Peeks pete by ae phi d peeererer ena TRE patter bed gst Peta Rue oer ey ag t ¥ ¢ ort ben eked | LIBRARY Michigan State University PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE 2/05 p:/CIRC/DateDue.indd-p.1 STUDIES IN DTH Heal neSisTau? ORGALTISuS Co’ COLD PaUarD vali) Fas Th2SsIs Submitted to the taculty of the .ichigan 4azricuiturul volleze in partial fulfillinent of the re- quirements for the degree of Master of Science. ruth wormington lay 1919 e THESIS the writer wisnes to acxnowledze her indebtedness to ..iss uae sorthrup and yr. ward -iltner Yor the helviul sugsestions tendered to ner durinz the course oc trese investications. 105189 ~~ ow I. Il. Iii. Wy worst UC Loads Introduction. review of Literature. 40 1. 4. thods of rrocedure. aXterior exaiaination of all cans of peas season. vonplete exadtination oi linited number. Jescription of organisms found. action of sinilar organisms on peas. Discussion of xesults. oummary. Conclusion, Sibliography. Canned during oe STUDIAS Il TER Koa acSISTL.T O2GL.Is..5 Of GOLD PacciD UAlLsD Plas Introduction. During the past few years imuch has been said about food conservation, the no.wie garden and hoite canning. scspecially has the de.:onstrators atte supted to help the housewife save garden veeetables which would otherwise be wasted. These efforts have been largely successful. In many instances, however, carned vegetables have spoiled. ‘Yhe reason for this has beer given as due to tne use of vegetables not fresh or failure to follow the directions as given in the vunitec states Departiuent of s«zriculture bulletin number 839 on howe canning. “rom this arose the ouestion as to whether the spoilage was due to either oi these causes or both, or if there might be some other cause for this trouble. It has been the writer's intention during the course of this investigation to solve this problem if possible and de- termine the cause for the very much larger proportion of spoilage from home canning in co:parison with commercial canning. review of Literature, Frrobably the first bacteriological examination of spoiled cannei zoods was reported by Russel (17) in 1895 in con- nection with the occurrence of "Swells" in canneries of Wisconsin. in this paper "vaseous fermentation in the vannins Industry" he cites the isolation frosi swelled canned peas, two bacilli which he did not identify. he recommended the increase of temperature to £42 decrees t,. and the pressure to fifteen pounds for twenty- eight tinutes. In 1904, Harding and sicrolsoni13) examined an outbreak of spoilage in canned peas. ‘Whey found spoilage in which there was Siuply sourins of the product and also spoilage in which there was gas present and malodor. aA suall rod or coccus caused the former and a plump rod with terminal spores Wnich zsrew vigorously in the presence of suzars at 37 degrees vv. caused tne latter. Linits for successful processing were determined. In 1905, Duckwall (12) wrote a book on "Vanning and -kreservine". One chapter is devoted to canned peas in which he discusses tne history of peas, their parasites, their coinposition and food vaiue, metnods of canning, and bacteria associated with Spoilage. .szhe organisms discussed were those found by hi:self and others. he mentions lactic acid bacteria, 3. butyricus, 3. mesentericus vulgatus, "A putyric acid bacillus which was a strict anaerobe with terminal spores similar to 3. tetani? 3B. mecatherium, 3. prodigioses, os. subtilis, 5. mesentericus ruber, B. mycoides and some organisms not namei put described. he recou- mended a longer sterilization period at a nigher temperature, for canned goods. savalla (21) publishes a book in 1916 on "Vanning of “ruits and Vegetables". In rart Lil, he discusses spoilage, siv- ing a report on two hundred and ecighty rive bacteriological ex- Qiuinations or canned roods from six camneries. wine spore formers were described but not named. vas was found in cans of peas but these organisms di@ not -orm gas in sugar fermentation tubes. Dickson (11) in 1917 investigated an occurrence of botu- lism on the facific Voast. de found 3. botulinus in three cases, ke then inoculated canned peas, beans, and corn with B. botulinus, heatine them at a boilins temperature in accordance with Govern- rent bulletin directions, These developed gas in three weeks. 