3G2 1% c n j a m i n W `i 1, Ii c niKl 0 8 w rb 1 cl 1`. A v c r y , to rcvir:\y t.ltc! (lcs~ri~tti~fts of othtr ttncilli found in soured milky 0C sil,I j j;:(. eftxrnctcr. Tltc first pltblishcd study in t,his conneclion is that. of i`,sf~~ :. tltc microorganisms present in Russian Kcfir. Since his obscrwti -' "I', e;ll*ricd ollt i1S early as 1.881, the nitt~tuc Of thC b~~t~riolo~i~al If!+`\ ' ' tl~;tt. t.int(! rrttdars his rcstl1t.s of do~tl)t,flll vndtlc for the pttrr)~s(? of t+clrnpi~ son. It, Set?mS I)roI)il,j)lt~, its I, ii It tt i S SUggCStS, thnt the b;~cillus C!itllCbiI rj i - spo1 . ;I, c il. II c ;L s i c 11 rri 11y K cr I1 hlongrd 1.0 the S II 1) 1 i I i S or M C !. +* :I - t (1 r i c 11 s groups. 01t i;ltc olhrr itwd it is quite lilwly tAtat tttc 13 :I c f, e r i it Ii, c 11, tt c ;t s i c tt m isolated from Kefir by 13 c i j c r i n c k wns the first indivi.. dtta,l of &is par~i~tt~~r grortlt t;o eomc under observation. A similar bacillus from tltc same source is described by v. F r c u d c n r e i c h who gives prcfercnce to t&c name I3 a c i 1 I u s c a It c a s i c ii s. The wry thorough irtvrstjgntiort of the f3gyptinn Lcbcn rni'b by R i s t and IC It o tt r y rcvcalcd the cxtstcncc of two somcwh,zt; distinct types of lactic acid b,zcilli, the S t r c p t o b a c i l- 1 11 s 1 c b c n i s and the Bea c i 11 u s 1 c b c n i s. The dctailcd study of their characters estnblisltcs their general relationship to organisms of the Cauca- sicttm group, and furnishes- nn nccurate basis for comparing allied bacteria. Three years Ini;er G r i g o r o f f isolated from Yoghurt two bacilli which correspond in many respects to the two Lcbcn orFn.nisms described by R i s t ;tnd I< h o u r y. These he designated as B a c I 11 II s A and S t r c p t o - b ;1 c i 11 II s C and it is the former which has been so widely known under tltc title of I3 a c i 11 u s b tt 1 g a r i c u s. Althottgft this bacillus, ttccording to t,ltc xtthor, produces a higher dcgrcc of acidity in milk. than does the strcpto- bncilltts, ;t,nd ~,lt~ltot!~lt it shows ctrtnin dthcr diffrrcnces in its chwacteristics, yet the rtt;uly striktrrg ~C?SClTlblilIlCt'S obscrvc4 wol1ld Fwnt to drttoic nn ittti- m;Ltc rcl;tt~ionship bctwccn thcsc two kypcrs. Unfort.itt4at-oly ;xettr;Lt,c corri- pnrisotts bctwccrt Gtcsc four or~~~ni~rns ttrc rendered itttpossibl~ by the lack of corr~sltondin~ details. Jn +1%X C o It c II d y described tmdcr 11te name -of B a c i 1 1 II s b tt 1 g n, r i c II s an organism isolagi,etl from Rulgnrian milk which is probably ident.iwl with ~.hr 13 a8 C i 1 I II s R of C r i g o r o f f. One of the most striking ftrntures of this organism wns the production in milk of nn ttnttsttally large nmount of lztctic a,cid, 3,23 per cent being found after ten days in~ttb~tion at 216~. In Armcninti Mazttn D ii g g e 1 i demonstrated tltt! presence of a somcwhnt similar bacillus, the protoplasm of which showed t,hc presence of more intensely staining granttlcs. It, was less xctivc! than B a c i 1 l tt s b 11 1 g a, r i f; TV s in tltc production of acid, yet; if, ~xltiltit~(l morphological and cultwal features peculiar to tltc C a tt c ;1 s i-f: 11 m group. .