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Introduction *** Pretent inTwtiK«tion — OMienil procwJure Project of invitttiKatioii Clawied of onraniKnilt inolntwl ^8" MedU employed *** llethodK 1*' Can* of Rardinea Apiiearaiice of earn* and eonditioii "f <'(tiitentit 189 Cana oxamitied ^^ Organiama of the ga!>-prod«einir type Detailed deacriptton **2 Oaa-producing organi»nii> Summary— Morpholodicttl and Biolo«jical fcatureo 208 Biochemi^ al reaotiona 209 EsperimenUl awelletl oan§ 210 Non-gae-producing organism* *11 Brief General Summary 214 Keferencea 2** • oeoROE V SESSIONAL PAPER No. 3U A. 19tt XII Tin lAOTEBIOIOOT OF iWIlLED CIMITID USDIXXI. tntroduriinn. »re ,i„ i„,,,.i„,i„„. ytz:^^'::rvL^';TT.:'i"'' ' '■■-■. '—'•■■k- ea^. .>.o. ...ei.,. H..e«.^ ........ :ilin:;:L;;r:;':^-"i^..:r bear a olce reH,.mbla„c* t ^on^ ^rd^d t „ ' 1"' ""'T' "-'*"'" "* ^^"^ "">»"'« were iiiocii ated nto sterile cang of UW^t^, ...j ■ , "" "'"■ "**"• *^«;n of the four ..ti.«ed. Some of the o'^iZ /h^:^^, '^ '^,1TZ?*" "'" t """' """ tain of the .trains de«.ribed by M.ophai a.td Br^e K.^P^^? •" ""^'li''""* **• '^'• a definite opinion a. to their mutual identity ' '* '* ""P°''"'>'*' »" "'P"*" K ed and ei»hty-«ven .Sed "e ean- '£"0,3 '" '?"'' '''""'•' '" *"" - .od and eiKhty.8even i.wlkJ;ardinrL,^- VJ "'*»«"'.•" '» a .,K,rn.former» The only „>feren,* I «.„ find t^ the bic lluf ol vSi "Tm ?"'' '''/ ^'•'''«'">'' detail*. In thn fall of last ve«r T l«. • « '^Wn* fa.U to ^,vo full cultural time her report wal „ot avSe as I haTrT'^L*'''" "'''' ^'' ''^''' »»'» »* t^** that it is noVyet publislr From 1 ret^„rc7ti":H-T \'"""'^^' '* "'"''""•' read before the Society of AmericarBaSCi t^l am unaW T " ' ""'"' ""^^ my strains with the " Bacillus A" Tl,« ,ltl T ""'''* *" <^mpare any of point in laboratory media but statl that the'" '^' ""* 7""*'°" **"« *''«"»•' ^*^^ of sardines surviZbathi^K in Sil'ltr^^^^^^^^^^ "S^iW 7"'^*'-" ?'" T' m my ri-port no experiments imdM ,V„™™I. 1 j • • '*" *"*' s*'*'''* descr l)ed For the p, ent I a^^t Wt fi^ in ZZ f' .»'°"''.'*'°''' ''"'^ ''"' ^"^" <^..ducted. informatio.. as is ava"laWe. il "brb clhl';' t" J'"" *" ?"*-, *''"* ^"'^ "'■ ""<•»' with the Bacillus A" of OSst '""'"^""'^ *'"" ♦*"■ "*">"' ""la-d by me are identical tbe.It'Sr^t'l^.abL'''""'" '" ^-"^""^ "- ''-^-^ ^y Auche^ (,S94). but '2^36^;;'"' ' "''' '■""""' ''"■" '• ~ '^■'-'"" °' .por« H;:7;;rn-,;„;;;r.;;;;;;^ is ij DKHHTMBXr or THK IfATAL BMMfWK w • OCOKOC V, A. «•!• Thr Miocialinii uf muM^U with food poiioiiing it citad bjr Viuchun, IMi* ; citinc fr»m Vaiufhan'* paper: — " That rhemiitil poicon* majr Im> tran«mitli< hiitory nt poiMiiiinff with nuiMttU uud with ftah. A* parljr a« tH97 Coinht' d«>M*rib<>d in ()(>tail thi< ayinptomii induc« aamn ■ubject ha* rwvntly hv\\\\ made by Rrhmitdmann, who hat found that non-iK)i*onou« muaacla plared in the water of Wilheluiiiharen toon beoame pni»»nou», and that the poiionouii muateU from the harbour Roon loae their harmful propertiaa whan placed in the open lea. Linder hat found in the water of this bajf and in the muMek liTinff in it a great variety of prntoioa, amoeba, bacteria, and other low formi of life, which are not found in the water of the open aea, nor in the non-poiionoui muiael. He hai abo found that if the water of the bay Iw filtered, non-poinonou* muaaeli placed in it do not become poitonoua. He there- fore conclude* that poiionoui mumieli are thoae which arc «ufferinff from disease due to reaidence in filthy water." In view of the dote relationship to niiiMeU of damn, a variety of ihell-fiah canned in both New Brunswick niiH Maine, I'.S.A., the obaervationt of Linder cited by Vaughan are of considerable interest. In the Mime pai>er VaUKhan describes the case of one of his own patients who showed poisoning symptoms after eating freely of canned salmon. The patient under treatment recovered. Vaughan submitted the rwnaina of the salmon to various teiit!< and found an organism which he describes as foUowa: — " The only germ which could be found, either by direct microscopic exam- ination or by the preparation of plate cultures, was a microeoccua, and thia was prwent in t. « salmon in great nurofcers. This germ grew fairly well in beef-tea, but the injection of five cubic centimeters of the beef-teu culture of different ages failed to affect white rats, kittens or rabbits. However, this micrococcus when grown for 30 days in a sterilized egg, after Hucppe's method of anaerobic culture, produces a must potent proteid poison. The white of the egg becomes thin, watery, markedly alkaline, and 10 drops of thio suffices to kill white rata. "Evidently in the preparation of the salmon this can wa« not steriliied; it was sealed, and for months, possibly longer, this germ had been growing nnaerobically. and elaborating a chemical poison." Savage, in England who has investigated many outbreaks of food poiioning, has isolated B, enterilides from tinned salmon. Griffiths, cited by Vaughnti and Novy*, claims to have isolated a ptomaine laordinin from sardines. In view of the types of bacteria I have isolated in the present investigation, it is of importance to note that Poch * in Rotterdam has isolated varieties of B. aoli from cases of food poisoning due to the eating of meat from a supposedly healthy animal. MvWct'ney * considers that meat poixoning outbreaks arc due to orfranisms of the following groups: — (a) The Typho '■'•'■ . mp, including B enleritides (Oaertner). (6) The grc p ot putrefactive aerobes (Proteus, etc.). (c) The obligate anaerobes (B. botulini»}. It will be seen, pages 192, 209, that of the organisms I have isolated, mme strains are varieties of the Proteus group, and some varieties of the B. coli group. Vaughan and Novy* describe the most common form of food poisoning that caused by con- tamination of foods with saprophytic bacteria; such bacteria either before or after the food has been eaten, elaborating chemical poisons. HirTgRHtUm- nr M Minis KH in MMIONAL PAPIK H: Ma PHEHKNT INVEHTIOATION. from the /.ctorie. I. . m^tfr of ooMid«.hl.. ..,„.w.. Th.. «Wir.bnitr of uiMTr »wHM .•onditi.m. .Kvurr..J to th.. priiM-iHil "f Mm-don-ld («1 !.«... |)r. F V lUrri-T i.. the .ummrr of 1»13. At th«l time Dr. If.rri*.,. w.« rnwK."d . th^- Lrin"Zio ' Ht Andrew.. ,„ the ex-mination . ' '..ddook .tfoked by «Kri.l dU^^ i^^d U wm while ^c^nduot.,., ,hi, i„ve.ti„ti.... .h.t the probIem^l..„^,,'\Hr. ;.l u„Ir' B..ar.l, and in due Pour«. it w.h my ipnmI fortune, on 'he nHH,mroend ,7„/ f^ Harrimm. to U. «.ked to take up the work. The i^roeedure "TTT • wll Si entirely „. my own hand*. Dr. Maeallum. and Dr. T^Trunt.ma! • ,.Ar „f S marine station at St. Andrew,, have thn,u«h«ut ^iven m^ i^vTZL mLt a.^ ?fX:Ser "' •""" '" '^-'^ "'' -'"" -""^ """^ tJa^IutT'the eKi d^^.':^or.:;rt%t„^rth7il^;:rKr^^^^^ ... th.. canning of herriiiK. .ardinea. haddo,* a ^0^™.. ^^'tTveW It i.TJSS e^r i.Ta?::.;tntt"artr '''•"' «""i-''"''"'' ♦ "«^'"^ ^.ii: i?3 thepackn"of "rdt^^L whilelth'!!''"'' ^'1"""* *'"' P"««iP-lly «>„cer„c^ i„ collaw awell-l r..„r ; ""^T'"'^ I^"^ «*""'»« t""* »ummer and «inoe returning to the In the majority of the factories visited, the rang are immersed in h«tL ^f K, i- tTat'o T T'f "' ''1 "r"- ^'"^ ^"'"''•«*- *•>« hrtirpr^ "bS; l tt^ lals of the treatment of the fish-which have been salted in the boats as taken f^m the weirs-on arrival at the factorj- is as follows: immersed in a mixture of leawZ and salt for 1 to IJ hours; spread on racks, termed flakes, in thin layeranTflr 10 184 DEPABTMEST OF THE SAYAL SERVICE 8 GEORGE V, A. 1918 minutes placed in flowing steam ; dried in room through which liot air is continually circulated, for 1 hour; heads discarded and the remainder of the fish arranged in the cans; oil automatically aHded, and tops put on, and fastened by either the "rolling" or the " pressing " process. The cans are then heated as speciiied above. In some fac- tories the preliminary steaming for 10 minutes is dispensed with, and a continuous progression through a bath of cottonseed oil at a temiierature of 200° C. is substituted, this occupying 2 to 3 minutes. In one factory where the fish are fried in oil for ,3 minutes or so, the final heating is done under pressure at a temperature of 22.')° F. for a shorter iwriod. It should be added that in all the sardine factories visited, the most careful super- vision is exercised in the fii.al packing of the cans in cases before shipping. Each individual can is rapidly passed through the hands of an expert "tapper" who discards cans displaying any irregularity, such being reprocessed or entirely discarded. The project of the investigation may be logically stated thus : "Essentially to deter- mine whether or not the swelling of the cans is due to the activities of bacteria." If on examination, and when submitted to suitable cultural methods strains nf bacteria are isolated, the procedure to be as follows: — 1. Purify and obtain in pure culture. 2. Determine the morpholojtical, biological and biochemical characteristics of the organisms. .■). InOi.ulate the strains obtained in pure culture into norniul cans and record condition at stated intervals. 