Ml M \ NIHWWI HHIHIUJHAWI Hll -'_‘|\]_\ ‘ Ioo ‘ .CDNOO SOME STL'DIES ON FLAT SOUR TYPE MICROORGANISMS PRESENT IN BEET SUGAR Thesis for the Degree of M. 5. Marshall B. Burt 1936 I In! “All" “T snag :3 .4“ ta] 4 1" --.- -5 In AV ‘\'n1 '.L t.“ .. ; w '13 .3 I‘ , 1.7.4 "I CH C CR. ' 5 - war‘ 1“ '~‘ "’1 L1. Y‘- 7‘— " f 7-1 '_ In 'j \‘y‘ r- . ’A‘rfl..:j UL-.LJ 0L \ .3...ng C... :JLIftL O('-.,-{ ,' _. ""d1\ . ‘ 'V'.'_~"'.f1 T.‘ ;‘:1w- In xv 'rw-Vrn ('1 Pi," ¢a_LV.LL(/L/ ~11:-..l~l- B.) - LL!)_. 1. I- AJILBJJ. OnyoiR rV'fiHT'“: ~~. m . WY“? "“' f"~‘-*FT"' "T "Tfi'T'Tfi ‘17 (IT-71' #1:“? fl 11.3mm 0L .2. -4..- .2 .LC 4.1.-..) f.L~/LL.L; (.21 -.-...U..... 1'3... u..;1-...'a u ‘ AL 313...: TV.’ l‘j._‘_."f"T ‘T YTIL .IL f'w'vffrq ‘7': mT'FT‘. 7V‘-';I,-.T~~,- j-jv Hm rPr“ .LJ.-. .‘L.L.L....£L.La L‘L _.. fi--..u...|. C'fi $3.2) «Lyn-6s. 2.12.3.3. .L‘J Q *1 ~ 1’3"“? p~11 11"1 ’3 )- C-L .LJ-‘A Ljh‘l VAL-J11 L I.J.iL:STf.'.LL B. W'ZLT June, 1936 THES'S A131“: 70 .3 5th D C}- LIT This oyportunity is taken to acknowledge my inaebtedness to Dr. E. D. Devereox for his constent guidsnce throughout this work. ‘ (3 z ‘ gr) 6.}! i. LT‘A“) 'fi “"7" , Tw—‘I:~-'.‘,q(‘ LJKJIAJJJ CI‘ Cb“-..n...o I. Introduction II. Experixental 1 3 - » 7. -'.-' o - . ° 7 A. 130.3 ion uni Josorigtion o. 0:33.15ms . dope Stuaies on 3011 Iroduction Ln 0 . pg C. Lest Assistance of spores III. General Discussion IV. Generrl Bongo y V. biblinrsphy I . I} 7321C DL C‘IICH Sgoilage of non—acid crnhed vegetables is often caused by the activity of bacteria or enzgmes which Pb J therhophilic sgore ‘hey elaborate. Three grougs o [I beetcria or xajor importance have b‘en ~. .._- O: L: e— *3 g): f.) :3 ‘ "a W Ho nitely associated with this spoilage. These are: (8) Th flat sour tjge, charQCterizei by the gro- duction of acids withctt s fron cerbohydretes. 1 :ilic an erobe H (b) The herd swell t; e, a the'hOb A. *‘x ‘ characterized CY the gr duction of ac U FJ. d and 395 (not hyderen sulphide) from carbohydrates. (0) The anaerobic therioghiles, charscterlzed by the groduction of h;dro:en sulphide gas. (1). Cameron and Williams (t) in 1946 reported on .the bacteriological examinations of the raw ma- terials used by a large cenning company. They were able to isolate the above mentioned groups of thermophilic syoilage organisns from refined white sugar. In this work a definite relation— ship was established between the bacteriological condition of the sugar and the thermoihilic spoil- :33e of non—acid canned roods. This led to a de— sire on the part of the canners to buy a sugar that had been tested for suitability for canning and started investigational work on the part of the sugar refining companies. This work wee followed by an announcement bv some of the com- panies that they were ready to supply a suitable canning suzcr as judged by their own standards (3). This, in turn, creeted 3 need for a suitable bacterial standard for sujer to be met by ell refiners. The Ketionel Canners Association in- troduced a beeteriel standard for sujar in 193 which they used in testing sugar for member canners. A revision of this stendsrd was nude in 1932, end at the present tine in "Bacterial dtanlards For Sugar” (4) a method is given for the testin: of sujer for the aresence of these .