` `1'1~~ cxist~n~c of (z r&cd bacterium is suggested by the obscrvattons of G r i - s 0 II i f who found but. f;Gled to isolntc a long ganulc-containing bacillus in the Sardinian sour milk G i o cl d II. ?V e i g m a n n , 1; r n b c r nnd 11 \I s s described a b;~cillus rcpc:ltcdly isolated from Maztrn, which is ttn- dottbtedly idrnticni with that studied by D ii g g o 1 i. ()wirtg t;o its origin tltc nantc 123 a c i 1 1 11 s M ;L z 11 ft w;ts qplicd to il. Directly cotttpar;~l~lc to l.hc 0lwrv;ktiotts of C r i g 0 r 0 f f , I2 i s 1` aritf Ii It 0 LI r y , I1 ii g g c 1 i and MT c i g m iL I: n , G r tt b c r ittlfl 11 li s s iw? the findings of 1, II c r s s c n and li tl h II who isolntcti, in ;rddition to it b;teillus o~~t~in(~d from st?v(wl ~~l~~ttcr~i~l preparations ittt(l called by tltttttt B a c i 11 tt s b II 1 g il r i c 11 s ) a second orgwtism which they dtisigttntn as the Kiirnchcnbacillus. `fltcy found thcsc two bacilli to differ from each I other in several important characters, and these differeItces we srtffi: c@tt, they consider, Lo cstnblish their id~nt,i~y as two distinct species. The l;~~t.tcr organism, W}ICW stained wit II n,Iknlinc mc:f.hylrnc blur: or with t.h(b y (` i 8 s r r st;lill, is SCPI~ f.0 contain b T;I,I~IIIPS which arfa not, oJwrvc!tl in thcb (:cJJ J.)otlv of the 13 ;L c i 1 I 11 s 1, II 1 g ;I r i r: 11 s. Althollgh II i,j g g c 1 i mcn- lions t.h&c granules 1,Jiwc nl~ttiors arc ihc first. to consider this fQCaturc! as ;Ln important. ground for difforcntixtion. Tn nddition to this diffcrenw, according t.o I, II c r.s s c 11 ;~nti K II 11 n , the T(iirnchcnhncilllls is to J)c distingllishc~d from tJlr 13 a (' i 1 I 11 s J) 11 1 g a r i c 11 s in 111(1 followiriq rcsJ)cc t.s ; a) 13~ its longer and more slcndt~r form and its g-calrr `tc!lcJcmy to the formni-iol; of thrcnds and chains. b) 13y the a~J~pc;~rnncc of the clolonics of t.hc: Iwo hncilli. c) `L'llC 1iii~IlCll~T~~~i~~ill~lS grows at, lower tcmpcrnturcls th;Lrl dew t,hc 13 n c i 11 11 s J> u 1 g a r i c u s , good at 37" and 4Y, n&t at all at b300 YY and only slightly at SO", whiIe the 13 n I g a r i c II s grows best at 45" to 500, hut dcwlop s slowly ;md feebly at 37". d) The former does not grow on pot,nto, while the' latter does. c) Milk is quickly ,and firmly cong&tcd by the Kiirnchcnhacillus at 37" to 400, the T; II 1 g a r i c II s ore the other hand prodr:ccs only a soft curd in milk and l,lj(~n orilj after scvuxl clays incuhntion. `rlirsc charactcrs nscribrd lo tlic 13 a c i 1 I 11 s 1) 11 1 g n r i c 11 s by the nuthors arc not in ont,irc qrccmciit with those dctscrilxd by others. This mnttcr will J$ more fully discussed in ano,tlicr part of t,Jiis paper'). \Wicn c,nc rcvicw and c,ornparcs the original dctailcd descriptions of the various J,acilJi mcntioncd af)oV(1 it is npp;ircnt that ;111 tJicsc organisms J)OSS(`SS ill COl111r1011, to ;L jilY':lt(`r Or JWS tJ?grOP; (`crt;\iTi (:h;lril(:tc~~iStics W]liCJl would swan t,o justif{, tJlc>ir' c:J;lssif`ic.a.iioII in ;I, c:ro~lJ) scJ)araI.o ;l,ntJ clist.inct from othctr more usuA organisms J)roducing Iact ic acid in milk2). It, will 1~: found furt,licr that many of thaw morpholo~ici~l fca8f.i~rcs arc included in the summary of distinct,ive (:hiIr;lCtPlT of the group Iz ic c t (: r i II m c a II c a - s i c u m (Kern) L. ot, N., as wvic~wctl hy I, ii 