4. Treat "control" normal cans in a similar manner except for the inocula- tion with the culture. 5. If swelling occurs in the inoculated cans, and no change is ni>te"♦'"" '"»« l^" Principnlly confined to the gas pro- fSflJ S!^i't^ '* '* •" "■' d-cription.s of these that the cultural part of the re^t of tS"!*^-'* "".Vf ""'"V^ «''"-«^' "rgani^nis included in Class II on the condition thel hfve h , '"''''• ] "•" ";'* •" " P"*'""" *° «'!"««« «"y "P""""- Many of 'Zi; "" l>a»?e* ^0« and 2()i» u summary arranged in tabular form is shown Ihe number of cultures described in Class I, and those more briefly referred to in thJ^ork ^: waslo^t'"''" V ^ T' •"'""'^' "* ""'*"'«' -•-^^ '" "»-"-- o" th! n tw ^- ^'"•«^*'''l' l"-<'l"ninary tests of a differential nature revealed r.lff!^ ■ ""T^ «tra>>'« were in duplicate, and sometimes even in triplLr By shin f« ♦i i ' f^ot^'P'^^a-fo"* were taken on account of their closer relation- ship to he abnormal condition of the cans. Some of the final culturrs describ^ represent the individual strains, after the elimination of as many a" four or fi^ c«h«rorc, "'t'^" '«""^, '" .'^-^•^ '""^ •""'" characteristics Teo^on ThrL priorTthe „r,r r'' f"f."' ^'""""'*"^ *° «''°'d duplication in description, just •n InH «r,'^T. ,"" ""* *''^ manuscripts, these being identical with ciUture;, 34, 'it, and 04, respectively. ' ' 184 Tt" i?Shpr***^ statement as to the project of the investigation, initiated on page ZX:^?:^;:^^:-^',^^:,-^^^ -"«- ^^^ ^-^ up to the present^an^d ?n '^T^K'^r^ ".rr ?"' ''"*" *"' «"'«n'""-' ^'- Huntsman whereby during a later season I shall have opportunities „f deterniinii* if ,ws.sible the source or soured T[hrcrusal organisms of the swelled .-ondition of cans of sardines The future scope of the laboratory work will mHx-ssarily include examination of ^Ige m"" °' °*'" ''"■'^''" "' '^''- ""'^"'''"'^ ^^- °^ -h-'' memlon'rmade^ l^r^**"^* ^^''^'"Aa^'' T""'"^ ^"'^'""^ '"*' *""""«•• the manager of n„e of the largest of these told me that a pressing problem with which he had to contend waT the frequent appearance among sardine cans of what are termed "sour fl^Tts" Tb! condition is one of which there appears at present to be no sSactTv explanation 2LfI^ ■^V'^'''^ unmarketable, and the condition is one which rn",ot^ detected until the cans are opened. »^nnoi 09 1M DXPABTMBNT OF TBE XAVAL SERVIOe 8 QEORQE V, A. 1918 MEDIA EMPLOYED. In this investigation I have need media prepared from fish concoctions, the ordinary laboratory media, and certain 'special media. In the early part of the work when experimenting with methods prior to the adoption of a de£nite procedure, difficulty was experienced in growing some of the strains isolated. The colonies developing on some of the plates at this time were too small to be subcultured. I therefore utilized the marine resources at hand and prepared media from fresh herrings, from clams, and from seaweed, using fresh sea water instead of tap or distilled water. It was found later that the organisms which necessitated this media were those I have put in the main Glass II, the non-gas-producers. After successive subculturing in the laboratory these same strains have grown moderately well on the usual standard media. The organisms of my main Class I, the gas-producing strains, have grown well in the standard media. The growth of some strains has been more luxuriant on herring media or clam media, but the use of such has grradually been eliminated for two reasons : — - (1) the satisfactory growth obtained on standard media, and the convenience of its use; (2) the necessity of using the standard media in order to compare the strains isolated with varieties already described in literature. Herring Broth. — Fresh herrings obtained direct from the weirs were washed in run- ning water and ground up, no portions discarded, through a meat grinder, mixed with sea water, 1 part ground herring to 1-2 parts sea-water, and heated for several hours in the steamer or autodav. The mixture was allowed to cool and the fat skimmed off; again heated, and strained through cheese cloth. The strained liquid served as the standard herring extract. Varying strengths of broth were made up, good results being obtained with the following mixture: — SOO cc standard broth, 1,000 cc. sea water, 16 grams peptone. The ingredients were heated together in the steamer, neutralized with n/20 NaOH to + 10 (phenol phthalein indicator), cleared with white of egg, tubed and sterilized in the usual way. Herring Agar. — To 500 cc. of the standard broth, mentioned above, were added 500 cc. or 1,000 cc. sea-water, peptone at the rate of 1 per cent and agar at the rate of 1-2 per cent; the whole heated together until ingredients dissolved, neutralized to -|-10, cleared with white of egg, filtered, tubed and sterilized in the usual way. Clam Agar. — Fresh clams were dug up on the bench, washed in running water, opened and ground through meat grinder; to this was added sea water at the rate of 1 part clams to 2 parts sea water, and the whole heated for several hours in steamer or autoclav. The stewed mixture was strained tiirough cheese cloth; this filtrate constituting the standard broth. To 500 cc. of the standard broth were added 1,000 cc. sea water, peptone at the rate of 1 per cent, and agar at the rate of 1-2 per cent; the whole heated together until ingredients dissolved, neutralized to +10, cleared with white of egg, filtered, tubed and sterilized in the usual way. I have also steamed clams in the shell in sea water, appr .nmately weight for weight; retaining the juice which has a typical "sheen" ; then after open- ing the clams using them as described above. In the earlier part of the work the medium was used successfully ^o some consideralble extent; and in comparison with standard beef peptone agar it appeared to exercise a selective action towards certain strains of bacteria BACTERIOWOY OF SARDIXES 187 SESSIONAL PAPER No. 38a S."i2"* ''T ?"e^..'""'r^- This in all probability would be due to the glycogen content While the use of this medium has for some time been dis- ZlZ '""'^** *" '"* '*' '"^"•' ^°' '"'"'^ P'^"- °f the laWato4 ™p„?.'!!'I11k ^'•'i?'' «* K'^J °» *»>« denitrifying bacteria used and recom- mends a broth of which mussels are the essential component. Beef Peptone ^l^ar.— Standard methods." Beef Peptone Oe7*/ine.— Standard methods." 0/ucow ^^ffar.-One per cent glucose added to agar prepared as above, immediately Loeffler's Blood SerumA'> Ldeffler'sTyphoidJolutionJ^-Thi, medium containing malachite green has been Ae»culin AgarU For specific reaction of organisms of the colon-aerogenes group- loops of a broth culture spread on plates. ^roup, MacConkey'eNeutrtd Red Bile Salt Lactose Broth^^-For reduction test of organ- isms of the colon-aerogenea group. Bouillon for V ogea-Proshauer Reaction."^* Bouillon for Methyl Red Reaction." Solution for Reduction of Nitrate, to Nitrites '«.-Giltay's synthetic solution was used. Dunham Solution for Indol Production.^^ Glucose Broth.— One per cent glucose in Dunham solution. Fermentation Broths.-For the fermentation reactions I have used ten test substances. It will be seen that m addition to the glucose salicin T have adopted the use of another gucoside a«,cui.n-used in conjunction with iron citrate by Harrison and Vanderleck--a8 a fermentable tef : bstance in Dunham broth. I have been using aesculm for this purpose during the last four months in connection with work on the gas producing organisms in the Ottawa river water, and find a correlation in the black reaction of the aesculin agar medium, and the pro- duction of acid and gas in aesculin used as a carbohydrate test substance. Litmus Milk.'^ METHODS. ™»*.Pj account of the comparative paucity in the literature, of descriptions of actual methods adopted in the isolation of bacteria from swe? >d canned fish, the procedure I r2 i Z T ^^^^"^ ^" *i«*''"»'"^ by experience as the work has progressed Th 8 procedure has been changed as better methods suggested themselves, and in the culturing from the many oans still awaiting examination I propose further changes tfra Sa?su'g;'^:iter -' "''"'"""^ """" -''-' -'" '^ "« ^^^ «<>---" Isolation of Bacteria from the Cans. sitat^^le'lLT/fn-'*''"!*'^"'*"*^''''!.'."."