3. botulinus and 3. subtilis were recovered fro; the cans. he con- cluded that the time given in the directions for sterilization in the Governient bulletins is insufficient. the Departinent of agriculture (19) answered this paper by saying that the spores of 3. botulinus are killed by one hour of heating at 175 degrees «. and that there is, therefore, no danger fron 3. botulinus in canned food. It was glso stated that the toxin from 3. botulinus is destroyed by boiling a few minutes. however, a warnins was given against eating 1:o0ds showing any Signs of spoilage. ae wv, 3ittine and 4. Gu. Bitting (4) published their bulletin "Bacteriological uxamination of Vannued #«oods" in 1917. LThey discuss leaks, springers, the proper method for opening tin cans for exanination, pressure in cans, swells and flat sours and orranisms of spoilage. -shey deternined by experiment that it takes a tin can of peas standing, over twenty isinutes to reach £48 degrees :. Cans are more uniformly heated when agitated. Or- fFanisas round were a large lactic acid bacilius, a coccus form which produced inmalodor and thermnuophilic forms in appearance similar to 3. tetani and 3. botulinus. They state that some organisms require aimuch higher tenperature than others to destroy the spores, Georgia Spooner surke (6) in 1918 wrote a bulletin, "The Lffect of heat on the Spores of 38. 3otulims." She states that B. botulinus toxin is killed by five minutes of boiling, but the spores are not killed by boiling for five hours in jars of fruit. They will also survive three and one half hours of boiling in an open kettle or fifteen pounds of pressure in the autoclave for ten ininutes. she concluded that the pressure cookins is the only sure method of avoiding spoilage by 3. botulinus. In 1918 Bushrell (7) experii.ented on the influence of cold shock on the sterilization of foods workins with both elass jars and test tunes. ke Tound that cold shock did not promote the xeepin= ouslities oz canned food. s&xclusion of air, however, prevented aerobic orsanisms Yrom developing when their svores were present. J. weinzirl (20) in his thesis entitled "The Bacteriology of vanned soods" ¢cives his resuits from investigation of one thous2nd and eighteen sanples of canned goods. seside olds and yeasts, he isoluted three hundred and ninety two bacteria, rep- resenting tnirty eizht species. she most prevalent organisms found were 3. ..csentiricus, 5. subtilis, ~. thermoindifferens, 3. vulgatus, and 38. cereus. He concludes that spores may be present in anparently unspoiled canned goods as vound on the market. ‘hese may be un- able to grow due to lac«c of oxygeli. Jechun is essential for the preservation orf foods under the present iethods of processing. ‘ood poisonine organisms, 3. vpotulinus and 3. enteritidis are not found in commercially canned food. .ethod of srocedure. In the summer of 1918 between the first and twentieth of July, in connection with an experinent in soils, thirteen lots of peas were picked, weighed, and later canned in accordance with the vovernient methods for cold pack canning, One lot was heated in stean; five were cookec by the hot water bath method; and seven lots were processei in tne autoetlave at fifteen pounds pressure. Lot aeiis». is the only one where the cannii.g was done the sane day that the peas were picked, In lot E.iW.5.1. and a.VI. the peas were kept at room tenperature over night. All others were kept in the refriz- erator. all of these peas except lot a.II.i. mentioned above, were partly shelled one day, then kept until the noxt day when the shelil- ins was finished and they