11.11 i s , "wo disl.iriguishcs Six ~l?ll(Til~l tyJ)cs, viz. ~- 1. hlilli. COil~llliLl;ill~, g:\S produciIig, Tj n c. c a s c i 17rcudcnrcich. IT. Milk c,oagulnting, non-gas producing, (R ;I, c. c n s e i Leich-' mann). UT. Milk non-coagul;lt.ing, gas producing, (13 a c t. c a u 6 n s i c II m). IV. Milk non-coagulating, non-qs producing, (R n c. II c 1 b r ii c k i). V. Slime prbducing iypc. VI. ,,~IZ;lniic~llt)iI(lc?nc~r" t,ypv. Among fhct bctt.cr known c~xi~nll~lcs of fhcso six types L ii h n i R men: t ions the va.rious vruict~ies of the I3 a c i 1 1 u s c iL s e i Frcudenreich. I, cl. C f, 0 b :I, C i 1 1 Ll s C :I, II C il S i C II S Ikijcrincli, 13 a c i 1 1 II s c R u - c a s i c II F .I~wr~tlcnrcic~h, 13 a c t. c r i II m c a's e i I-411 Leichmann etj Iii~zarowslti, 13 :I, c i 11 u s D 0 11, r ii c Ii i I,cichmnnn, S t r e p t 0 b a c i 1 1 u s 1 c b e n i s and 13 ;rcillus 1 cb enis of Ris t and Kh oury, and t.hc I3 a, c i 1 1 u s K ;I, z u n of D ii g g (f 1 i. Similar organisms men- tiorzcd by I-I r i n (1 m a n n arc 13 a c i 1 1 II s 1) a n i s frrmcntati ;~nd 1, c JI t 0 t. 11 r i x 11 II c c a, 1 i s. I< II I1 t % e IliLS 1'ccc111 ly si~gp~stcd the probable rcl;ltionship existing hctmccn some! of the bacilli of t.his group and the 0 J) J) 1 c r - 13 o R 3 h a - cillus, 13 a c i 1 1 11 s ;I# c: i d 0 J' Jl i 1 u s ) 13 a c i 11 II s g a s t r o p h i - I 11 s ) and 13 ;I c i 1 1 u s b i f i d u s c 0 m m u n i 9. Even mqro recently It 0 d 11 11 ;I ass&s t,h;lt thcsc five last named organisms a,rc identical. 'J'hct rc~lationships csistiiig hctwoc~n these va.rious lactic acid J)roGiCTng h- Cilli as might Ix disclosed by nn accurate comparative study of their morphological, cultural, and biochemical characters offers an interesting ficlld for furthfir invcsfigation. Such iin investigation would undoubtedly osta,hlish thn idrntity of scv'cral of thcsc bacteria which at, prcscnt arc wnsidcwd a8 dist.inc t varictitts, ;tricl frut her would yield a more satisfactory basis for il dcfinik clnssificntion. Ry no means the! least service crendercd would hc fhc abolishment or ch;Ingc of some of the wnfusing and in- iLpprOpri;ltC Ilq g:I es. According to prccedcncc t.hc name 13 a c t c r i 11 m c a II - c a s i c II m c uld bc used to designate the group. but this name as well 2s the name 13 a c i 1 I 11 s iV a z n 11 , 13 n c i 1 1 u s b u 1 g n r i c u s \ Rncilli~s l~hcnis . and S t r (a 1) t, 0 1) a c, i 1 1 u s 1 c 1, c n i s Iiavc scan1 jast~ific:il~tiOll when d)rlsitlc~rwl ll]""l t ilV ground of t,llc:ir iI(tCllI'ILCy 01 dcscriptivcness. It has I~cLrl the purpose of the- prcwnt irIvc:stiption to ohscrvc undc~r idcnticnl conditions in a compar;~tive way t.he salient features of a series of lactic acid producing, milk curdling bacilli originally isolated from oriental milks. Tn ordc:r fhaf t?l~ wsult s obtaincld mighl, scrvc as- it ~bnsis of compa- rison in further studies of thrsc and nllicd organisms the ,znnlyticnl schcmc of L r h m a n n and K c u m a n n , and the plan cndcirscd hy the So- citltv of I\mcric:m 13actcriologists wcrc adopt,od. Such omissions and vnrin- tion's were made as sccmod to 1)~ indicatctl by t.ho condit,ions met, brat thcsc wcrc of slight, importance. `I'hc Organisms. - `J'ho various cultures chosen for study wcrc of following origins; hlg. ltrccivccl from lh. .li (h .q I' c (1 k n of lirc Inst,itut I'mtwr, hl:mh 1908, uniler tlic nilrnc ,,I% a c i 1 1 0 An originnl' culture rnnrkrti t) II 1 g n r 0 C 0 h e,n d y." 13. 