^ **•" ••""^ ^''^ pronounced swelling neces- sitated the use of a disinfecting agent which would disinfect, and remove the oil. at the 1M DBPABTUSXT OF TBS NAVAL Sffi.riwK 8 GEORGE V, A. 1918 game time. Absolute alcohol has proved to be simple in application and quite satis- factory. The cans were first cleaned with a weaker alcohol (70 per cent to 90 per cent) then thoroughly treated with the absolute alcohol Can openers, forceps, and dissect- ing scissors were immersed in alcohol «iid flamed immediately before use. When a sufficiently large aperture had been made in the can, pieces of fish and a portion of the oil or sauce were removed with forceps and pipettes and inocukted into tubes of liquid medium. At the commencement, it was at once obvious that direct plating from the cans would not be at all satisfactory on account of the oily nature of the contents; liquid media have therefore been used for the first inoculation from the cans, the procedure having the additional advantage in that such media serve as enrichment fluids. I farst used peptone broth (Dunham), herring broth, and nutrient broth; later, the addition t.. the series of glucose peptone broth proved to have advantages. As a result 01 the additional knowledge provided by a study of the strains of organisms already worked out. It will be desirable in further work to use media having differential quali- ties tor the hrst inoculations; in addition to the broths already in use. The tubes were incubated at 37° C. except during the six weeks spent at St. Andrews, when all cultures were kept at room temperature. The broths were examined in 18-24 hours for growth; if no growth were apparent, further incubation was resorted to; if growth could be noted, series of plates were made. The preliminnrv incubation in broth tubes had the additional advantage to those already mentioneu. m that the oil had risen to the surface leaving the sub-surface li ,aid comparatively free, i-iney drawn out pipettes with the finger over the end were passed through the layer of oil. and the culture fluid drawn up. After suitable dilutions had been made, plates were poured using herring agar, clam agar, beef peptone agar, and glucose agar; in K »^"? '***'"* "^"'^ *'•'""'** "«"'■ '^•"« "««• "l™""* "olely. The plates were incu- bated-temperatures as aforementioned-and when growth was suificient. those colonies most common were streaked on agar slopes; from those the necessary purifi- cation by platea Viing made. nlw,J,"H;;n-''^fJ""*""l'"'"i^ incubation in broth tubes was in some cases, but not always, duplicated aerobically and anaerobically. The following apply to the main Class I:— Microscopic examination».-The microscopic preparations were uniformly made from beef iwptone agar slopes incubated 18 to 24 hours at 37° C. •Grams Stain.— The gas-producing organisms. Class I pages 192-207, display an unu- sual degree of resistance to decolorisation with alcohol in the Gram method of staining. When treated by the usual method,— decolorisation with alcohol un- til no further colour can be washed out,— each of the eight strains recorded would be classified as Gram positive. The shade of violet is not as deep as that which 18 typical of the classic Gram positive reaction, but the result is much nearer positive than negative. On prolonged soaking in absolute alcohol, 30 to 40 minutes, the reaction is definitely Gram negative. Films made from a typical Gram positive lactic acid producing organism withstood the decolor- isation with alcohol for 40 minutes The organisms herein discussed should therefore be described as Gram nega- tive, displaying an unusual degree of resistance to the decolorisation with alcohol. J/o^7i7i/.— Hanging drops for these tests were made from the water of condensation, .ig.ir slopes; young cultures incubated at 37° C, never longer than twenty-four hours. Inoculation of Media.— All tubes of media used for the determination of cultural fea- tures and biochemical reactions were inoculated from young peptone brotk cultures of the particular organism. The use of peptone salt solution instead " BACTERlOUHir OF HARtH\E8 ,-. SESSIONAL PAPER No. 37a of nutrient broU, eliminated to a minimum any ride due to the pH^ence of mu«c e sugar I nwy be mentioned that repeated te.t. for the ^IZ of tnton^ ""''' *'*' *"•" '^"* '"•""'•*'^ *'""' -" •<''"•« strain of ttfl Prior to the inoculatioua of the series, peptone broth tubes were inoculated 20 hor'the wh r^ •"""'"r' "» ^^' C. After 18 to 24 hours "^uaTaCt f*. ulJ -1 ' S.arops of culture to each tube. Slopes of solid media were fZu^ }.u '*'""^"'^ •I"""- '°"P "'"*"»»" needle, "^he numberii tub" hiSout"thl rr"";5 f *"' '"'**'""^* necessitated have been consiLr"£ it« ul ♦• ^^\:''''^' J"'^ t" '"«"'« «=onomy of expense and time, strictly quan- mearor riunL™ ft'^"/-'.^"! »'?- "ot been carried out other ThaTby ^!rZ *''!.^"°^»™ *«•>*.• In v,ew of the method noted above, however, the results are truly comparative throughout. Moreover, for the p;rtieul«r pur- Xn of tle'^t"* r,' *'' """*'•' ^'"^ *" ^ ''"'••^^ regardinSet^'enl Jas or dSs iflr -S"**' '"fr ' *»''«-«lo^ «' Particular culture produce mu;h nr^^L.! /"^'"'^**"'* '* '* "°* ""'y ^' considerable interest but of Tr^uo^Tn • »"'*,.«^»^^"'«'«'t?'y value to know whether the amount of gas o^fm^ sSnh ?nf ^'* " '^'T ^^•"P*'-'"'^ fron* a given substance is great Dunham tub^ "•^o^afon --an be comparatively well shown by the use of the Indol 2°f«f^°'--The tubes to be tested for Indol were incubated at 37» C. for 7 days; the Bohme Ehrlich test being used. deduction of ^^rates.-The Giltay solution was tested after .3 to 4 days incubation „t reagS'ts were uS'"" "''"'"• ''''' ^"'''''"""''' '>-'*' «"'' a-naphthylamin ^'""'£"d'wft'/'r"''"'-~i^**"' *' *° "' ''""^ incubation at 37" C. the culture ttSpTcaf ^^Sr -"^r °' "^T '^'•^ *"* '' P**"*"'^ l'- "«"ally shown the typical eosm shade m the upper layers, within 2 hours at room temperature. Methyl Red Reaction—jyetermined after incubation at 37' C. for 48 to 72 hours. Cans of Sahdixe.s. (teneral Descnption. Appearance of Cans and Conditions of Contents. torii'Ti'* *"• *^^ ''"i^^}^ °' "brands" of sardines produced bv the canning f,c- S^^ Sv^tTfl"^? S "' '"f '•* "^''"i^' ""'^ *^« '^'«"-" -b^tances'tiZed ?:r tb«n r. r and consistency to the finished product, it is not possible other than .n a general way to described the conditions met with in my examirttns. ing'-^Thelr^nZJ'^r*'""^ appearance there is a complete absence of anv "bulg- haking. ^eTe^f n^^rTttte'-Tn'r '"*?"* "" "'"""'' imperceptibly concave, o'n heard Wbpn nl. J fi ll ^ '^"f"'''-^ ""''' n'°v«''nent of the contents can be Hnt A- ^^^ 'y,'*'' *'■*' *'"""• *•»"« '-^ no expulsion of air or with little It any exuding of the 3il or other material used in the process of pacV; ev.r A ''"T ! "^ ^""' "°* "'aeerated. and often white in colour, last how- Hiiiaiy cnaracteristic of the fish, qu.ilihcd by the var etv of oil or tomato sauce naP.) l)"\y:^ appearance and odour a complete absence of putrefact'oT The fish are usS' W \t ^?^''- °' I'^^Z "^'«"* "'*'» "- »>'• — . - other flavourlgagens used but without losing th.ir firm and solid condition. The oil or sauce will be reen w thilTh-erdieT' " *^ ■"^^"''''^''^ '"^'^"" '""^ •"•''^"^-' «^''' "^J'- *''- nc^uaS 190 DEPAKTMllKT OF TBS XAVAL SBMYICB a GEORGE V, A. 191« Swelled CaiM.— Outwardly the cans vary from a alicht " bulged " appearance to a more pronounced swellincr. The top and bottom are forced out as a result of the pressure, and present a decided convex surface. As the swelling becomes greater the oil or sauce will be forced out between the soldered parte of the can, and in pronoun- ced cases the outoide surface is greasy and wot, and possibly covered with the oil or sauce. Swelled cans, when shaken, have a characteristic " rattle " on account of the extra Space within, resulting from the swelling. When the cans are opened, gas is ex- pelled, accompanied in advanced swellings by portions of the liquid contents. In ad- vanced cases there is a tendency for the oil or sauce to pour out over the durface of the cans. The condition of the contents varies considerably. Usually the fish are macerat- ed, disintegrated, and soft, and are intermixed with the oil or sauce; they have lost their entity. The odour is variable.— frequently it is not unpleasant, resembling to an accentuated degree the natural smell of normal sardines. In other instances a pronounced putrefactive odour is evident. It may be that thz putrefactive odour is present at all times and is masked by tht spices or other ingredients of the sauce. That ia a point wh-'-h can only be definitely pronounced upon after a more extended investigation. CANS EXAMINED. Up to the present I have examined forty cans, normal and swelled. The cans have been obtained personally or by express; (1) direct from various canning factories in the province of New Brunswick and in the State of Maine, U.S.A. (2> From the Health Department of a city in the Maritime Provinces. (3) From retail grocery stores. Many of the normal cans, representative of the various factories, proved to be Sterile; from some have b-en isolated spore forming bacteria, inactive on fermentable carbohydrates,— see page 211, Culture 21 and in no instance have gas producing organisms been found. From certain of the swelled cans I have isolated a variety of • strains of gas pro- ducing bacteria, none of which show evidence of spore formation. The cans from which these strains have been isolated are representative of three of the factories engaged in canning; and for the sake of clearness these factories have been specified as Packer A, Packer B, and Packer C, respectively. Further, from swelled cans I have also isolated strwins of bacteria which fail to ferment any of the carbohydrates used as test substances (pages 212-213). It remains, therefore, to be added that from some cans apparently " swelled " I have failed to isolate gas producing bacteria. As already stated (page 185) the organisms isolated from the various sources have for the sake of con"enience been arranged in two main classes : — Class I. — Gas producers. Class n. — Non gas-producers. The gas-producers (see pages 192-207) have been isolated solely from swelled cans of sardines. Of the swelled cans examined the majority were obtained from sources 1 and 3 (page 190). Some were submitted by source 2. Under the circumstances it has seemed desirable to use some means of differentiation. Accordingly the swelled cans obtained: (1) from the canning factories, and (3) from retail grocery stores have been designated "Swelled cans. Series 1"; those submitted by (2) a certain City Health Department, "Swelled Cans Series II." BACTBRtOWar OF 8ARDISK8 ^ SESSIONAL PAPER Na SSa Swttttd Can$. 