‘vi three types of sooilege crinnisrs and a llmlt is set for their tolerance. Five sa:gles from en; one ship ent of sugar are exegined bacteriological- ly. The li it of tolerance for the flat sour t31e \s s oilage or;enisms beisfi a mBAlAUA or not gore than 75 spores end en evera;e of not more then 50 shore per ten qrexs of sugar. aletin: numerous samples of beet sutnr to detect the presence of the sgores of "flet sour" Tzscterie, es reconnended by the Kotional Canners zissociation, it was observed that colonies of acid i“roducinj organisms of the flat sour type developed on plates incubated at bfio C and 500 C. It was rrimerily with the orjanisms isolated at the 1,. above tenreretures that the ex erigentsl work to be discussed in this thesis was conducted. Flat sour tyres of orronisxs capable of growing at these temveretures are of particular interest because of their possible reletionshi; to this ty;e of s oiluge of lgfrOUeer ‘rocessed canned goods held in storage. - U ‘ -—I m"*.7 '1'“. s ""1 ‘L .L O .L'JJLI L’JL .LJ"...J..'~ 1.41 A. Isolation and Description of Urgenis s Historical The research laborvtories of the Litionsl Can- ners Association (4) give the following method for the detection of he spores of the flst sour tyne bacteria. ”Plece 20 gr ms of en: r in a sterile 156 ml. nrlennerer flask marked to indicate a U volume of 100 ml. Add sterile water to the 100 ml. KETK. firinf re:iCly to boiling and boil for five minutes. Replace evejoretion with sterile water. Into each of five getri dishes pipette 2 nl. of the boiled suqer solution. Cover and nix the inoculum I b with Eecto dextrose t yptone agar. (This Leer was ‘LJ develored by Difco Laboratories, Detr01t, hichigen, end conteins Mn indicator, broncresol purgle.) Incubate the pistes at 550 C for from 36 to 48 hours. The combined counts for the five plates represent the nuxber of spores in two gregs Of sugar. Lultiply this by five in order to extress the results in terns of number of s ores yer ten irons of st; r." The colony formed by flat sour bacteria is round, meesures fro; two to five Kill- reters in dimneter, presents e typical cycque central srot end by reason of acid production and I ‘3 ILI‘ it the presence of the innicetor is usuelly surroun ed by a yellow halo in a field of purple. In 1925 Ceneron end Esty (5) reported on studies "1""I‘: A ‘ 4" Vi~~" -'2 " . Q (~ L" w *F‘ o ‘r' . 0‘. allied out on olbenlsns isolated iron sociled cen- then into the followin" érouos: ‘y‘ 711", _\ , rsn, 1-- . . ‘Ir’ . . .," . n” 1 GTOUg I--lne aciOolC b'Oie lelneis. v" Group II--The lucultetive theruophilic orcnnisns. Grrue III--The strict thermophilic orgenisms. hembers of Grou, I are not considered as causative agents in food spoilage and no further mention will be made of them. Organisns in Groups II and III are considered as typical in the production of ilet this work gives the essential characteristics of these ortenism in the form of index numbers. Table II (Cameron and Esty) Essential Characteristics (Index numbers -- Soc. Am. Bect. Descriptive Chart) Group II. 5221, 52220, Group III. $212, 52120, 1 Briefly the orguiisms in Group II were cram ENDSitiVe rods producing terminal endosrores, «than: “1N i 11 -n W - .A. k. (F s.) A /\ V. A .3 .L . . A .l C O . a L. 11. o B S l 4C .m u S S S 0 Wk tL . .Iu .M. 3 no 4 .P. A 2 .Tu .1; n. , .. u 3. C e . u T G T ._ u Ab .., A_ 1» .V .L. k; .r u t C 3. , m . e n a. J6 r 1.. S a r. n .._r o. . A ._ v,» «u NH .l C I i L. o si— .. A LU .. . n l n m. U 3 , ,u C .l . :u .l .u l .l S m t 1.4 Br 1 J .x- ml— . .74 v a.