31. .,T3 n c i 1 1 0 b 11 1 g n r c" silpplid by the Societo Lc l~crrnent of Paris to their hncricnn representnt,ivc, August 100% Ld Il'ronl the L e cl c r 1 e I,:Ll)orntnries, New York, August. 1908, originnlly fr0111 I'rof. 31 c 1, c II n i k 0 f f. 13. K. I+OIU li r L 1 , Odolw 1!108, nuwkc(1 ,,I< n.c i 1 1 II s b II 1 g IL r i c 11 24." I'. I(. I'rom I< r ;i 1 , October 1!)0!3, mu-kcd ,,I< n c i 1 I II s p 0, r n I n c t i C It S". h--l lsohtctl Octolm 19OR from ,,Iincillnc" prclmretl by the I,nctol~c~cillirlo CO.. ;;c:w York. 7lL-m I 13OliLtC!tl fro111 I,r~ctohcillinc llldt Ol~t~LiJlC(1 iTl'aris ,lUly l!)OH. 7-3 Isolntc41 September 1908 from Lw~ohcillino 3lnlt lmptmd l)v the l+mw- h~erirnn J'ern~ent Co., Kmv York. IS-1 19olntctl froni Ihcilhc in hlnrc:ll I!lOS. ~h~crvntion~ on certain Lactic Acid T%actcria of tllc :;0-dletl f%ul,gnricw typC!. I.65 SS~T~-I Isolated ilfnrcll 1908 from hour milk obtained from native Syrian8 in Brooklyn. \lT. Isolated 1)~ and rcccivcd from Prof. K II n t z c of Jdipzig marked ,,B a c I. Y 0 g Ii II r t . V n r i n 1. ii t W". 35iC I Isolntcd October IO08 from ,,Zoolak", a commercial 3Iazun prepared hy Dr. D a d i r r i a n , Xew York. 3 Gib-- 1 Isolated October 1006 from a commercial Armcninn Rour milk. il IL--l I~olat~ecl October 1008 from Mlnzun propared by native Armenians in New York. 4L'u-1 Isoln.ted October 1908 from sour milk prcpnrcd I)y nnt.ivc Armeninn~ in Now York. Ii. 13olatcd by and rocoivctl from Prof. I< u n t z 0 of. I,eipig marked ,,Kiirn- chonbacillus". For purposes of convonioncc the cultures from Bulg. to W. -will be referred to ns Type A, llrose from :jli~---I to K. inclusive a~ Type 1% While it is qi1it.c prol~nhlc t,llat the above list includrs strains .lirl,ving a common origin yc t thcsc hayc~ ~V.WI subjcctrd lo vnrying conditions of culti& environment. In ardor to Ie;Lrn t,he influctnce of long continued RO- litary cultivation, of symbiosis with yeasts, bacilli and cocci, and of culti- -vation in different incdin th,e possibly identic;il stra'ins were therefore in- clndcd. Thcsc sixteen eulturcs wprc cultivated and studied simultaneously dnd always under itlcntic;tlly annlo~ous conditions. In addition to the nbovcl some twenty differrnt strains of similar hncilli were isolated from various sources, comprising commercial ppnritlions from Gcrmnny, bnce, Eng- la,nd, Scot.land, Switzerland and t,he Unit,cd St.ntcs, also spccimcns of sour milk obt.nincd fror?sont.hcrn Sihcria, Sofia, and from the natives of Armc- nia,, Syria., and Egypt living in i\;rw York Citv. Thcsc isolations wore ax- complishcd durin, u the COUI+~P of 1.11~ invrst,igniion, .;l,nd t,hc organisms thus koIxtxr~ wCrrx conipxx~d :ls t 0 i htxir rxr~nfi;d fc?af.nrcs with 1 hr? skains nlrendp st.udied. No new types wcrc found and since none of thcsc orgnnisms showed iUly significant varln,tions from those listcltl no rncntion will be made furthrr t ban to asxocixtr~ t,Ilcrn wit Ii t hr> t.y~)rls incI1ltfctI in t,hc prwcnt, study. 