8trie$ I. Can. I, Packer B.-Obtained direct from canning factory; packed with tom.tn f.bo«toS °^nch '■ T^'-lf "'""'^ "r "' »''•' ''""»•»"• *«"' .trewn over th^ laboratory bench. The odour was pleasant, though pungent, and mar b^t be described as the natural smell of normal .ardine, .Suat^ j^j, „J interest to note that the pintes made, using herring agarrwp'Sy dereloVd at room temperature a putr.d smell resembling, as «p*«W^r7 lablrK colleague, that of an "oriental latrine." "pressea hy a laboratory See Culture 32. Class I. Can II. Packer 4 -Obtained from a retail grocery .tore; packed in cottonseed 0.1: same brand as those of "Swelled Cans. Series II" This can was^S " ht:ttVl're"S S " "^" -"'^ ^"^Vr "' •"•' °- I>e-nrex?min^S 01 mg stock I retamed it as suspicious. I have no knowledge as to the date wasTo evfden " ?T'"? '^"^ """ "" '^"'^'^^ Bwollen. con.eV but t£« was no evident of oil exuding due to pressure of gas. On opening a nercen- colour slightly darker white than normal ; odour an accentuation of the normal See Culture 34, Class I. ^"^ la^dt^X ^-TSo""* and brand as Can II of this series. This can submitted to me by the salesman. The appearance of the can. the appearance condUtion and colour of the contents identical with description appS to Ca^ Tl See Culture 35, Class I. Can IV Packer 5.— Source and brand as Can I of this series In t>,;. „»„ *\. T^ry s ^'"^"T? "' '"'' '" ^"^ '' --^ - ^p^" g tCs wa" Lot ^tu^o of ^h ^""f *""""' ^•'^•"Pt'on there applied to the contents and to the nature of the subsequent plates is equally applicable in this instance. See Onltu-- 36, Class I. Can F Parser B.-Source and brand as <^an I of this series. The extent to which the^can had swelled, and the further description used above for Can ivljply See Culture 37, Class I. Can VI, Packer C.-Ohtained dirett from canning factonr; packed in tomato sauce; characteristic "swelled " appearance top and botto^ To^Z. On o^ mg a sman amount of gas escaped. The odour was not unpleasant, and may ^described as the natural smell of normal sardines accentuated. The Zl tents of the can were not nearly so much disintegrated as noted in some pre- :::tstfir;Tis^"^ ■'"'"^^'" •^■^' --^ - '^'^ '^- ^-^^ ^•^^ *^-" See Culture 64. Class I. Swelled Cans, Series II, Packer A. A cargo of sardines exported by packer A had been sunk in a harbour remainin,r were vi.„hiy swrlled. The local Health Dopaitniei.t submitted a number of these cans for examination, as a result of which the ear^o was condemned Such cans, of course f^'tuTrfThVr' rf"' ""'","' '"""r*'^- ^^- •*— ' their condi ioTaS f^n, ntW rf "^^"*^^ somewhat similar to the swelled cans obtained frudSln ZrZT '^•'"-^"■**- °^ -- »^ *h^ »--'- i-lated have been 192 DKPA, rUeXT OF TBK XAVAL KKMrtOK S OEOROE V, A. 1»1t To differentiate from the «welled cans obtained direct from the canning factories and from retailer* I have designated the salvaged cant ai " Swelled cant, series 11." The brand of sardines of which this cargo consisted is one of the least expensive brand* on the market ; cottonseed oil ii used. Can II.— On shaking, i»erceptible "rattle" characteristic of the awollen cans. On opening with the cutter escape of gas and pronounced putrefactive odour; contents soft and disirtcgratcd ; colour dirt.v white with tendency to redneat in inner portion) See Culture 24. Class I: Culture 14, Class II. Can //f.-^Charactiristic "rattle"; ci>cape of gas and pronounced putrefactive odour on opening of can: contents soft and disintegrated, and of a dirty whit* colour. See Culture 26, Class I; Culture 16. Class II. oRot.NmMa or the c:.*s-pBoriiTiNO type. Culture ik- Source: Can II, Ser. II, Packer A. Morphology. — Microscopically: coccus forms to short thick rods twice as long a» broad; average length -8 — 1 ^ •Gram negative. From old agar cultures no evidence of spores. Motiliiy.— In hanging drop occurring singly, in twos and in chains; some individuals with rapid movement, some having slow undulating motion. Cultural Charaeteristicn. — Agar slope. — 36 hrs. 37° C. — growth luxuriant, raiseU, glistening, iridescent, yellowish-white by transmitted light. Loefjlet'a Blood Serum.— H hrs.. 37 C. moderate, yellowish-white, no liquefaction. LiiefHer's Malachite Green Sol. — Green precipitate or weak coagulum at bottom of tube; this very slowly changes and within 14 days partially digested; liquid portion assuming brownish tint. Gelatine Stab.— Boom temperature; liquefaction begins in 24 hrs. crateriform; in three days liquefaction on surface and. along track of needle, crateriform to infundibuliform ; growth very slimy on this medium; in 7 days yellowish, cloudy stratiform extending 1 cm. from surface, remainder infundibuliform with heavy yellow flocculent sediment to bottom of tube. In 18 days .'iquefaction not yet complete; upper portion heavy milky even cloudiness, merging into layers of semi-transparent cloudiness, the lower portion a heavy ferric-yellow ma^s of precipitate. Nutrient Broth.— 2i hrs. 37''0.— heavy clouding with bluish rim; in 3 days floc- culent flakes of bluish tint on sides of tube; in 5 days very heavy dense oven clouding, watered silk appearance; this condition persists. Herring Broth. — Condition similar to above; very heavy growth; in 9 days a loop of the liquid lowing decided iridescent bluish sheen. Milk.— In 24 hrs. unchanged, except tha'. much froth on shaking; in 3 days coagulated, soft cunt some whey expressed; in 9 days yellow digested fluid 2/3 of tube, remainder white soft curd; in 14 days ropiness noted, and medium almost entirely digested with slight amount of flocculent curd at bottom of tube; in 5 weeks almost wholly turbid yellowish digested fluid with slight jelly-like yellowish iridescent flocculent curd on base of tube. BACTemouwr of sAmnsEii in ■ WMIONAL PAPER No. Sla Litmu, Milk.-lni4 hour, mut-h froth on .hakin^. viol.oou. for 1 cm from J-^Mlf?*'"" P""^'""- fluid .vollowi«h: in 14 dav, bluo rin, IL»hZ' Aesrulin agar.-l l.ov from |M>ptonP l.roth ruhiiro stro.,k«J on pkte. In 24 Aetculin broth.— In 24 hours black reaction. MacConke»-» X.R.B. flro/A.-No nnlucti n to o«nar>- .vrll„w i„ 24 hour,. Gelatine «fo«.«.-(l«t appearance) room temperature, in 72 hours li„urf«ctinn shaped entire edgea; liquefaction typical of the proteu. ^un cerro, c»^ny dark white apot -25 mm. diameter, remainder of colo^ v«rv?, ^ f rom clear space to fine precipitated Rranule.. Tender the low ,V,wer WectU^ opaque centre, edge, entire: medium tinted gr..n. and di.tin.Telrt.^TJ Agarcolonie,.-20 hours at 37°0.. growth moderate, surface colonics round con- care.glisten.n^ raised, distinctly radiate: by transmitted light v. unrdn^L bluish, older colonies becoming whiter, more opaque and dn;keh. cent- Sub-mirface colonies small but well defined, while. Under low , . w r obW ive surface colon es distinctly ye'lowi^h with entire edgcj o fL.uS.: through, dense a..d dark: structure cannot be defined; .mailer co on es dTrk centre, then pale yellow, and near the ..Iges almost transparent Sub^ surface colonic, well definod. edges entire, yellow to dense. Temperature Relations:— Thermd death point.-W minutes' exposure in nutrient broth at eO'O "''zz':Tt:::-::S£Zr-:r^^^^^^^ — -'^ -a ^e arc. '"tSi^m.'jS': Sr"'^ ^"'""' -''''''- ---' "'"-•>^ - "-'«^'"' Relation to Oxygen.-The culture is a facultative anaerobe: incubated for .36 hours under anaerobic conditions moderate growth on glucose agar as discrc e .t^lon^s along track of needle 1-? mm. diameter; by transmitted light convex Tarkwhte centres, paling to blue at edges. Growth is not so luxuriant as under 'acr"bict,n Biochemical Reactioiw: — Indol production : Indol not produced. Reduction of nitrates: Nitrates to nitrites. Voges-Proskauer reaction : Positive. Methyl red reaelioii: Alkaline. Fermentation of Carbohydrate,.