were canned. in two weexs, sSnoilave oefran to occur. she following Chart stows the data upon this: 4o.of spoil- Lot var yize Sterilization Time Gans age. asl, “@Son rts. autoclave 15 lbs. 40 min. 7 7 a.1I.0, 2S0nN tts. autoclave 15 lbs. 40 min. 17 1 ALIA. aSon rts. autoclave 15 lbs. 40 min. 33 4 4A,iil. seal fast its. autoclave 15 lbs. 40 min. 30 20 aeiV, ..aSon utselrt. Autoclave 15 lbs. 1 hr. el 19 eV. iu&30n «tsel1 Pt. autoclave 15 lbs. 1 hr. 14 14 a.VI. .880N wtS.1 tt. «sutoclave 15 lbs. 1 hr. 4 1 fiee del, wiason tts.,ats. uot water bath 4 hrs. 4 1 ie wed lI. wason Pts.,ats. hot water bath 3 hrs. 3 o h,w.o.,l1l1l.Seal fast rts. not water bath S hrs. 21 18 E.o.5.1V, uwason its. not water bath S hrs. 27 19 Heu.b.VY. 5821 fast Pts. Hot water bath 3 hrs. 17 5 Sel... ason kts.,yts. Steam © hrs. 15 11 Fercent spoilage oz those autoclaved 50.9 percent Percent " " " cooked in hot water bath 63.8 " Percent " " " cooked in steam 73.3 " rive cans were selected for exanination from lot a.l. and Yive from a.V¥. where there was entire spoilage. shese cans were ex@uined for swells, ¢as, leakage, and all evidences of spoilage before opening;tnen the cans were care- fully wasned with mercuric chloride solution of a one to one thousand dilution. alcohol was poured over the cover and-then burn- ed of2. whe can cover was then iifted enouzh to taxe sainples of the juice, zive cubic centizneters of tie juice were titrated to deteri.ine the acidity. ‘swo sets of dilution plstes or 1 to 100, 1 to 10,000 and 1 to 100,000 were made. ne set of these was clac- ed under araeerobic cor.ditions in a.uovy jar, filled with hydrogen. Une hundrecth of a cubic centimeter was seared uniformly over one Square centimeter on a slide and stained for a direct microscopic count and also ror the deter.ination or the vezatative forms present as much as possible. ‘wo sets of sShaxes were nade by the loon dilution «.ethod into selatin agar. Une set was heated to 80 degrees LG. for the deterii:ation ov the vresence of Spores. ali cultures were incubated at rooi tenperature. ws2ch orzanism isolated was inoculated into a tube o: dextrose gelatin acar. u.chen this was proved to be a pure culture, transfers were made into two tubes oi sterile pes in distilled water. tne set was made anaerobic by vourinz sterile white paraffin oil over the toon of the tube. after tre action in this median: was deteruined, cultures were made in all media usec comnonly for identification purooses. vultures were also .ade ln a one percent starch peptone Solution to detriiine the reduction of starch to surar and the production o- acid. -«zhe thermal death point of eack orranisi was oo G4 g tT C ; SUIS TUBZIN VOV' LD, 9GL°TTO'T, O08'S26'Sh, 000'F80'69G, OOZ*TZZ°tT, JUNOD oTdooS ‘ \ ' \ ,TOLOTU YOOITC O00 OG, LOpeslds, OVl LOD Fe, Letyendty , cLepeerds , oTqoLeeug I { j ft i 999 G6Z, OOF 229, GES SEP g, OGL L , OGh 2, oOFQ0L0R i 1 1 70° ated gunog A i a 1 a TBtslozoeg Teuzror ATavey, Teuroy , Tswx0y , Tewro ATIea;7, TBULOT , LOPO i i = | ig I N/Sv0° . N/Sgo° , m/eeo° , N/8E0° , T/8Z0° , Ay TpToy L. i I _~ i i = : - \ - , PpeABTOOINBOdY, - ' wear 4 _, _- i f - ' - i - ' - ' - i TTEMS 8 8 | 4 ft + i ¢ 1 + ' - ' + 1 SBX) I i 2 i _f + ' + : + ' quoultTpes +, JUOWTpES +, Apnoto ' woser JUTgG —osen GUTa, wosey 4UTa, wosern yaTT, osen QUuTa, Ie ~ } rf , } | ' G°T°v , P°T°’v G°rT°vy. ss, o°r°Y ss, T°I°’y¥ ' mB) { j _j A i *arnsseid spunod ¢cT 48 oaetTooyne of} UT soeqnutu A¥aZOy pezt[Ttzeqye *y *y 4OT *s4Tnsoer ‘IMTpBW STUY UT UOT OB ATS, SUTMI91Eep 04 Sved TO Ssoqnqy OTAOCLTEBUe pUBe OTQoIEsB OLUT pegBTNOOUT Way. eLOM SOTASTIOZOTIVYO ISTIWIS SUTABY susSTtuesi1o UMOUY eu, *‘Sq4Tnsez fo uostaeduoo A¢ *HOUTILZO1V9D 9LAM UBO 9UO Wey aeLOW UT sUTAINOOO SUWUSTUBSIO ‘opal SYWoweATNSBoeUl PUES SUTSIS Pues POeTITWIS4ZEeDP late Il. Lot a. i. ye ~ ay _ sy ~ en" f ~ _ ° 7 ” * ~~ — on Lr an oe ‘eo \ o &” a “oe Pw \ ~ } ~~ ° 6s ~ a” yan A.lele van Aelede =D \ \ v7) \' \ \ ~~ a ® \ re ee ~ Noe fm . “4 . ‘ . os VO delece Can wh Can a.1.5. aw Uy vo. G4 o 4 oo SUSTUBSIIO { Jj | I } O08 244 62% 2,000 2T9 608,000 &28 89G, O00 SPR B89G, 000 968 ¢¢0 &, 4UNOD oTdooS . _~—! . 