1. Whey :Lpr. `) Th& following charnct&&x are common to all- strains; wide variation in length, from 3 p. to 50 +, breadth about 1 p.% Nearly all individuals are in- tensely Gram pcsit'ive and these show regularity of outline. They are straight to slightly curved, with rounded ends and show no vacuoles or gmnulcs. All strains R~OW invo- lution forms exhibiting vacuoles, ofton appearing a~ empty cell membranes. These forms are Gram negntivc and vary grqatly in both dimensions also in form. All show a tendency to chain formn,tion, ,somc being nrranpxl in chins of 0 to 2.5 segments. The chain may contain both Gram positive and Gram ncpativo individuals. Gram negative spherical hodies, varying from 0.26 (L to I p arc srA3n adhnrinC: tlircct~ly to the 8ides of some GraIlI negntivo indivi(!ualf3 in &7CIs of `l'yp B. 2. Wlicy. Tn their bcha,vior in whey thrw? organisms cshibit some fc;lturcs of drkdcd int crest. `I`hc: icndcncy tolvitrd dcgcncration and involution is ninrlird. In 111~: c>arlv st:~gzcs of iricllh;ltion at. 37.5" or 44" t.lic bacilli arc uni- - form in sizrb xrid intc~nsc~ly G r a m positive whil(b in tli(b succeeding stagy thr irregulnr. vacuol;~fxd~ lnflatrd and ruptured forms predominntr. `Fhcsc Iill t(!r forrtis illVilTi:~l~l~~~ rlrw~loriw. Iktwrxn 111rl 18th xnrf 24th hour of in- C!lIl)ilt.iOIl at, -14" tllv slrairls of t,yI)t` A Clc~VOlOj) il ~tlor~~ll~~logi~~lI pecldi;uity first noted by I< u ii t z (`. `I'his is thcb formation of oval t,o Iridnrlv-sh;lf)cd -~~. -__--- 1. Whey ;lgar pla tcs'). `I'hr! following description is :~pplicnhlc to all 111~ organisms studied with l,hc cxccptions as noted: Growth u'n.q fir& noted at, the end of 24 hours nt 37,P. Co1onie.s nro then minute: a) Surfnce; nt 48 hours about, 0.25 to 1.0 mm. diameter, irreplular, filamentous, curled, flat,, weakly rcfmct,ivc; wit,h low mngnificnt,ion t,he structure is found to consist of n mnss of cnrlcd filiLmcnt.s, often in pnrnll~l strantln, ns in Ant,hmx colonies, often streaming; are trnnslnccnt,, t,inged slightly bluish by reflected light nnd showing hlui.sh white flocks ,or iHlnnd3 more or leso recifqplnr in form. Uie me&um is slightly clouded immet?ia&y nl)out tlic point of growth. No tenncit,y to the ~urfaco iR shown. 1)) Subsurfncc; hIinute woolly t,uftR, delicate not dense, composed of hair-like p-ocesges radiating from the center which is yellowish by t,ransmittcd light. Exceptions: !CjtrainR Ba--1, 7a--I, 7&--2, IX--l ~Iiow u distinct, entire, cvcn periphery. At times tho growth produces no clouding of t,lle medium. ,Frequcntly the colony appenrv to be nucleated; the center is darker in color and hns n slight c;levntion, being umbonate in character; This feature becomes more itppnrcnt, ns drr-elopcmcnl propwc.~. The nucleusmny 110 wrrounded by n compnrati- vplv clpnr zone and t.llis in turn 1,~ II bluish white filnmrntous bordor. NO chromogene.si!+ is observed. .