“ , r. U .1 e. C n. l w 0 T .. u C 2.. M. u u . Q S C 5 fix. .U. ,3 kn. C S to. m; t .31 :4 m0 “u.— ..L. rum. 2. . h 00 ,u .w l. T, c. .1 r. ,_ 2... ...:.. um“ H H S I N. t a: e . r. P -w u . L. .. Mn“ 3 . 1. hi. T. +9 ”I "L Wml I“ «l 01.x. J!‘ ...vU .H 01 7h .3 .. l t . . r .YH.“ H. . n. . |+ a w v . .1— ‘rn ‘ . 3d .AAAK'CU: O O C x K L tiles. 1 1‘ I J. A. Ct tile'it tri c: 5.4 TO 2 ' 4 V 1 ”477 l ‘J .\ \ AA t ‘3 24 ' .L n! r .. VLV ,\ -0- Exterinentel ..Q-L_ O ' ‘ 1 1“ ‘ , “ 5° — < r‘ anile jlsti. su er s les eccc1dln, to tie previously "entionei nethcd four sets of "lztes a" . H 0 .~ ‘ , . ‘ rz'w ‘. .100 C, .37 C an? 0;; C. ht the end of ‘l—C‘ hours incubation these «lfites were BXHHlflEu for the ~~. . \ ,q 4-) f‘ ,- ' w ‘,r \ 5-, ' I‘ "r... - 1.. . - colonies oi ilqt sour cecte-ii. l ble I hives -- n 4.7,. ,. 7, A , p A - ,1! -. “ 3. _..‘-I -. .3 .',. 7, e CKNnfit or the run obi oi cellulies noveirn “L: at 3, ~ - _I ‘.' ‘ O ._ ‘ W“ _I ‘ .. _ K ‘ r _"4 . ‘1‘. . I ’I _ ‘ '3 t.m3 veltrons inlcuusdiron.in;1,ei.atuiess(is con: free. I 7L. . ‘. 4.. ._ ._ i - ‘ - 4— _ ‘ '“ro ‘ ~‘ 1. r I, F ' . t."' “ ‘ .LUA‘. L1 it, Luanulr'l l CCLVAU zuu LIL) Co rv-u . €_ ,- ‘f - L.) ‘7-.-'_..v o 'E . - r ~\ , ' "1 r\ C“ ~ fl" ~'.-‘ 12". 'v\: .. o o-e Count of flgt oour 3,,e _wcteil (H H :3 (i) (1‘ C+ Se yle Io. Incubation Iegpercture o 270 o C) C ,‘ r?“ th U UU and) 5 5 407 10 20 270 5 20 2 IO 40 a: O , O 39 170 505 8 10 210 46 25 40 i 298 55 150 250 ° 5 35 120 295 Leo u 85 100 150 L5 —-- 175 {\JP 1\ CR '. \‘ICE‘F—‘(P 10007010 C l\ U \7 01 O .9 C (f 01 9 (‘0 O 5 Q Q a p (J It )1( x“ 3 O O EXpressed es scores her 10 grsns of sufigr. Acid producing colonies érouin; at 230 C and f2 0 ‘ - r i- in. " :1" I "Dr'\ ' ,‘Vb ‘fl QC C were closen for this horn. A trunsfei cI a well isolated colony was made on a plain a er slant and incubated at 30° C. In all 28 cultures were isolated from fifteen samples of efine beet sugar obtained from factories in Lichifian, Ohio, and Illinois. Cultures nurbe rs one to e1 3ht inclu- sive were isolated at 230 C, numbers 12 to 23 were isolated at 300 C. Inasmuch es colooiitiv stucies were to be nae e at 300 C, 570 C and 550 C, stock cultui es oi these organism: were carried in the la‘ooratory at 30° C. Three additional cultures, nugbers 29, 50, and 53; and two ty;icel flat sour cultures, numbArs 51 and 52, ( he latter two ob- tained from the Rational Gunners Association) were used for corporative purposes. All five cultures were isolated at 550 C. In the future these will be rred to onl;r by nu; Seer. As a check upon the )4: (I: re purity of the cultures they were periodically elated and examined for contagination. Of the 23 cul'ures originally isolated, 21 were satisfactorily ca‘ried tniou “out tris work,t1e eXperimontel data being reported only on these. The or anisms isolated ell conformed tithin reasone ble li:;its to the yreviously described group II of the flat sour type of Cameron and Esty in that they were all gram positive rods N1 .Ill F" 13 averofin3 about 1 by 4.0 nicrons size, pro- onetines (.0 ducinq ter inel endosreres which caused a bulging of the rod. They were all facultative anaerobes and produced acid without J; T28 in dextrose and sucrose broths. In ei.ition k to the above grouping they were divided into 0 ~\ coordln3 to their ability to grow on (T) irours P potato sl nts. Ihose