1 i 4 7 ,TOLOT JO98LITC OOS 649 8, OOS 964, Tapesrldac, oleres O00 098, oTqQor10buUB j i A a | A 008 8%, OOF 9LL, 000 00%: 00d, CER TIE, OTQoLae i I i i ‘ ' \ i © D5 ied 4unoo 1 1 ¢ 7 1 TBTt1l9a40B_g TBwIO0'T , oTrIAgna, [TBULIO'T, Tewrorr ATIVE, Tewu10'T ATAIVOM, LOPO one =} A A i wd 1/EL0°, 'T/SOT", T/E80", 'T/980°, T/9L0°, Ratproy i ai J <é j + ' + , PeZec'{T AOU, peysoy 4o0u, + ' YBOT ' , pepTsoeser, peTvesoy, a ~ i + ‘ + \ + i - t TLOMS i j i. a | t ; ¢ ; + ! + ' + ; SBD i J I i i + \ + ,guavirpes + , + \ + \ Apnoty j L. lL. i A uosey *4q , Bose” °4" , UWost *4* , Wosey *4r | wose7 *4P LBC —_ a -_— —_ j | | PE°A°Y , ST PA OV, OLA eK, TT°ATY , OT° ACY UuBo i wf LL j j ‘ainssoid spunod wee_ZsTy 4@ Annoy suo paasToogny "A ew 407 - 10 clate Lot she \ ™= / } 7” “ ~ XN f ‘ ( - . ~— a ” van -he Ve LO. \ ~~ ° ~ “A ~ ° ( sn ™ 7 NL ~ ~\ “ ~e CAN ze Ve le Li. e -/ “ey #) Dull,finely wrinkled. (c) vream colored ,becoming tan. (d) water in bottom pinkish. Oo. vVelatin agar Volony: (a) Growth - capid. (b) lat. (c) form - Spreading. (d) Senitransparent. IIt, Physical and Biochemical Features: 1. fernentation tubes, neutral red broth plus (a) Dextrose: (b) Lactose: (c) saccharose: (Gag - Nherative. (Reaction - acid. cas - legative. (xeaction - Alkaline. (eas - lecative. (Reaction - acid. 2. teas in distilled water: (a) Aerobic Fubes: G2. AS e Ureamy ton growth becoming deep red ey ye oe, (underneath; more readily produced at (37 degrees C. (b) Anaerobic tubes: (Gas. (liot much visible growth, no color. =~ 13 - 4e Peptone Solution: (a) Indol production, slicht. 4, witrate reptone Solution: (a) Litrate reduction. (b) Amsonia production. 5. otarch reptone solution, neutral: (a) starch reduction. (b) Slight test for sugar. (c) slight acid. 6. Temperature relations: (a) crowtn at room temperature. (b) savored by 37 degrees C, (c) iiot killed by heating at 120 degrees UC. for ten ininutes. (d) «Killed by heating at 120 degrees C. for twenty minutes, Bacillus B, (Conforms to description of 3. subtilis as ziven by rord) “ound in cans 4.1.1, A.1.2, 4.1.3, 4.1.4, a.1.5, A.V.11, A,V.1 I, «-orphology: Be Vede. 1. vegetative vells: (a) xod. (bd) Size - 2.5 = 4.5 x 0.5 - 0.75 microns. £.* undospores: (a) Fosition + ventral or nearly so. 5. -Otility: (a) Actively motile. ~14- 4. otaining: (a) Grain positive. (b) stains with ordinary dyes. Ii, vultural vharacteristics: 1. welatin stab: (a) Top growth. (b) Liquefaction - early crateriforn,. 2. wutrient roth: (a) Heavy tov crowth not easily broken. (b) Sroth nearly clear, yellowed. Se Litmus milk: (a) Feptonization. (b) slkeline. 4, wlycerin rotvato: (a) Very abundant growth. (b) vull rugose. (c) Vvreaa colored. 5. velatin agar volonies: (a) Growth-Bapid. (b) Granular. (c) xhizoid. (ad) «white. lil. rhysical and 3iochemical +eatures: 1. fermentation tubes, neutral red broth plus . (Gas - lone. (a) Dextrose: ( | (Reaction - Acid. (Heavy top growth. (Gas - iione. (b) Lactose: (ieaction - Alkaline. ( (heavy top zrowth. - 15 -~- (Gas - one, (c) Saccharose: (Reaction -~ Acid. ‘Zor growth. £e Feas in distilled water: (a) aerobic culture: Gas production. ( ( (Cream colored wrinkled, growth, darkens. ( ( slime production. (b) Anaerobic culture: (Gas production. (Slime. (Yellowish liauid. 3. reptone Solution: (a) Indol formation. 4, witrate :eptone solution: (a) sitrate reduction. (b) ammonia production. 5. Starck reptone solution: (a) Complete reducticn of starch. (b) Large quantity of sugar. (ec) solution, slichtly alxaline. 6. renperature nelat ions: (a) Grows readily at room temperature. (bp) savored by 37 degrees U, (c) ilot killed by heating at 120 degrees C. for ten minutes. (d) Killed by heatinz at 120 degrees UC. for twenty minutes. Bacillus ©. (xeseibles B. botulinus) “ound in can 4.1.2. I, ..orpholory: 1. Vegetative Cells: (a) Large rod with rounded ends. (b) size - 4 += 6 x 0.75 microns. 