\ sour odor is noti&l,le after 48 hours incubation. (See plnte~ V. Vl.). J\`liilc thr gro\v:th on whey qnr furnishes no points of' type differcntia- tion yet it scrvcs to df$initcly separate these erg-nnlsms from all bnctr?r;\ not included in thtb group 13 a c t c r i II m c n u e a s i c II m. The resrm- bla,ncc to I3 a c i 1 1 II s ;I II t h r a c, i s , andinawayto I3;Lcillm sub- tilis, although borne Out'to a' sligkt dcgrrc by the .morphology is no more Ihan interesting. I, II r r s , t n nnd K u h n consider that the growth of' the 13 a c i 1 1 u s b u 1 g ;L i * i c u s in the form of round regular colonies having a smooth periphery is diagnostic of this type. Tn the present instance it was ob&vcd only in those s&ins which hnd been snbjeoted to cultivation in pure cult,urc for a pried of ;I y(`zr or niorc. Since it was ncvcr obscrvcld in lli~ other nrganisnls nl' !?fl)~ A if, niirhl. s(`(`n1 f.h;et. t.liis JY;;;IS 3 c:asr~;ll or acquired charxcter/ 2. Whey xgnr stil~l~s. The growth first nppenrs at. the end of 24 hours in the upper prt of the stab a.7 n woolly filnrnent. At the end of 48 hours the development progreascs downward, delicate hair-like procccsses extend lnternlly from t,he line of growth nnd the medium becomes sligt,hlSy clouded. After 3 days incubation the growth is rpnrked along the entire line of inocnlatlon and becomes nrhorescont with dense clbnding of the qpr. The surface is wxnty will1 littlc spreading nnd iR nearly tmnspnrent~. Further inwbntion intcnsifiw t,hesc c*ll:Lrnctcri~t its without causing the nppenrnnce of further distingui.qhing fenturcs. None of fhc st,rains studictd showed ;Iny marked. vnrintion from the al,ove, This growth would seem to constitute R group characteristic. 3. Whey gclntino ~)la~tcs. (10% gela.t,ine.) - Owing to the comprati- vcly low incubn,ting tcamprraturc t nccxssitaicd, this medium is poorly adaptrd for the cultivat.ion- and differentiation of this class of bacteria. In several ocpcriments the onlv st,rains showin[~ b growth were Bulg, B. M., C&---l, 7a--I, 7 am--2 and IS-I, rkd in cnch case aftrr long incubation at 25O to 27O the colonies were niinut,c. ~~lt~hou~h a microscopic examination showed their Stl'u(:llIrc~ to \~c idrntit:al with t.IliLt, Of tllr whey-ngar COlUIlitX No growth U':IS O~lSc~rlf~r1 ~)rfOrP Six tfiLyS \Vlli~O l1.11 fr(Tllly iSf~l:ltlY~ StrilillS fClilPd f0 d(`\r(`JOJ~. 3. Whev pl:Lt inc `: t :11x4. -, :I1 I'ir-st no ppwtll what,cvcr WilS ohcr\~cd in t11v (`:lsf' of 111P or~;lllisIlls of' typo 13, ilIt 11011~1~ If'J)Cil~t,Pd at~tc~rnpts wcrc mndP lo c:rrlti\`;Lt(b ~III~III in ttli!: III:~IIIIP~. l,iltflr, aft (`r coritiriuc~tl cllltivi~tion in rttillc ;1 slight, tf'JldPllC)' t ow;lIYl crf)X th WiLS rn;Lnjf(3tc!d, which ncvcr PXccPtlPcl :1 `) In thi.7 scrics as ill it11 otlmrs liriles~ ?ip&iI menCon is made the &dium was inoculated from n. 24 hollr \vhey cultiire I)lrmted in turn' from n 24 hour milk culture. Tile n~etliiim contnined 2 per cent, n,rrnr. 172 By I< c n j a m i n W i 1. 11 0 and 0 x w a 1 d T. A v 0 r y , 7. EJl%ymrs, `l'hc addition of wlcium c;rrtibiii~fx, calcium chloride, and zinc chloride in CSCCS~ of the amount required to ncntralizt! the acid pro- duccd, falicd to prcvcnt, coagttlntion of the milk. Wtis might w~ur! for the prwxw of ~JI cnzymi~. 8. Acid Iw)duct,ion. It. is hoped t0 cont.inrtc stntlios in this tlirwtion. a) Milk : hlcthods: Tubes containing IO cc, each of dccrenmcd milk were stAri. lized and then incubated for 48 hours at 37.5O and allowed to stand for several days at room tempcraturo to influro fltcrility. AH .tubea in oac!l series wore filled from the same lot of milk. Those tubes were inoculated with 0,oS.c~. of a 24 hour milk culture and in- cubated for the determined periods in a moist atmosphere. At the end of these periods. 