-This culture does not rapidly ferment many of he carbohydrates. In 24 hours lactose is but feebly fermented to acid saccharose, mann.te and xylose are fermented to acid and gas with prof,.; frothing; arabinose and inulin give slight gas; while gas apJLrs in glyceS 1M DafAHTMBST Or TBM VAfAL ttMrWM Dulcite. > - Salicin. + ♦ ■ aCONOC V, A. ittt onljr after a pwiod of 7« houn. Tha nroaininc aubaUnoea uaed are femwntod modmrateiy well in 84 houra to acid and to faa. Oluooae. Laotoae. Baocharoae. Mannite. ♦+ +- ++ ++ Adonit. Baflnow. Arabinoae. Xjrloae. ++ ++ ++ Aeaculin. Oljrcn-ine. Inulin. ++++++ •»■ = acid. ■H- a acid and gat. CuUurt te Souret: Can III, Ser. II, Padcer A. Morphohpy -Microacopieally, roda U to 1) timea aa long aa broad; arerage length 1-6 /t with many longer forma eren in young culturea. Oram negatiTe*; from old agar culturea no eridence of aporea. Mioroacopio preiiarations made from culturea of thia organiam incubated at the aame and at different temperaturea haTe shown much rariation in morphology; iucceaaive plat? culturing, howerer, haa failed to ahow impurity. Motility. — In hanging drop occumng aingly, and in twoa, nometimea side by side; longer forms noted; non-motile. Cultural Charaeteristiet : — Agar Slope. — 86 houra, B7*C., moderate, along track of needle, glistening yel- lowish-white by transmitted light. Loeffle/i Blood Serum. — Growth slight after 72 hours. No liquefaction. Lbeffhr'» Malaehxte Green Solution. — 24 hours, 37''C., coagulated as soft junket- like curd attached to sides and bottom of tube, green, with pale green liquid expressed. After 14 days ro change. Gelatine Stab. — Room temperature — in 3 days scant growth, filiform, no liqut- faction; in 18 days no change apart from increaaed growth, no liquefaction. Nutrient Broth. — 34 hours, 37°C., moderate clouding, no pellicle, no sediment, no ring; in 3 days watered silk appearance; in 9 days no change except slight sediment at bottom of tube. Herring Broth. — Similar to above, but much more luxuriant growth. JlUk. — In 24 hours at 37°0., no coagulation, much froth on shaking; in 3 days coagulation beginning; in 5 days firm coagulum, no gas, no digestion; in 16 day i curd slightly split by gas. In 5 weeks shrinking of curd, Hut no digestion. LitmuH Milk-. — In 24 hours violaceus, much froth on shaking, no coagulation; in 3 days liliaceous with weak coagulum ; in 5 days curd slightly cracked by gu. In .'i weeks no digestion ; pale lilac to isabclla. Aeiculin agar. — One loop from peptone broth culture streaked on plates; no reaction. Aescvlin broth. — In 24 liours. Slight change but no black reaction; later medi-im darkened slowly in several days becoming black. Mar.Conl-ey's N.R.3. broth. — No reduction to canary yellow in 48 hours. Gelatine Coloniei. — (list appcaraiitt;), 72 hours at room temperature. Surface colonies yellowish white by transmitted light, J-IJ mm. diameter; a charac- teristic depression immediately around edge of colony could be seen on tilt- ing the plate; no bluish appearance; no liquefaction. Under the low power objective colonies pale yellow, with paler rim, and entire edges, structure finely granular. MACTKItlOLOOr OF «4«Df.VC« 1M 1 SIMIONAL MMR Hm. Urn Agmr colMJM.— 90 houn. »7*C. Oruwth ibw, puncUform. tearoriy Tiiibla to tlM «r«. ExamiiMd S tky*; bjr trammittcd licht turfM* ooloniw gNyiih white, r'' ptieal and round, the brgrr coloniM 8 mm. diameter. Subeurfaoe ooloniea pre««d: rololw "'"«"''"" •• PwilMtatioii on .ide. of tube, no r«lu«tion of *''''Ii!'.fl!'''r"r'? »t"»f"»«':^" ho"" filif..rtn. ..« liMUefa..tio,.; in 4 day. growth abundant: in l week no li.,u..fa.tion and no change in medium, growth er tt*U require to he madp; t^t* iHrform.^l up to th» pmM>nt indi and at .IT * C trow well: numt natisfartory irrowlh at 87° ('. ViMiln on ruUurr mfdia.-l\w cultun. nurvivea •<>vithI month, un artificial medium, ayar or gelatine. Relation to Ojygtn: — The culture i« a farultative anaerobe; incubated for 3fl hour* at 37' C. under anaerobic condition! moderate growth on slope of gluooM> acar: medium cracked and <|ilit by km bubblcn, much froth in tube and heavy clouding of c«>nden»Htion water. The organiiim appeam to grow equally well in the prenence or in the abnence of oxygen. Biotkimieal Krartiont: — Indol produrtion: Indol not produced. Reduction of nitrate*: Nitrate* reduced to nitrites. Voge* I'ro*kauer reaction : Negative. Methyl red reaction: Alkaline. Fermentation of Carhohf,drate».— The action of thi* culture on lact.»*e i* feeble and »low. ga* not ar>|>earing until the second day; dulcitc i* but slightly fermented to acid and no gas i* produced. Aew'ulin is fermented to acid and ga* ill 84 hour* and in 9 day* the Andrade indicator reduced to a lemon yellow turbid iridescent colour, while no reduction is noted in the case of sahcin. All the other test suimtances are fermented to acid and to gas rapidly with profuse frothing and heavy turbidity within H hour*. Obicose. Lacto*e. Saccharose. Mannite. Duleite. + + Xylof" + + + - Salicin. + + + + + + + + Adonit. Raffiiio»e. Arabinose. + + + + Ae«c>ilin. Olycerin. Iiiuliii. + + + + + + + = acid. ■H- = ncid ai:d bd*. Culture 3|. Source: Can. 1.'.. Ser. I.. Packer A. .¥orp»o?offy.— Microscopically varying from coccus forms to short rods; the majority ^1 ^ long and twice a* Icig as broad, many thinner; stains unevenly with Kilhnes methylt;.e blue; Gram negotive*; from old agar cultures no evidence of spore*. Motility.— h\ hanging drop occurring singly and in twos; actively motile. Cultural Characieristict : — Agar slope.— Se hours at 37° C. moderate along track of needle, glistening irides- cent, bluish by tranwmitted light, gas bubbles in medium presumably due to fermentation of the muscle sugar in beef extract. In agar culture 2 months old distinct sliminess has been noted. 2.^365— 2iK 1M DEPABTMBJIT OF fBE If AVAL 8BKVICE 8 GEORGE V, A. 1918 Btmna agar-iO hours 32 C, growth abundant, contoured, yellowish white ^wth along trade of needle, spreading over slope as blSh film of dis^r^tl /- ^!TZ' ^•'c'""'*' '"**'^"*= ''^"^•y '^''^^ of condensation waL ^•CfS^nfftrrZ'""" ''''' ''°^'""^^' ''^'^ -»ow growth, no ^''"li!I!l 'i^rJ'^'^ temperatur^24 hours fiUform. no liquefaction, equally good on surface and in stab; in 4 days growth abundant; in 7 days no Hqu^ faction and no change in medium; no liquefaction in 21 days 7™!l^'f-~'' ^^r-K, ^^° C-.^l««di„g moderate, slight pellicle; on shaki,« ?rhL« 7 P^'<^ept,ble m medium; bluish rim; slight viscid .edin.ent; i* 72 hours cloudy waves, as watered silk, some flocculent precipitation in .us- pGnsion* Herring fcrott.— Similar to above, but heavier. ^^''Z^t hours 37° a. much froth on shaking, no coagulation; in 48 hours, weak coaguium beginning; m 72 hours coagulated with gas and expulsion of whey, -urd later splitting with gas holes. in 48 hours weak coaguktion beginning; in 72 hours coagulated, gas. whey expressed, later bleaching to Isabella and much splitting of curd by gas :Note— Milks and litmus milks incubated for 2 months have appeared to ' p slowly digesting; up to the present I have been unable to verify this and further tests must be made to establish the final condition of th^ clot. Af»cuhnagwr.-1 loop from peptone broth culture streaked on plate. In 24 hours at 37 L.. reaction brown-black. Atsculin hroth— In 24 hours black reaction. MacConk^ys N R^B. Broth. -In 48 hours. 37= C. slight reduction to eosin tint, out no Imal reduction to canary yellow. Gelatine colonie».-Room temperature (Ist appearance) surface ...lonies up to J^^M-"""' ^ *'""7'*¥ ««ht bluish-white, glistening, almost transparent, resembling more the description of the B. typhosus colonies than the typical a. C oil colony ; flat ; subsurface colonies smaller, white to yellow-white, dei.res- sion around edges, see Culture 32. Under the low power objective sulfate colonies pale ye low paling near rim with hedges entire; structure finely «T«nular with clearly defined border around more dense central structure- subsurface colonies similar. u^^ure. Agar colonies.-20 hours 37°C.. growth moderate, not so rapid as other cultures; surface colonies 1 -- IJ mm. diameter, round, concave, glistening; by trans- mitted light bluish with pin-point dark white centre, distinctly radiate. J."bsurface colonies dirty white ; organism growing better just under surface. Under low power objective surface colonies dark centre, remainder of colony tainUy discernible as finely granular lemon yellow, with edges entire- sub- surface dark, compact, too dense for structure to be differentiated, 'edges Temperature Relations: — Thermal d^atk point.— iO minutes exposure in nutrient broth at 60'C. Optimum temperature.— Cuhmea incubated at room temperature and at 37°C grow well; most satisfactory growth at 37''C. Vitality on Culture medium.— The culture survives several months on artificial medium, agar or gelatine. BACTBRIOWQY OF TARDIVES 1M Mannite. + + Xylose. + + Dulcite. + + Salicin. + + SESSIONAL PAPER No. 38a /?el«i hours 37°C. Coagulated junket like coagulum clinging to sides of tube, gas; in 72 hours reduced greenish yellow in 14 .^v., reduced to yellowish-brown slimy looking liquid, partially digested' arlat.ne .Sfafc. -Room temperature, in 24 hours filiform growth equally good sur- face and stab, no liquefaction, slight gas-presumably from muscle sugar- growth hixurmnt. No hquef.ifti.m in 21 days. Nutrient Broth-18 hours 37° C. clouding even, 'no pellicle, no sediment bluish rim at surface; in 48 hours hea^-y clouding, viscid sediment at bottom on shaking; in ,2 hours flocculent suspension, later sediment increasing, medium beciming clearer, and flocculency. meaium aoo DBPABTUmiT or TBE NATAL BBBTWK 8 QEORQE V, A. 1918 ^"^^t.l'*''*"";?*^"^*"'* "^rf"'' """^ "«" •* ••"'««• R«»ic'e. viscid pre- rC Lt'^'loud.