2. tndospores: (a) Position - rolar, iuaking the rod club 3, .otility: (a) actively motile. 4, steinines: (a) Gran positive. (b) stains with ordinary dyes. II, Cultural Characteristics: 1. wutrient sroth under oil: (a) Sroth cloudy. (b) Odor of butyric acid. £. Gelatin ager volonies: (a) Anaerobic. (bp) “urry. (c) jhitish, becomes somewhat tan. 3. Dextrose velstin sgir shake: (a) Gas at 25 degrees U, (pb) nwo gas at 37 degrees vu. Til, rhysical vharacteristics: I. ‘temperature xelations: (2) vrowth best at 25 degrees UC, (b) slow crowth at 37 degrees Uv, Shaped. ~ 17 - (c) ot killed by heatin= at 120 degrees v. for ten minutes. (d) Silled by heating at 120 degrees v. for twenty minutes, Bacillus D, 1, ..orpholosy: le. Vegatative vells: (a) Slender rods. (b) size - 1.5 - 3 x .5 microns. &e undospores: (a) Position - Ventral. (pb) sSlicht enlargement of the rod on (c) spores - Oval. 3. »Otility: (a) actively motile. 4, stain:. (a) eran negative. (b) Stains with ordinary dyes. II, vultural vharacteristics: le. velatin stab: (a) Liouefaction,infundibuliform. 2e wutrient Broth: (a) wo top growth. (b) Oloudy. (c) seiiment. 5. Litmus milk: (a) Peptonization, (pb) Alkaline. sporulation. 4, Glycerin rotato: (a) lat, smooth elistenins growth. (o) Color creauy tan. en e velstin sacar volonies: (a) #lat. (b) Spreading. (c) white, turnine tan in forty eizht hours. Iti, thysical and +iochenivcal seatures: 1. serwentation tubes, neutral red broth plus | (sas = lone. (a) Dextrose: ( ( Reaction - Slightly acid. (Gas = Lone. (b) tactose: ( (xeaction - alkaline. | (Gas - iione. (c) saeccharose: ( (xeaction - acid. 2. reas in distilled water: (a) acrobice culture: (Gas. (Creany top srowth. (b) Anaerobic culture: (Gas. Se ceptone olution: (a) to indol production. 4. witrate reptone Solution: (a) .itrate reduction. (pb) amnonin production. ee wtarch septone solution: (2) lone or very slight reduction of starch. (b) io production of sugar. (ec) Slight acid. 6. Temperature relations: (a) Growth both at 25 degrees and 37 degrees C, (bd) wiot killed by heating at 120 degrees uv. for ten minutes. (c) Killed by heating at 120 degrees C. for twenty minutes, Bacillus &, (esembles 3. ramosus) hound in a.¥.10, and «a.V.12. I, worphology: 1. Vegetative vells: (a) Thick rods. (b) Size - 2 = 3.5 x 0.75 - 1 microns. &. undospores: (a) Position - Ventral. Oe --Otility: (a) ..otile, not active. 4, staining: (a) Gran vositive. (bd) stains with ordinary dyes. Il, vultural vVharacteristics: 1. Gelatin »~tab: (a) Liquefaction - vraterifora to stratiforn. (b) whitish sedinent Which turns pinkish, 2. sutrient roth: (2a) Zop crowth flaky which settles on shaking. 3. Litinus milk: ‘ (a) teptonization. (pb) alkaline. 4, wlycerin sotsato: (a) Growth abundant, raised, contourec. (b) .-oist creany to pink becoming ourplish. Do. Felatin agar volony: (a) lat. (b) sometimes soinewhat spreading. (c) white, translucent. Ill, thysical and 3iochemical «eatures: 1. #ernentation tubes, neutral red broth plus _ (Gas _ 1. ONC « (a) Dextrose: ( (xeaction - Acid. (Gas - lone. (b) Lactose: ( (Reaction - Slightly alkaline. (Gas - one. (c) saccharose: (Reaction - acid. 2. teas in distilled water: (a) derobic culture: GAS. — oan oe Souewhat rose colored top growth. (b) anaerobic cyjture: (cag. Se reptone solution: (a) Indol formation. 4. Witrate teptone solution: (a) itrate reduction. (pb) aanonia formation. 5. starch revtone solution. (a) Slicht reduction 6f starch. (b) slizht test for sugar. 6. vemperature xelations: (a) Grows readily at either 25 degrees or 37 degrees Uv, (b) kot killed by heating at 110 degrees UL. for ten minutes. (c) Killed by heating at 110 degrees \. for thirty minutes, Bacillus 4H. Found in cans Aa.1.4, and a.I1.3. I, +orphology: le. Vegetative vells: (a/ Rods usually in long chains. (b) Size - 2.5 - 3 x .5 microns, 2. -otility: (a) Slowly :otile. S. sndospores: (a) Central. 