that showed no apLerent *rowth on potato slants were desi nated as group A while those thut showed an abundant yellowish brown butyrous frowth were designa- ted as Groun B. Being interested in the ability 0 f these organisms to produce acid from carbohy- drates and the heat resistance of the shores of these organisns no further work was done on their classification. . Surrurary Exrnination of Table I shows that refined beet sujer contains spore forming organisms thrt have the ability to frod ce acid at 30° C. This leads to the fossibility that these organisrs Light 'prove to he a scoilaue hazard in the canning in- .i . J ) dustry. The organisms isolated conform very Closely to Group II of the flat sour bacteria described by Cameron and Esty and due to their ‘d‘u ability to produce a non3aseous acid fermentation it is the Opinion of the author that they should be considered as flat sour tyres. Their abilities to nroduce acid, to grow in concentrated sucrose solutions and to Withstand high terpera ure are noints thet will be considered in sections 3 and C of the exterirental part. -10- 3. Studies on Acid Iroduction Historical The typical flat sour type spoileqe is that of a nonjeseous acid fermentation with slight if any 4‘ odor and the pH Va ue not markedly above 5.0. (o) O a (I) h re is an agperent lack of inforgation con- cernin acid production by flat sour type organ- isms isolated from sugar; However numerous papers have argeared concerning acid groduction of flat sour type orjcnisns isolrted frog other sources. Wyant rnd Tweed (7) studied a :roup of these orranisms in refrrd to their ability to produce the flat sour conjition in canned {oods. n this work they reported 5.6 as the lowest pH value that was obtained. Cameron and Katy (5) found that their group of facultative theraoyhilic orienisxs would groduce acid in corn, gees, horiny end other non-acid conned vejetables relucinj the pH value in all cases to below 5.0, and in most cases below 4.5. -11.. 1 .11. A A . v7- 1.. aan‘d‘ If .‘S’. 3 '_ L...“ .. u; QMfi (has... 1“ - '-~7‘\"r\ 6-H: « x~ "t!“l “4-4; 'Cx.L .L4-L6 (l (J Acid :)iod uction in dextrose broth--nerooic fie ability of tne OICJfiiSMS isoldt ed in port A to ferment dextrose use de ermined as follows: Tubes con dining 10 ml. of one per cent dextrose bzo th were inoculated with C.5 ml. of u 24 hour broth culture of the oresniszr's and alone with un- °,, 4. ‘ 0 q 1, w - , n O "1 inoculated coutiol tube 3 we e incubated at 50 v, r ,1 r—.'“0 ‘ a . ' ‘- . 570 b and 55 L. nfter a Six our in cuootion period a five ml. sample fro; each of the inoculated tubes ‘ mes diluted with iive ml. or distilled water and titieted with K/BO FeCE usinr rhenolphthslein indi- cator. The uninoculeted control tubes were titrated in tne sen manner. The difference between the ti- retion figures 3ives the total acidity produced by the microorfienisns. ILese data are reported in Table II under nP (acid orOduction). Grou> n bein3 the or enisms slowing no srowth on potato slants, éroup B x;e organ M1 is s} owing rrowth on the toteto slants. Acid production in dextrose broth--nneerobic Tubes containing 10 ml. of one per cent dextrose broth were heated in flowing steam to eXpel the air, -12.. - a . . . n .° ._ , 2 ,3 . . cooled to about 400 v, anCUlacud mith 0.5 ml. of r‘~ \‘ 1‘ ‘ -'. ‘V V‘. A 1.\ \ ‘ --. \4" +1 .-, - ‘. x" -. wx- ‘ ('1 r ,- 0. ‘64: 110 L14. v.1. wt“; 0141 till 8 CU. vllb 0.3 _.L.1.11.LS-.