^^" ^^'^ ''•^'- •'-''-"-^ *«^^--' '-' «- ^"^^^™i? -^"^ at 37' C frothy but no coagulation; in 72 hou« coa^lation commwicinK. g«8; m 4 days gag holes in curd, frothy; in 10 days clear whev on surface of soft gassy curd. In 2 months no dig^tion. ^ ^Th™!!!"~'" ^,1^'""? ^r '•!•««"''. »'«'»> froth and gas. no coagulation; in curd pinkish to Isabella; no digestion in 2 months. Aescultnagar.-a7' 0. One loop from peptone broth culture streaked on plates- in 24 hours reaction brown to black. Piaies, 4e»cuZ«n {.ro< v^. ^*'"of gela"tine"'""" ^'^''^-^"""''^ ^«^""1 «>o"ths. in artificial media, agar Belation '"^^^' •""«»' froth: medium throuZut • tube riddled with gas l.uW.los. The org.nni.m appear, t^ grow c.,uX w.^ aerobically or anaerobically. '^ k|u.iii> well Biochemical Reactions: Indol production: Indol not produced. Production of nitrates : Nitrntes reduced to nitrites. Voges-Proskfluer reaction; Positive. Methyl red reaction: Alkaline. SnTif "'-i^ ^"'^':'''-^i™**"'-7T»'^ "'-tio" of t»'e --"Iture on dulcite is vari- able but It evidently ,s able to ferment this aleohol to gas. some tests being BACTERIOLOOT OP SARDINES an Mannite. + + Xylose. + + Dulcite. + rt Salicin. + + SESSIONAL PAPER No. 38a positive, some negative; the alcohol adonit on the other hand is fermented to acid and profu..e gas with frothing in 24 hours. The action on inulin ia somewhat characteristic, fermentation to acid and gas with frothing in 24 hours; no other strain isolated has such pronounced effect on this test sub- stance. Withm 24 hours all the remaining carbohydrates are fermented to acid and profusely to gas with very pronounced frothing. In general this culture u much more active in its fermentation reactions than any of the cultures hitherto described. Glucose. Lactose. Saccharose. + + + + + + Adonit. Raffinose. Arabinose. + + + + + + ■ Aesculin. Glycerine. Inulin. + + + + + + + = acid. ++ = acid and gas. Culture 36. Source: Can IV. Ser. I. Packer B. 3^orpAo/opy.— Microscopially v.rying from very short stumpy rods to forms twin, as long as broad; the majority .8-1 ^ long, staining unevenly with Kuhne's methy- lene blue; Gram negative*; from old agar culture no evidence of spores. MotUity.— In hanging drop occurring singly and in pairs; extremely active motility. Cultural Characteristics: — 4ffor »/ope.— 36 hours ST'C, moderate along track of needle, glistening iridescent. rorcelain to yellowish white by transmitted light. Herring Agar slope.— id hours at 32°C., growth moderate, slightly raised, dry but glistening, some discrete colonies, by transmitted light blue to yellow. Loeffler's Blood Serum.— 'Z^ hours a"" C, moderate, Klisteni.iK. No liquefaction after 7 days. LUefHer's Malachite Green Sol.—U hours ZVV.. coagulated as Culture 34, much gas; in 72 hours reduced to greenish yellow. In 14 days coagulum not further reduced but precipitation on sides and bottom of tube; ferric-yellow liquid expressed. Gelatine *i 'I hours niiforra growth equally good on surface and in stab; in 48 hours ,.o liquefaction growth ..ii surfa.c showing, moist; in 4 days growth lu-xurimit; in 7 days gnnvtii becominfr brown, medium elightlj tinted; no liqueftetion artor 21 days. Nutrient Broth.-18 hours 37''e.. moderate oven cl..uHing. no rK^llide. bluisl, rim at top, no sediment; in 48 hours heavT ck :ding watered sill< apin^arance, later sediment noticeable; no pellicle even after 10 days. Herring fcrot/i. -Moderate growth, clouding Hoc-ulent suspension, bluish rim. no pellicle; in 48 hours brown viscid setlimeiit precipitated; in 10 davs ring on surface, very heavy floeculent growtli, black sediment. il/t7A-.— 18 hours 37°C. Much gas on shaking, with froth persistent, no .••mgula- tion, in 14 days weak coagulum commencing anfl en„gulatioii sl.nviv rrm- pleted when examined at the end of two months. Litmus ililk.-ln 18 hours no coagulation, much froth on shaking with froth persisting; violaceus merging into heliotrope; no further change in 10 days; in 14 days lilaceus. no coagulation; when examined 6 weeks late coagulation complete, lilaceus. 2oe mVAmUBST OF THE yAVAL HCBVICE 8 QEORQE V, A. 191S AucuKn agur.—iluf loop from peptone broth culture streaked on plate«- in 24 hours 37° C. brow.i to black reaction. AeseuUn broth.— The typical black reaction not given after 7 davg; change only to brown. MacConkey', N.R.B. broih.-Jn 48 hours 37° C. an eosin tint but no .-eduction to canary yellow after 7 days. Gelatine colonies.-Eoom temperature (Ist appearance) in 72 hours surface colo- nies small average 1 mm. diameter, glistening flat, round; bv transmitted light bluish white, almost transparent; characteristic ring in g.-lntine as noted, c-uiturc 8:J; surface colonies yellowish white, small, round. I'nder the low power objective surface coloiiics round distinctl.v granular and dark yellow centre surrounde,! b.T pale border and edges entire and hyaline; on gelatine, the colonies unlike those previouslj- desorikd. N.B.— On referring to the notes made when this culture was originally isolated SIX months ago, I find that on agar the colonies were tharacteristicalb' different from the .•olonjes of Cultures 32, 34 or 35. It is of interest to note that this individualit.v has been maintained throughout a period of this length, iiiid in spite of having niiiny times been subcultured on labora- tory media. Agar Colonies.-SO hours, 37°C. growth rapid; surface colonies Mi mm dia- meter; flat, glistening, iridescent; some colonies extending as thin blue pro- tuberances over the medium; by transmitted light colonies bluish, little darker and more opaque in centre. Subsurface colonies up to 25 mm. diameter. Under the low power objective surface colonics coarsely granular, j lediate centre slightlj- darker and well defined; remainder same structure throughout • edges entire ; subsurfs .'e colonies compact, grumose to "mound-like" structure ' often the surrounding medium a light ferric colour due to precipitated gra- nules with no definite outline. Temperature Relations: — Thermal Death Point.— 10 minutes exposure to fiO° V. in nutrient broth. Optimum Temperature.— Qrowth satisfactory when incubated either at room temper- re c at 37°C. Most sptisfactorj- growth at 37''C. Vitalit « Culture Media.— The culture survives several months on artificial ^la, agar or gelatine. Relation to Oxj/flrpn .—Facultative anaerobe; incubated for 36 hours at 37° C. under anaerobic conditions grows on glucose agar as pale bluish thin film along track of needle, transmitted light; spreading over slope as discrete colonies; heavy cloudy growth in condensation water; much froth in tube, gas bjbbjcg j cm. diameter throughout medium. The organism grows eiiually well aerobJcBlly or anaerobic- ally. ■Biochemical Reactions: Indol production Seduction of nitrates Voges-Proskauer reaction Methyl red reaction Indol not produced. Nitrates reduced to nitrites. Positive. Alkalinu. Fermentation of Carbohydrates: The culture ferments lactose to acid, but gas in not produced until 72 hours after inoculation; the amount then is small and no increase is observed on further incubation; glucose, saccharose, xylose arabinose, and mannite are fermented to acid with protuse evolution of gas within 24 hours. The action upon raffinose is feeble. The Andrade indicator BACTERIOVOOY OF SAItni\gs SESSIONAL PAPER No. 3t, ij^^^^^ dr"'°"r'^ i" '»>«. ac8ouHn. amiming a lemon yellow tint ,nn>. pe«,«t>„K; th» oolour i« partidly ch.e to th. ^iLside hself ' "'*• Glucose. Lacto*. Sacx-haro^-. Mannite. Dul.it.. ■^"^ +* -^^ ++ -_■ Xylow. Salioiii. Adoiiitf>. Aesciiliii. + - Lactose. + + Raffinoso. + - f'l.vcfriiK'. + - Saccharost'. + + Arabiiioso. + + Iiiulin. + - + + + = acid. + + = a<'id mid Ka-*. Ciilhirp .jr. Sourrr: Cm V. Sor. I. Pu<-k.r B Cultural ('liaracter'.sticn: — 'iumably from muscle sugar in meat cxtrnrt \f ,• " *V^'"n> P'e- i-oeffiers Blood Serum. — 24 hours 37° f liivi„;„„* • j liquefaction in 7 days luxuriant, ra.«ed, white, spreading, no Loeffler's Malachite Green Sol —In 24 hn„r^ „, • •» * , ,. , on sides of tube; in 48 hours r^i^tiS^to vr"''^ '"'''* ^"^" '""'"f"'"'" to yellow and aimo«t eSy digeS "^ " ^'^""""*' '" ^ -^"^^ '-'"'•«» "'frf;:^;;;Sr ^=-i:^ - --^^^^^ —-t^i r form; in 7 d«ys liquefaction complete and medium shTr^I^^^^?,:^"*?"'- layers; .mmediately below ..rface liauefaction appeal the Jlu of tu*^ Nutrient Broth.— 1% hours, 37''C.. heavy elondino. ..„f ki,.»i. • -11,. cioudinir. surface iridescent, uellicle ^emHff Hroth.-Vety similai above but heavier growth ■ i. ± A . ing and thick bluish white pellicle; later floccufent ''' '^ ''^''''^^ directly from surface to bottom. aT a lat^^d^t^ ttT2e":li:iZ'Z *H DKPAMTMKyT OV TBM NAVAL BEMVtCM • QEORQE V, A. 1»1t been in pure culture for vivenX znonths, • decided ropineM wae noted, milk tubes being dictinotly tlimy within 24 hour* after inoculation. This feature appears to hare developed under cultivation and has since persisted. Liimut Milk.~In 18 hours violaeeus, no coagulation ; in 4« hours gas. heavy pellicle, coagulated and digestion proceeding; in 4 days a yeibw digested fluid extending 2cm. below surface, remainder vicJaceous; in 10 days i digested, remainder soft gelatinous curd; in 14 days except for tint, appearance very similar to milk as noted above. Aetculin agar.