4, staining: (a) Grain positive. (b) Stains with ordinary dyes. if, vultural vharacteristics: l. Gelatin stab; (a) Liquefaction - vrateriforn. 2. wutwient 3roth: (a) ito top growth. (pb) -ledium cloudy. (c) tine precipitate. 3. Litmus milk: (a) Peptonization. (b) Reduction. (c) Alkaline. 4, ulycerin rotato: (a) Growth - -.oderate. (>) Dull creany yellow growth. 5. selatin agar Colony: (a) Granular. (b) «white. (c) Lacerate edges. Ill, Fhysical and siochemical «eatures: Ll. sermentation tubes neutral red, broth plus Gas = None. Nn ( ( (a) Dextrose {Keaction - acid. ( (s uwreen iridescence,. N ; (Gas -— lione, (b) Lactose: (Reaction ~ Alkaline. (Gas - lone. (ec) Saccharose: ( (Reaction - acid. 2. Peas in distilled water: (a) aerobic cultures Gas. Very inoist growth which developes a ( ( ( ( (touch of bright tan color. eS Oe (b) anaerobic culture: (Gas. 3. teptone solution: (a) Indol production. 4, litrate reptone Solution: (a) witrate reduction. (b) amuionia production. 5. Starch reptone Solution: (a) Starch reduction. (b) Wo test for sugar. 6. Temperature relations: (a) vrows readily both at 25 degrees and 37 degrees C. (b) Kot killed by heating st 120 degrees C. for ten minutes. (c) Killed by heating at 120 degrees UC. for twenty minutes, Bacillus «& #ound in can a, I. 4. Ll, »-orphology: 1. Vegetative Cell: (a) Large rod, usually in short chains. (b) Size - 2 = 4.25 x 0.75 microns. £. indospores: (a) Position - ventral. 3. .otility: (a) Slowly motile. 4, Staining: (a) Gran negative. (b) Stains with ordinary dyes, we peewee ~ 24 « II. Vultural Characteristics: 1. Gelatin stab: (a) Liquefaction - Urateriform. Nutrient Broth: (a) Top growth finely wrinkled. (bd) tedium clear. Litmus milk: (a) Peptonization. (b) keduction. (c) alkaline. Glycerin Yotato: (a) Growth moderate, controued, raised. (>) Dry chalky white growth. Gelatin agar Volony: (a) xerular. (b) white with thicker creamy spot in centers. Til. Fhysical and Siochenical features: Lb. #ermentation tubes, neutral red broth, plus (Gas- lone. (a) De-trose: ( ( xeaction - acid, iridescent. | (Gags - ivone. (bd) Lactose: ( (Reaction - Alkaline. (Gas - lone. (c) Saécharose: ( (Reaction - acid. £e teas in distilled water: (a) serobic culture: (Gas. (Keavy creamy wrinkled top growth. (b) sanaerodic culture. (Gas, - 20 =. 3. Feptone Solution: (a) Indol formation. 4, iLitrate teptone solution: (a) Nitrate reduction. (b) Amnonia Production. 5. starch «eptone Solution: (a) starch reduction. (b) 0 test for sugar. (c) keaction - Alkaline. 6. Yenperature relations: (a) Growth at 25 degrees U. (b) Favorec by 37 degrees C. (ec) Not killet by heating at 110 Desrees C. for forty minutes. (a) ) Liquefaction - apivorm becoming stratiform. ee sutrient -roth: (a) Yop growth, rine. (b) ..edium very cloudy. 3. Litmus milk: (a) Seduction in twenty four hours. (bd) voagulation in thirty six hours. (c) neuction - acid. 4. ulycerin rotato: (a) Growth vigorous. (b) 2ull wrinkled whitish crowth. DS. selatin agar vVolony: (a) Tree-like spreading growth. (b) white in color. Ifill, thysical and siochenical reatures: 1. termentation tubes of broth with neutral red plus (Gas - lione. (a) Dextrose: ( (xeaction - Acid. (Gas - lone. (b) Lactose: (reaction - ieutral. (Gas - ivone, (c) Saccharose: teaction lheutral. 2e teas in distilled water: (a) Aerobic culture: (uas ( (..oist growth with touch of bright tar (color. (a) anserobic culture: (Gas - sone. 3. septone solution: (a) indol forination. 4, wzitrate teptone solution: (a) iitrate reduction, (b) Ammonia formation. 5. Starch «eptone Solution: (a) Starch reduction. (b) Lo sugar. (c) xeaction - «cid. 6, vemperature nelutions: (a) crowth at both 25 degrees v. and 37 degrees vu, (bd) wot killed by heating at 120 degrees U. for ten minutes. (c) Killed by heating at 120 degrees vu. for twenty minutes. Sacillus <4, #ound in van a. «. 