mice. This work was con-‘ firmed by Dawson (1930). This method of reversion was tried on R types of tie Brucella cultures. The suspension of living and killed organisms were prepared in the usual manner. The suspensions of killed organisms were plated out to check sterility. Equal amounts of the living R reanisms ahd killed 3 rgeninms were mixed together, diluted with sterile physiologiCel salt solution.se as to give a turbidity reading of 7 mm. on the Gage hephelometer. Living R suspensions were prepared in a sinilar manor. Guinea pigs were inoculated subcutaneously with the Lirtures of killed 5 and iiving R organisus. Guinoe pigs were also inoculated‘with suspensions of living R organisns alone.. A series of four injections were given.the animals, at weekly intervals. The guinea pigs were killed and outspeied. Table 17 gives the data concerningjpostdmortem results, tissues from.which o QFHiSLS were isolated, action of isoletel organisms on ;;eo , hydro3en sulphide production or isolated or3nimi 1.1s er a agglutination tests sith smooth abortus anti-scrum. In the guinea pigs receiving the living 3 or3anisms and killed ; organ 5L3, 2 strains Vere isokmted from tho l.iver, opium llunjs. 3 or313 cLs were also isolated from.t e liver. spleen and In 35. S or3nrisns were also isolfited from 3uinos pigs that receivod living K or51niozs, althon h the or3 1.1533 were not isolata 6. fr on all tissues t at 1-: ere on- tu"od. Study of reverted strair- It will he noted that al 2‘. types of the Brucella croup reverted to their 5 ooth homologous otrai s. The R col nies reverted in all instances, to the S type by injecting subcutaneously sue .nsion of killed 8 and living R or3nnisme. These cultures reacted the some s did the nerunl smooth types. in.re3erds to action on dye plate, hydrogen sulphide production and agglutin- ation. It will also be noted that none of the P. typos zero stat le rou he. Tee R types of Era molitonris were the most stable, since they did not revert to their homolocous :3.1eot’.1 tvpee h" rapid tm 11$ orzing. but did revert on eniual passe o usirj a suspension of living R o d kills} M 0333 ~ age. F3 "“ :‘ ‘f r. 84' 3- “)SY 9“» ‘13‘. ': .11: 233$ {3103417. :.'-L.‘ 5., '. r Isa-(.1 ., 1': 3;? '._" If .‘x ‘1 ‘ 1. j: ‘1‘: 1 J V' ’ “‘y '1 '5, 1.- '1 t" . .1.‘ ,, uL ‘ A V o M. J. ’1} L . 12;“? L' Min} , a. 1.2 2.3U1' n.) . . 1. 1111-1- 1.1.5.1141“ «1911 3.14.11. L» 1.1:??? :. -r - 1 a 1‘, 1: ~ . .-. 1. A. .. g was 47.1 11.1.1” 4 . - .11. .. . . . .. 3 .1 . « 'I<.‘ 1 - . .. , - ...- - . c 1.. . ». - .1 , . . 1. . ,1. g ‘r- 1' ‘1.‘:';~“ [111: v.1 {1'2‘f3‘1’ n‘ .112 "1"?“ -:‘ {v} -_~_.; V. 11' v. . . 3" l. ’ :J I; .- 1:. i“! x E: 1: ._ x 1 _.3' .‘ . .' ‘ V, \. 1 1" ... ‘. . IS hJIJsluLth V 5.3 3.33. if .5. L: :5 J31 J1.“ .. 33:41:) . .155. £3151- 3“? " -» ’ =11 1 22 1.31. -.L . 1. "1. 11.1%.» .11. 1.4- ;- . .1 - ' ’ «v ' ~11- +31"! 0 Poet mortem fiaéin3e ‘Gglturee ‘ TL1t'e3 of ‘T73fi3e3c3 eelfihiéE‘ I 3 t 1 i 3 .Guinee pig '111 tissues cultured ‘ieeletee firem 'ieeletee eqlgaeee 'yrefimeuiema ‘.Agglutinatififl test using ebortus : . ' ‘ 3170110111 :1. 