—l loop from peptone broth culture streaked on plates. In 34 hours black reaction. MacConkey'i. N.R.B. Broth.— la 24 hours heavy growth. No reduction to canary yellow. Later colour slightly changed but no definite reduction. Gelatine CoUmiet.—iUt appearance.) Boom temperature in 72 hours liquefaction well advanced ; individual colonies up to 3mm. diameter, round, saucer-shaped, characteristic of the organisms of the proteus group; centre of colony dark white spot -26 mm. diameter, then clear space, then semi-transparent rim. Under the low power objective opaque centre merging into myceloid filaments, then clear space, and heavily clouded borders with entire edges; medium unchanged, no characteristic smell. Agar colonies.— 20 houre at ST'C. growth rapid, surface colonies concave, IJ- 2ivam. diameter; very slimy after repeated sub-culturing drawing out on needle 10-15em.; grlistening; by transmitted light distinctly radiate, whole colony bluish but slightly more opaque in centre; subsurface colonies bluish to white. Under the low power objective surface colonies brownish with dark opaque centre in some, finely to coarsely granular; some colonies same struc- ture throughout; edges entire hyaline. Subsurface colonies distinct, grumose to mound like. Temperature Relations: — Thermal death point. — 10 minutes exposure in nutrient broth at 00'' C. Optimum temperature.— Cnltutea incubated at room temperature and at 37'C. grow well. Most satisfactory growth at 37° C. Vitality on Culture Media.— The: culture survives several montlis in artificial mi>diHm agar or gelatine. Relation to Oxygen. — The culture is a facultative anaerobe; incubated for 36 hours under anaerobic conditions moderate growth on glucose agar slope, bluish tint; very heavy clouding of condensation water; on the sloi)e seen as discrete colonies varying from a thin bluish film to converse moist colonies 1 mm. diameter with ferric yellow centre paling towards edges. The medium riddled with gas bubbles i - 1 em. diameter, much froth in tube. This organism appears to grow equally well aerobieally or anaerobically. Biochemical Reactions: — Indol production: Indol produced. Keduetion of Nitrates: Nitrates reduced to nitrites. Voges-Proskauer reaction: Positive. Jrethyl rod reaetion: Alkaline. Fermentation of Carbohydratea.—JhU culture ferments Inctnso feebly to acid the Audrade indicator showing reduction in 48 hours, and no gas is produced. Raflinose, glycerine and inuHn are fermented to acid with slight production of ga? ; the gas in glycerine not appearing until the second day. The remaining fermentable substances are acted upon rapidly, evolving gas profusely within BACTEBIOtAMir OF HARDISBH aoB SESSIONAL PAPER No. 38a + + Xylose. + + Salicin. + + 24 hour.. It wiU be wen that of the two glucosidet UMd. talicia and ae«>u- an, tne former only ig fermented to Kas. In the later cultural experiment, a di.tinct .limineM appeared in all SS^'o.i!!''"^*'"".!'" ,*'*'* ?** *'*•«'"* "•'^^ •»«•'•: • P-l^^hite rim a surface obserred to be .limy after sereral days at 37* C. Glucose. Lactose. Saccharose. Mannite. Dulcite + + + - + + Adonit. Raffinose. Arabinuse. + + + + Aesculiu. Glycerine. Inuliu. - + + + + + + = acid. ■H- = acid and fras. Culture ei. Source Can. VI., Ser. I, Packer C. From this source four strains have been i8olated-64. 64a. 64b aerobioallv and e4c anaerobicalbr. The similarity of the strains in culture is such that neS'd.^rS^ Si ,„7^ " "**' warranted There are. however, certain cultural differonces i^Wa T. rrh^ri^rsrthrfoSSjdt -s^i --^ »^ -'-• -" "' culture has been recently isolated, and no evidence of spares h^sS obtained- this feature cannot at present be finally reported upon/ oDtained. Jfo'•• """^"«*«" "'"'* ^----k »f needle, flat, slightlv con- ^"'?r'thti.t7er''°'""''- "" """^'^""*'""= - '•' ''-- -'X'--. --^ ^"^co?J/l"1''''-?'r"Ki^/'--'' •'""" •■^'^ ' ■• "''^'P''«t« "t lK.tto,n of tube, no fluTiThf""- *"'?'^' ^" '^r" ™'''"'- '" ^ -^-y^ ■^''»"^'^»' b^'W" turbid fluid with ferric precipitate at bottom. Gelatine Stab --R,^m temperatur(^24 hours, filiform, no li.,uefaction- in 7 days no ...uefaction. growth luxuriant, surface and in stab; yellow growth i^ stab '' '""-'/'•'"''■-24 hours ..7; C. even clouding abundant, ' watered silk " ap^ri ance no pel hcle. „„ sediment; in 7 days clouding even, no pellicle h^vv viscid yellowish white sediment at bottom of tube ' ^''^giTlu^Tf °^-' ^T^' "" ^^'''''"•^- "° -"''"lation; in 72 hours soft coa- gulum, much gas. whey expressed, curd shrinking; in 7 davs white turWd whey, curd shrinking and split hv gas »-. ' uajs wn.to turbid Lit,„us mUk.-lu 24 hours liliaceous." much froth on shaking, no .angulation- ir .2 hours soft coagulum, bleached to isabella, curd pciceptiS "hrinki'n^ 7?cTiMZirt'''' '^"^'1 ^'''^ heliotropenim'a?ift"tpth gas hoTes. ''^ '""^ ""''^'^ disintegrating and permeated with N.B.— This culture is violent in its action upon milk. DKFAMTMtKT Of Tat VAfAL aSKVtOM • OCOROE V, A. 1»1t AfevKn agar.—\ loop peptone broth culture streaked on plates. In 84 hours Z1*C., black reaction. Afeulin hreth.—ln 84 hours. 37'r., black reaction. MaeConkey't N.R.B. Broth.— In 48 hoars, 37T., t diced to canary yellow. Oelatiue Colonie*.— Room temperature (1st a^, aiance) — identical with Culture 3S — A4a presents some variation. In 78 hours irrowth rapid abundant, more luxuriant than any of foregointr cultures; surface colonir to 1 mm. diameter, compact, white, opaque with tendency to c roun'l; the smaller colonies bluish to bluish white. Subsurface co .mall co&.pact. Tndcr the low power objective surface colonies have appearance identical with the literature descriptions of the B. eoli colony, edges eati centre dark and opaque; subsurface colonies pale yellow in colour, very finely granular, slightly darker in centre. See Culture 36. Agar Co/on w«._20 hours, 37°C., growth rapid, flat, surface colonies 1J-8J mm. diamc'.er, round with tendency to spread; by transmitted light distinct bluish appearance, glistening, iridescent. Subsurface colonies up to 0-25 mm. diameter bluish to white. Under the low power objective surfoce colonies have small well defined dark centre, remainder lemon coloured; structure coarsely granular to grumose, edges entire, hyaline and well defined; pale radiate filaments — star-like rays — emanate from the colonies into surround- ing medium. Subsurface colonies dark grumose to " mound-like." Agar Colonies Bia. — 20 hours 37°C., growth rapid, surface colonies bluish from 1-3 mm. diameter, glistening, iridescent, tendency to run together, forming blue film over agar. Subsurface colonies up to 1 mm. diameter white to yellowish white; some force their way to surface and appear as yellowish- white in centre, spreading on surface to 3 mm. diameter, blue, flat, concen- trically ringed, contoured, edges undulate to lobete. Under the low power objective surface colonies (majority) finely granular at centre to grumose near edge; in some instances characteristic protuberances over agar as in culture 64, edges entire; subsurface lemon yellow, edges entire. , Temperature Relaiiotu: — Thermal Death Point. — Exposed in nutrient broth for 10 minutes at 60"C. organ- ism survives; exposed for 10 minutes at 70''C. no subsequent growth; exact temperature not yet definitely determined. Optimum Temperature. — Grows well at room temperature and at 37° C. More satisfactory growth at 37''C. Vitality on Culture Media. — Not yet determined. Relation to Oxygen: — The culture is a facultative anaerobe; incubated for 36 hours under anaerobic conditions at 37°C. the medium — glucose sugar — is split, riddled Tvith gas bubbles and upper portions blown to top of tube, much froth; heavy cloudy condensation water permeated whole medium. The organism grows with extreme rapidity both aerobically and anaerobically. Chemical Reactions: Indol production: Indol not produced. Reduction of nitrates: Nitrates reduced to nitrites. Voges-Proskauer reactions: Positive. Methyl red reaction: Alkaline. Fermentation of Carbohydrates. — The culture fails to ferment dulcite and adonit to acid or gas. All other test substances used are fermented within 24 hours MAvnKiouoar or aAKDissa UMIONAL PAPKR N«. Sta to aoid. and profuMljr with much frothioff to gat. In th« sluoow, laotoae. MCotaaroM, mannite, rafflnoM. and arabinoM tubes the Andrade indicator is comp etely reduced within S4 houn. the reduction in the xylose, .alicin and aesculin tubes being slower. Compared with the other cultures described liwein, the rapid and violent action upon the carbohydrates is both distinctire and characteristic a. also is the rapidity with which the Andrade indicator "L-^ T^- The decolouriied tube, when tested with methyl red show decided alkalinity. The rapid reversion to an alkaline rea tion U « pointTf considerable interest. *^TT'"' '!'^*"''*'?" «'"<'♦'»«• •"> identical with those of the above culture, noted'i'n thl"f t- "'"""^*^^'•*^'""/ ^ ••"* temporary however, ha. be«i noted in the action upon the Andrade indicator. No reduction of the indi- cator in any tube, was noted within 24 hours; in 72 hour, glucose. m.nn"to. ««b nose xylo«, and salicin had chained from the scariet tint of the l^d I h«l^ « ^? completely reduced givi^r an alkaline reaction to methyl red. toZLtCA^SlT-Z'V^".'