6 cc. of the milk was diluted with about 46 cc. of water, then brought to t.he boiling point a.nd titrated with n,/Io NaOII, with phenolphthalein as indicator. Each serieR includd two or more control tubes of t.hc same sample of mill; incubated and titrated under si- milnr conditions. `J'ho titre of the conbrol in each case WM. subtracted from the t.itre of : tlte corresponding inocul&C!d tubes. Whey culture8 vyere f3imilarly inoculated, cont.rollfxl and titrated. The fignrcs in the tnh!es represent the quantity of "/1o N&H, rcqnircltl .to nc?tlt.rnlizc 10 CC. of, inoculn.txxd milk. Of)servntionR on cort,njn Lnc:Lio Adid Bnct,erin of tile So-c!&lletl DUlpriCllS typo. 173 :ici(l cq~ml to it~botlt iwicc the aniorlnf, prodwxx1 by tfir strains of Type 13. `I'llis would seem f.0 constitx tc c'm inl~)0I%itnt l,ypc ctiffowncc hitttc?rf;o tinnotrd. E. No significant diffcrcnces bctwrcn the types or strains are shown in the acidit.y produced in whey. The small nmout of acid formed in this ~lcdil~~ is in marked contrzt to thr quantity found under analogous con- ditions in milk, 1.0. Acids produced. ' A. La&c acid : `l'hc statc~rncnts of various authors regarding thn natttrf! of the ln.ctic nc,id produrxd by this group of organisms show a discordnncc rcfcrnblc prtly to the methods of analysis employed a's well as to inherent differencrs cxhibitcd by separate specks. Grigoroff reports that both the B a c i 1111 s A and the S t r e p t, o b a c i 11 us C produce inactive a,cid, hut the method of d~tcrm~n~~t~on is not given. From a 5. d:iy cultlrrc of the Bacillus bulgaric~~s B.crtrartd and Wciswcillcr isolated the lactic acid by mca(ns of the zinc S&S and concltldc that the acid produced was n mixtwo of the lacvo nn,d dcstro modificbkms wit,h a p-v- dominance of the Irttkr. In quoting frorq the work of L 11 c r s s c n and IC 11 h n certain authors have erred in !tnting that they found only the dcxtro- rotatory modificstion in cukrlrcs of t,he I3 a c i 1 1 u s b II 1 g a T i c II s and Kiirnchcnba~cillus . In the original pnblication it is specifically mentioned t,hat "the whry turns the plane of palnrizcd light to the right". This can scarcely bc int~erpreted a,s indicating the prcsencr! of dextro-rotn- tory: lactic acid. EM r t r a, n d and D WC h a c c IC find that the I3 u 1 - g a r i c 11 s produecs t~~c&y tqiitd ~~I~lol~nt,s of both the right and left acids, and t.hat. this action is tltjc to an .i~l~r~~~~~~ll7~~~,r I;tct;w. `I'hr! st.rains stadittd by 11 C i It (! 111 il8 I1 11 tivc acid. :jll prnct~rd i I?(> itr;trtivck' t.yl)c without, iL tr;wc* of ;L+ In the prcscn1, invcsiigntion the following method was adopted; The whey obt,n.ined from 500 cc.