11 ’ 1:111 11131111 ’11 11.1213 ‘ 3 serum . . ‘ 'tieeuee_ . ’ZhiemiL fincheinn ’; Q 3 1 5 6 ? i 1 n '33. enieraed numerener ‘ m ‘ 1 4 ' ' ' 1 1 1 . G. pig.fio.l '3reynehifie ioei. ' Liver ’grewth grenih ‘~ ~ - - - - . ‘leéO'1—10’1~80'l-160'l~380'1~640 1 {Vaccine 3163 'Ep. flamereue white 1 ' Pyleen ’ ' ’ i313 ’ 1 ‘ 1 T r Bacteria 5168 'foci. ’ ' 1711:1113 ’ ' ‘ 3*; " 3 ' + ' + 1 3 v 4, 1 : 'Qeetieleeunoamel ' Lteeeee ’ ‘ i ' ' ' 1 1 1 2 . 'Lungmgreen~bleek foci' ’ 9 1 ' ‘ ' v t 1 a 'Kiéneynncrfiel ‘ ’ ' ‘ ' ' ' 1 t z . _ 'lerge ebeeeee et ‘ * ' I ' ' ' 1 1 1 n ‘ 'acint of in ectiea ‘ _33 3:3 _' ' ' 3' 1 1 v 1 ' I ' 1' V 1‘ 1 i 3 ' ' ’ 1 3 Ge pig E0. 2 ' Lie mafiml ‘ (‘1 @1QQTZ 9 PGY'EE‘I :53; $63331 3'? 14;" ‘5' '9” ~11 us #- i 1 2. 3 + ' + ' + ' '5. ! {Vaccine 4303 ’Sp.~ white foei ' 3333 ‘ "" 1 I + ' 1 1 x 1 . iBeeterin 4008 ’Eeeticlee - normal ‘ gheeeee ‘ 1 1 v 1 1 z 1 1 3 'fiung ~ greemieh~ ’ ' 1 I I a 1 1 1 , t ’ b11111}: £0133. ' ' ' ' ' ' ' 1 I 1 . ‘Kiduey n wer331 ’ F 3 1 ' 1 I 1 1 , 1 'Abeeeee at point 03 ' ' ° 1 ' 1 1! I x x 1 ' in ~ctien ‘ 3 ‘ ' 1 1 v a v 1 . ' T a; I a? w E I 1 3 I i 1 t Gapig $0.3 'Li. men? ‘eerlfiuwhite. 1111 1* “W 111 1%. . 1 - 1 1 1 t 1 . .vao cine 4R 1 £04131 3' p a” ‘ :3gig: 3 1213 H (31,- 1:11; ,5. 1 oh ' .11. + ... ... ... m m l +1 1 4. ' + ' + v + ' + ' 3538103633121 4:3 F313. '- 1311113131116 1 3,3110 , , 1 1' a I I t 1 g . {2.111111% - 11ormel . 311111111113 . v I .3 . 1 1 1 1 , . . bunge- normel . . refiefi. . , '» 1 1 q 1 . . , 'Oon3eeted aree en . ” . 3 ‘5‘ , , , , , , . ‘left side of 119 1 , ’ ‘?; , , , , , , . 'humeroue, fillea , 1 ' V“): , , , , , G? . -1 'with fibrinene 3 § : "* . : 1 , , , .3.“ ' 'exufiate ‘ ‘" 1 , ' 1 . -46- The data gmeeented in table iVIII denonstnetes the comparison of agglutination of R and RS antisera, when homologous rcurh and smooth types of antigens ewe used. It will‘be Observed that the entieers obtained, as a result of injecting living H and kill- ed 3 organisms into animals, produced antibodies for both the R and 8 antigen. The R antigen was agglutinated in low dilutions. The R antiserum contained antibodies for the H type ant13en. It will be noted that the R tyye of‘gg. ebortus was agglutinated by its own specific antiserum. R [3. 9.2.13.9. and 53;. molitensie one not age-’lutinated b:; R '12:. ebortus anti eera. .4?- weapons :3 ... c 333309 in I can .g. 098 . 8* 3.35 ooh» smack I m sawdust 09m» 5093 I a b hrvrp-b br-r hr-p bh-r br-b F I O O U I I 4 q I +«OI 4 ¢ ¢ 1 +-I +‘I k Ph-p Link hhnbLbL LI-L LE-b b 'II‘O IVII 5 . vNOHIn.O.: ’f IIJI § rr-F O I -+ £0 F Lh-L FP-b 4 H‘LLLL ¢ I! § LL-L #+-» p bh-L b 1 L #‘ ++ I» +4 .9 #9 O .0“. Dom - .lb {by Itb.¢¢ pofld - #9 Fr. P1 4+ - III >M¢++mr III+P1+++p+I¢+P4++§F I » P L If? ‘P. LT: .b I b I P} 0 b»! p 0+ p 4 b I P .49 . L J p b F‘ \Jb b‘l .mflnv IpItwurowpzu—ortI §§OP.00$\F»§U§&PW‘thQHN§hO§§+b C bpb* b wags ME? L441? » r p t +++p I . 04+»HW¢+¢.I¢++PW++«. I -+.b '___- r-b -.LP- - . P . D- by-tpp-b- b b b in I- b - - - - .b I.b LW L} Lr » I ooob 9+4 b ¢¢++p+++wb++¢+h++++p I +0 b,¢I¢¢» +04+b+++ebmdflvp+++ub I ,L - wP, P . p ,P . .iP I n I P‘ -xfiy I > I by I P}- b +I+ib ¢++ . oo++.+++fi?++++»++++b I + ¢++ ++++ 904+ §+++ ¢+++ ++++ I .I ..WI ...m... ‘10 I 19...... -..mIe r Hr!- b IDH b} P b bp-bL-b bite p+~b r odmdmnaad cam dwmwmo MBHE amfla mauafiaHaDH01< .HHH>H.MAm