13. I. «worpholory: 1. Vegetative Lell: (a) slender rod. (bo) size - 1-2 0,5 miLerons,. Til, -q 7] yy “4 = e 2 LGOLLPOLCES? ~~ 0 (a) rroducei subteruinally. (b) shepe - Oval. Oe --Otility: (2) .etively u.otile. 4, stainins: (a) sraa nesative. (b) stains with ordinary dyes. vultural vheracteristics: Ll. Gelatin stan: (a) Liquefaction - Infundibuliforn. Ce nutrient 2roth: (2) Top erowth soft and heavy breaks to a cloudy pre- cipitate, Litmus milk: O (2) Increasei alxyalinity. (b) ito peptonization, curd of recuction. 4. “lycerin .otato: (a) rotato gray. (b) Growth flat yellowish gray. oD. Gelatin acar volony: (a) naised. (b) Glistening. (c) Yellowish white. ili, thysical and ~iochenical seatures: 1. ser:entation tubes of broth neutral red plus (Gas = sore. (a) wextrose: ( (Reaction - leutral,. 5 a G ( 13S = one. (b) Lactose: ( (Reaction - Leutral, (Gas - sone. (ec) sacenarose: ( (reaction - seutral. Peas in distilled water: NM e (a) serobic Culture: Gas - Lone. heavy creamy wrinkled top growth, liauid “~~ = en oor (yellowed. (bp) saneerobic vulture: (Gas = .one. € - reptone solution: (2) Irndol formation. > » Hitrate teptone solution: (a) u.itrate reduction. (b) -smsonia vroduction. 5. starch teptone soluticn: (9) otaurch reduction. (b) Test for sugar. 6e sempcrature melations: (a) Uranus at 25 degrees C, (b) «savored by 37 degrees U. (c) sot killed by heating at 110 degrees U. for twenty minutes. (d) silled by heating at 110 degrees \. for thirty minutes. P2Gillus «a, g ~ ~ dy Sacillus vu, ap rlate 50 = Til. me ~ o” = ¢? Pacilius , 4 q - © i" 7 o - e l-- ~ e a” Bacillus Be @& 4 ~~ vfs 4 ~~ Vs Yu - Bacillus D, slate IV, “~~ ‘ r ' . » \ ~ \ y ™~ \ : @ , 22eililus u. “aGcillus i. ~~ wh s Rw ‘fc eo “#4 sacillus Y. j3acillus 2. vultural vharacteristics in Yeas of vomion Spore searing o2cteria. The followins spore forming bacteria corresponds quite closely in all cultural and ::orphological characteristics to certain of the bacteria just described. I1n the first two instanc- es , even to sas formation in peas. «as far as can be determined this is @ circunstance hitherto unnoted,. I. 3. subtilis. 1. Aerobic Culture: (2) Gas after forty eight hours. (b>) abundant tor srowth. (c) srowth dull, wrinkled crear colored. (d) savore®’ by 57 degrees v. (e) Liauid yellowed. 2. anaerovic vulture: (a) Gas formation - sone. (o>) Growth - »elow surface of oil. Il. 3B. ranosus. 1. aerobic Culture: fe) Gas production in twenty four hours. (b) abundant top growth. (c) srowth - ..oist, white, turning pink then deep red below surface or top growth. 2. anaerobic vulture: (a) Gas production in twenty four hours. (b) Growth - Below surface of oil but no color. III, 3. mycoides. 1. serobic vulture: (a) vas = lone. (db) top erowth slow in developing. (c) srowth - --oist, white. (d) nindered by 37 degrees U, 2. anaerooic vulture: (a) Gas = lone, (b) srowth - 3elow surface of oil. iV, 3. mesentericus vulgatus. 1. serobic vulture: (a) vas - sone. (b) Top growth slicht. (c) srowth - Lizht tan in color. (a4) vrowth hinderel by 37 degrees U, £. Anaerobic Culture: (a) Gas - ivone. (b) white growth in tube. Discussion of kesults. This work upon spoiled carnec peas has necessarily been limited. ior more complete data bacteriological examinations Should be made of cans sterilized by the several methods and the work Should be carried on for seversl seasons. ‘This has been in- possible in the present instance. However, some very definite conclusions may be drawn. Since the orzanisms found in this investigze*ior resenble 3. subtilis and other soil oreanisns, it is reasonible to suppose that 211 are fron that so.rce, ‘the means of contamination may have been from one or all of three sourses: Just on the pods may heave peen transferred to the peas in shelling; there may have been dust on the hands o- trose shellinze the peas which would be transferred to the peas; dust fro. the air may have settled upon the shelled peas in the roo or refrigerator when left over night. «as the peas shelled and canned the sa.se day showed 2 much lower percentage of