`of the milk culture was acidified wit11 phosphoric acid 5 volumes of water free' other wore added, then thoroughiy ngitateci in u shaking t~ppart~tus. This step QXLS repented five times and, after an exact separation *from the whey, the ether ertrncts were united and ctw, por~ted to dryness in vn~no at 450, Tfle residue wns taken up in water, tho solution clarified by fiItrnt,ion t.hrouph animal charcoat, nnd after the nnfditiort of nn OXCORR of zinc cnrhonnto it WLS boiled &d filtorcd, -4fter concont,rstion on n wnt43r bnth the solution \vu9 nllomcct to stand. `I'he crvst& fornleti Wvgre removed by filtrntion. I"twthcr crops of cry,qtlLIg fornling in thcj .Wcc&.qivc filt,rlhtcs were preserved nnd ex~mineci scptwntcly. WIS nccornplished in the usual mnnner. WlC detorniirlrLtion of the water of cq3tr\llisntion The: nnnlyscs of th[? zinc salts obtaint4 from the whey of 24 hoilr milk cultures arc a,s follows; SXfV---I lhllh' B. 3%. Led I{. Ii. 1'. K. W--l 78-l 7n-2 1x-1 IV I Culture / Per cent, `I`ypo If. / n-ntcr 12.77 I" 01 U.. 13.04 ' 13.05 12.88 Cntculnted for Zrl(C,II,0,)2 `- 2 I-I,0 1" 90 I. c-i 1' 0 w c q II a I 1 5' I\' P 1 1 II 11 a f? 1 a c r 0 11 i c a n tl ;I, n ;I, (t r 0 1, i c c 0 n d i -t i 0 II s. 0 p t. i m II m t. r m p I? r n t II P 11 f 0 r ur o w t h is 44*-45* . 17 a i r E r o w t h it t, XV, s I i g tl 1. ;I b 25* n 0 n c 11.t 20". 3 N 0 g r 0 IV t% 0 n p 0 t ;t t' 0. iI 1 0 It ,g t tt (1 s t a h t 11 c growth is ctlncifornt, t Ii r e 5 d 1 i k c 0 r h 0 ;1 d (I d , rarely showing hori- zontal projections. Agar stab, ;LS a rule, wc;Lk- 1 y d c v e lo p (3 d. N a r r o u r i h tt 0 11 0 r d 0 t t e d g r 0 w tS 11 iL 1 0 n g S t il b. `V h e p r t: - scncc of sIrgiLr in tfttl agE:.r or gclcztinc causes clouding. I n b 0 11 i 1 1 0'11 , when growth ta 1~ es place, 0 nl y a w h i- tish sediment is obser- ved iin the clear fluid, while the ;1ddition of dnxtroso or lactose pro- d 11 c c s a w e a k 1, 0 s t r 0 11 g clouding rzs well as an increase i tl the sedi-. ment. On potato as a rule there is no dcvclopmcntY at b e s t i 1; i s s 1 i g h t,. ,hl i 1 k i n g c n (! r ;I. 1 i s c 0 :L g 11 - I a t c d III 0 r I! s 1 0 w 1 y t 11 it, 11 t) y s t r 0 p t cf c 0 c, c i. S 0 m 0 i n d i v i tf If a. 1 s g r 0 w fi !I 1 y s 1 i g h t 1 y i II 1: h i s m r d i II m. `F II c 0 p t i tn II `m f 0 r f: 0 n - g II 1 iI t i 0 n I i c 9 In 0 s t I ?' f, e t w e c Xl 400 a I1 d 50", f 0 I s 0 rn f! 30o. `I` 11 r 1 ;I, c t i c a c i d f o r m tt tl i s mostly libcvo-lacC,ic acid, m 0 r c! r n r e 1 t! y t h f' i n n c t i v c 0 r d c x t. r 0 rn 0 - d i f i c a t i 0 n. 0 n 1 y PL trace 0 f vo 1iht.i lc aei d s nr c prod II c P d, i f a t a f 1. ,, S 0 v e r tt, 1 0 r g a, n i s ttl s 0 f t h t' `g r 0 11 p 1) 0 p t, 0 11 i % 1% (7 ib s 1: i I1 1' a t, h 0 g c n i c i t, y h ;L s 11 1' v (' I' b (5 r II 0 11 s c r v c d. \T 11, r i ;I, 1 i 0 n s i n 1. I1 c c h a, r ib c i c r i s t i c s 0 f 1, 11 c v :I, r i 0 11 s s t, r ;L i n s p 0 i n t, i n g t 0 t y p 0 d i f f (' r (1 ri t i a t i o 11 a r (' -- I Type A. 7' y p c IS. IV i t h L 0 e f f 1 e r `s m I) t h y - W i t 11 t 11 c s c s t a i 11 s t 11 c ,,_ . 1 c II c b 1 11 c! 0 r tv i t It N 0 i s - p r c s e n c c of intcnscly serfs S t ib i n t h 0 `11 r 0 t. 0 - staining grItnu1cts map 11'1 as 111" i s 11 0 nt 0 g r n f; 0 II'S - ft e d f: m 0 11 s 1, r ;I, t. 1: tf i II t, tt f' 1 y s 1. iL i 11 e (1. . p 1' 0 1. 0 1' 1 a s m. 1' r o d 11 c c 2.7 to 3.7 11 c 1: c c fl t! I' r 0 d I1 c t? 1.2 t 0 1.6 1' e r c e 11 t 1 tic t i c n c i d in m i I k. lactic acid jn milk. The lactic acid formccl is `f'hc! lactic acid formed is t. h e i It ib C t i V (! m 0 tl i f i - ;~lwnys the la&