^"^''''' '^'" 'PP'«»* -elective act^n rsIt^r'^^rn;^^^^^^^^ r.^rstrs.Kocr '^^ '^'-•^ ''- "-" - -^ •" -" ^- -^ »? '" *ilir'"T-''"' i- ""^••f "^ '"»*"*» '« ™«"'»i°n that for .ome month. I have i^ter anawS Thf ?""" "" " T "''"•"'°' '" «""»-"•"• with iS water analyses for tho colon group; these experiments are as yet not suiB- ciently complete for publication ; I have used this indicator in sS b«th" as the Andrade indicator, a selective action. Glucose. Lactose. Saccharuse. + + + + + + Adonit. Raffinose. Arabinose. + * + + Aesculin. Olyoerine. Inulin. + + +± + + +<• = acid and gas. + = acid. Mannite. + + Xylose. + + Duloito. Salicin. + + m I DKrdKrMWT or rat s^tal tmntoB It rt t' h ^i ~n '•< i" — 4 k :j 'i si ii el U • MONM V, A. If !• •■*»o«n«»ji»i^ ■»«ik I » I •m i iJiJii^ 1* 1' 1^ l^^l 1I!W II .' Ji 13 i I j>i I R i 9 "m^n loauviM ' . ^ I hi tn^i n n Jill] J il BACTK»ioi4mr or itAiinixKa UMIONAL Mnn N» ••• ■«l|n"I MiMMMii ii tio DBfAimtMKT or raa watal tBMftoa • MOWW V, A. ttit «XPr«iniE!fTAL RWELLED CANS. Having iaolatod ttniiM of (u-prodttrinc bcctori* from iwrlM rani of MrdinM, ■itd htTinc determinKd tiwtr rullunil fniturM and biochemical naction*. the itrat tlap wa» to >ropt the eaperimental twelliiic of normal cant bjr inoculation of onraninn* •Ireai' olated. Up to the pmcnt I hare UMid three culturtw for thia purpoM— cut- If 37 and 64. Thaae three cultnrm on the basi* of their biological and bio- «•! reartiona are tuilciently differentiated (page* l»V-Si07) to warrant indiri- durfl trial*. A number of normal cam of tiardinea were moat rourteoualy supplied by the manager of the Chamoook factory. St. Andrews, N.B. Some of tlic can* weru of •ariiiiiea periled in cf>tton*eed oil. olive oil having been UMy. 'n two. an.l i„ long /„,«. Cultural CharaeUri$He§:~ '"VdJ:; ?«r -""'•'' "" ^''•■«- "• ^ -•'•• ^*"- -rbi., .,iK...i.... „...„.,;..« .till prooeedin^ ^\t\^ L'JmnJZt . ""= 'V " <'«y- Ji'iuefnctbn f.'rri.. lemon. »*" "' "*"'' " •'"^''•'t^' """"' Kr-.wth; milium Biothrmical Reaetionn: Indol; not prodiufHi. Nitrat<>«: not reductHl- Olucogo broth : acid, even clouding, no km excroL'" •"""" '" ''' '""♦•""" " ^^P'-l "' "-y "train, i.„l„ted from herrin. Culture HI. Source: Normal Can 8ardine«, Packer A. .VorpAo/o.7.,/.-KxtrcmeIy long thin rod*, forming ^Dores^ in h«. ■ a «.ngl.v and in two,, motile. Oram ponitive. *"* '''"'' "<•'•""!•« Cultural Characteristicg.— ttnck creamy, medium yellowish brown ' '*' '""""""t- ''•''hiii^cot'isri.rio t;^ leirsr"'"'- ^^'■''""•- "■ « "-^^ »-»- medium firm h;rd curd * *'"" "^"'-^ '^'"P'"*-' '•'"■ainder of LoefflerB Blood 5er«m .-Rapid li,,uef«ction 23366— 3k 212 DBPARrUSyT OF TBE NAVAL BBXTICB a GEORGE V, A. 1918 Gelatine Stab;— Room temperature, in 24 hours crateriform liquefaction beginn- ing; proceeding slowly in 7 days to 6 cm. from surface of stab; in 18 days not complete, layers of yellowish precipitate. Biochemical Reactions: — Indol: not produced. Nitrates: not reduced. Glucose broth: acid, chiefly at surface, no gas. From the same can. and other normal cans, strains were isolated which according to the reactions noted proved to be identical with this culture. Culture IS. Source: Swelled Can I, Series II, Packer A. Morphology. — Large coccus, occurring as staphylococcus, no sjiores, Gram positive. Cultural Characteriaties : — Nutrient broth.— 24 hours at 37°C., moderate, cloudy; no pellicle, ilft'tt.— In 6 days no changi»; no change in 1 month. Litmus milk. — As milk. Gelatine Stab. — Room temperature. In 2 days no liquefaction; in 5 days scant growth filiform to discrete; in 14 days mediimi faintly browned, growth in stab discrete and ferric yellow tint; no liquefaction, growth better under surface. Biochemical Reactions: — Indol: not produced. Nitrates: ? Glucose broth: Acid, even clouding, no gas. Culture S8. Source: Same Can as Culture 13. Morphology. — Long rods many times longer than broad, oval spores formed; Gram negative; in hanging drop appear singly; in twos and in long chains; motile with gliding movement. Cultural Characteristics: — Nutrient Broth. — 24 hours 37° C. Moderate, cloudy, slight pellicle; in 1 month cloudy with flocculent yellow sediment. Milk. — In a days no change; in 1 month digested completely, yellow turbid fluid. Litmus MUk. — In 10 days dark purple fluid with no previous coagulation; un- changed in 1 month. Loeffler's Blood Serum. — Rapid liquefaction. Gelatine Stab. — Room temperature, in 2 days slight liquefaction noted; in 5 days liquefaction progressed to depth of 2 mm., stratiform, remainder of stab dis- crete colonies; in 14 days liquefaction 1 cm. iK pth, stratiform yellowish layers. Biochemical reactions : — Indol : not produced. Nitrates: not reduced. Glucose broth: Acid, upper part, pellicle, no gas. Cultures 13 and 26 typical of several strains isolated from such cans. BACTERlOLoar OF I^ARDiyES SESSIONAL PAPER No. 38a 213 Culture 1],. Source: Swelled Can II. Series II, Packer A. """ttr, dtp^:i:;'Liri„' 3 ^d^rt-^ ^- «-- -«ve; <„ but prolonged examination r;veairsW?«K^" J! "*' '"^" "* *'"' '""""»•"« appearing to pu«h themselves Xi ".ovement, wme individual. Cultural C nrar^-rigties — biochemical reactions: — Indol : not produced. Nitrates: not reduced. Glucose broth: acid, more particularly nenr the surface, no gas. ai4 DEPAIfTMBXT OF THE XAVAL SESVICf! BRIEF SUMMARY. 8 QEORQE V, A. 1918 2. CottoiMeed oil. and the excreta of fresh herrings have been examined. UoJl^. l""" «*™'f«' Cultures 24 and 37. liquefy gelatine, and fail to ferment iS^m^VIm ' " '" '^" '''■'"'"' *'°''P' ^- ""'^""^ (»»»*•«' ... /?2 '^^^ 'fmai""* six strains are lactose-fermenting types. I consider that these include typical and a-typical types of the c'lon^rogeneTgrZ (Escherich) ; but for the present an individual classification is not offered. n.«f" Jb^ ^^^^o^ ""^ reactions of the gas-producing bacteria have been summarized; Pages £\}o &nci zUtf. 1 . *• ^*P«"'?«'ntal "swellings", typical in every respect have been produced in the laboratory on mocuation with Culture 35. 37. and 64 respectively. The^ganisms subsequently isolated have been proved culturally to be ide^cal wUh those ^^^fTr inoculation; thus satisfying the " Postulates of Koch." 7. No bacteria have been found in the cottonseed oil. 8 Non-gas-producing bacteria have been isolated from herring excreta from sweUed cans^and from a small percentage of the normal cans exam^ed ; br ef nZ are presented on pages 211-213. «"iiijeu, oriei notes 9. No gas producing bacteria have been isolated from normal cans of sardines. I desire to express r,v indebtedness to Dr. A. B. Macallum; to Eh-. A. O Hunts- ^; to Dr. F O. Harrison; to the Maine Inspectors of the " National Canned Association of America ' ; and to the proprietors and n.anagers of the various c^nW factories which were visited with their permission. canning BACTKRWLOr.r OF 8ARDISE8 21S SESSIONAL PAPER No. 38a HErERENCEH. 10. 11. 12. PfMcott 8nd Underwood, 1897. " Micro-organisms and Steriliiing ProceMes in the Canning Industries." Technology Quarterly X. 1. P. 183-199. Macphail and Bruere, 1807. "Discolouration in Canned Lobsters." Ottawa Supp. No. 2, 29th Annual Rept., Dept. Marine and Fisheries. Obst, 1916. " A Bacteriolitgical Study of Sardines." Abs. Bact. 1. 1. P. 50. Nielson, Ivar. 1890. " Eiti Stuck moderner Bakteriologie aus dem 1:! J«n- rundert." Central, fur. Bakt. u Parisit, orste abt, 7. 267. Auchi, F., 1894. " Comptes rendus de la Soc. de Biologie." P. 18. Vaughan. V. C. " The infection of Meat and Milk." Trans. 7th Inter. Congr. Hyg. Vol. Ill, Sec. III. P. 113-129. Savage, W. G., 1913. "Bacterial Food Poisoning and Food Infection." M.O. Rpt. Local Govt. Bd. Food Bpts., No. 18. P. 46. Vaughan and Xovy, 1902. loc. cit. P. 209. loe. cit. P. 188. McWeeney. " Meat Poisoning— Its Nature, Causation and Prevention." Journ. Meat and Milk Hyg. Vol. I. John Bale, London. P. 1-31. (Note. — Separate not dated, evidently about 1909.) Baur, 1902. " Ueber zwei denitrificirende Bakterien ou* der Ostsee." Wissensch, Meeresuntersuch. Neue folge. Sechster Baud. Abt. kiel. P. 21. American Public Health Association, 191.5. "Standard Methods of Water Analysis " P. 77-137. " Cellular Toxins." Lea Bros., Philadelphia, P. MS. Longmans Green, Lengtnans (ireen. 16. 16. 17. 10. Besson, 1913. "Text Book of Practical Bacteriology, etc." London. P. 53. "•"sson, 1913. "Text Book of Practical Bacteriology, etc" London. P. 410. Harrison and Vanderleck, 1908. " Aesculin Bile Salt Agar for Water and Milk Analysis." Trans. Roy. Soc, Can., Ill, Ser. IL P. 10.5-110. Savage, 1906. P. 215. "Bacterial Examination of Water Supplies." Lewis, London. Contrib. to Canadian Biol., 1917-18, "Bacterial Destruction of Copei)ods." Ottawa, 1918. Ref. 6, P. 227. Clarke and Lubs, 1915. "Differentiation of Bacteria of the Colon-aerogenes group." Journ. Infect. Disea.se8. P. 17, 160-173. criltner. Cited by, 1916. "Microbiology." Wiley, N.Y. P. 365. "Bacterial Destruction of Copepods." Contrib. to Canadian Biology, 1917-18. Loc cit. P. 218.