spoilage than peas partly; shelled and left until the next day be- fore canning, this last nethod of conta:rination would see. an im- portant one. The percentage of spoilxce was much lower in peas auto- claved then those sterilized by any other uwethod. whis doudtless accounts for the hizther percent2se of spoilaze of home canned peas, usually canned by the hot water bath method, in comparison with the commercially canned product. The consercial canner is especially careful to have his peas canned as soon as possible after cutting. He is also very thorouch in washing the product before canning. Doubtless here is the secret of the low percentage of spoilage as his process con- Sists of heating the cans at lle - 115 degrees ©, for 35 to 40 minutes,a temperature withstood by the organisms described in this paper. sne ability to form gas in peas seens to be due to the action ov organisms upon some protein or proteins found in peas and not in artificial media. It may be possible to further differentiat: between Sililar organisms by use ot peas as a medium since organisms not classified as zas formers were found to produce gas when so tested. Summary. 1. the lowest percentage of spoilaze was found in peas sterilized in the autoclave. 2. keas canned immediately after shelling had a compar- atively low percentage of spoilage. 3. Ureanisms found were all spore forming bacilli. 4, sll organisms withstood from ten to rifteen pounds pressure in the autoclave ror ten to twenty minutes. 5. early all orzanisms reduced starch to sugir. 6. seven of these organisms caused peptonization in milk. 7. sut one organism found failed to produce indol from peptone, 8. signt of these organisms found in peas produced zas in these medium but not in other imedia, 9. B. subtilis and 3. ramosus produced gas in peas but not in other media, Conclusion. The spoilaze in cold packed canned peas is largely due to the presence ot resistant spore forming organisms which are not killed by the prescribed method for sterilization, theretore, oefore canning vegetables, the product should be very carefully washea to remove all soil or dust and thus re- move a greater percentage of organisms. the time for sterilization of vegetables should be length- ened so that the center of the can may be at a high teuwperature suf- ficiently long to kill these more resistant organisms, Sterilization of all cold packed carned vezétables should be carried out by the pressure method. Bibliography. (1) Saxer, bk, a, 1918. ‘he vannine industry - some accomplishments ‘and Up- portunities alonz vechnical Lines. Jour. ind. and sng, vhel. Vol. 10, so. 1, p. 67. (2) Bigelow, «. D. . 1916. whe Inspection of vanned soods. Jour. Ind. and «neg. vhenis Vol. VIII, cart II,p. 1005. (3) Billings, *. 4H. 1916. w#acteriolozical uxamination of vanned Goods. sansas State soard of Health. 2ull. lo. 4. (4) Bittine, «a, W. and Sittine «a. G. 1917. Bacteriolozical utxanination of vanned s/oods. xesearcn Lab. watl. vanners Assoc., washington D. vu. Bull. iO « 14. (5) sitting, «a. w. 1915. «ethods followed in vom:nercial vanning of oods. U, D. Vvepte Or azr'l. Bull. LiO. 196. (6) Burke, Georgia Spooner. 1919. The &Sffect of Heat on the Spores of 3. sotulinus. JOUr. AM. w@O. ASSOC. 72, Le (7) Sushnell, L. D. 1918. she Infiuence of vold shock in the Sterilization of Canned woods. Jour. Ind, and une. Uhem. vol. VI. p. 452. (8) Uhester, #. D, 1901, «A+.anual of veterninative Bacteriology .iac..illon vo., lew York. (9) Conn, 1. we, Sten, We uu. and stocsing, We. a. 1906. vilassification of Dairy Bacteria. sept. storrs (Conn) der'l wxzpt. sta. (10) Dieudonie, 4, sacterial ood Foisoning. Translated by Baldman, U.i’. &, B. treat and vo., iiew York. (11) Dickson, w. C, 1917. sotulism: the Danger of roisoning from Vegetables Canned by the vold Yack siethod. Jour. am. ..ed. assoc. Vol. Lalx p. 966. (13) (14) (15) (16) (17) (18) (19) (20) (21) Duckwall ’ ie We _ . 1905, Vanning and rreserving of ood «