-S , u'v‘txlekl 7 IV 4' 4‘ ‘ If} "V‘ . W‘AJ I ~W'\1 \ ; ‘ I . .‘ “-.. “ . W vii +0110 U0 bruise Cclitlhib‘ 2.-.; (J: Etfii lle pznlgl...ln, end ulonC W‘th ' ninoculated cent :01 tubes were incu- bated at 300 C, 370 C, and 55° C. These were titrated at the end of a six day incubation period, the acid production being determined in the Same manner as under aerobic conditions. TAG results of this work are reported in Table III under AP. Acid pr olu ction in pea juice. Twelve organisms were studied to detenuine their ability to produce a flat sour condition in pee juice. This medium was obtained by pouring he juice from sound canned peas. Tubes conte ining 10 ml. of this juice were autoclaved at 15 pounds pressure for 20 minutes, cooled to about 40° C, inoculated with 0.5 ml. of a 24 hour broth culture of the organisms, sealed with about two centimeters of sterile melted pa reffin and along with uninocu- lated COILtIOl tubes incubated at 300 C and 550 C. After a six day incubation p riod the pi: values of these tubes v.ere deteinined elec tr cmetrically. Table IV gives the pH values oi the inoculated tubes as combared with the uninoculated tubes. -13.. :5... E!“ A II‘II.‘ Anaerobic conditions were emglcyed in this work to Simulate the conditions found in canned food products. nCld production in and inhibitorv action of sucrose. Twelve cult‘res from ¢roun A and five cultures iron iron; B of the orjanisns isolated under Part A were studied in regards to their ability to produce acid frou sucrose and to deternine the effect on acid groduction of various concentrations of sucrose. ‘his work was carried out aerobically to ‘3inulate the conditions found in the canning factory when tanks of syrup have been held for a prolonged oeriod. Tubes of broth containing 10 ml. of one per cent, 10 per cent, 20 per 0 n , and 40 per cent sucrose broth were prepared and inoculated with 0.3 ml.-of a 24 hour culture of.the organisns. Cne set each of these along with uninoculated control tubes were incubated at 30° c, 370 c, an 550 c. At the end of a six day incubation period a five ml. sazgle of each tube was diluted with five 41. of distilled water and titrated O y-a {‘3 with h/ CaCH using phenolphtnalein indicator. These n Table V, the acid H. excerimental data are re orted - ~ ‘- II Ho production being calculated in the same manner as Tables II and III. -14- TABLE II Acid Pr cducticn In Dextr e Broth Under nerobic Conditions. Incubation Tenp. ”00 C 570 C 550 C “P* DB‘* 3P D3 a? “B Orja nism h tber l .15 ? .10 ? .60 ? 2 .15 ? .25 ? .70 X 5 .15 X .25 X .55 X 4 .25 X .20 X .00 - 6 .25 X .50 X .25 X 8 . 5 X .40 X .5 X 15 .20 X .15 X .40 X 17 .15 X .10 X .55 X 21 .0 X .55 X .40 X Group A 22 .05 X .55 X .40 X 25 .05 X .15 X .60 X 26 .05 X .25 X .20 - 28 .5 X .50 X .55 X 29 .00 - .50 X 1.70 X 50 .05 - .45 X~ 1.90 X 51 .00 - .10 X .70 X 52 .00 - .10 ? 04:0 X 5 .55 X .50 X .10 - 12 .45 X .80 X .45 X 15 .75 X .70 X .50 X 14 .65 X .90 X .65 X Group B 18 .50 X .85 X .40 X 19 .55 X .65 X 40 X 20 .45 X .50 X 55 X 25 .60 X .65 X .55 X 24 .65 X .85 X .45 X 55 .60 X 1.00 X .45 X *AP denotes increase in titratable acidity. This is the DJ ifference expressed in mi. between the titration figures for the eX;ei nental tubes and the control tubes when 5 ml. ortions 1r r these were titrated with 3/20 FaCH usin3 Pher olehthalein indicator. **DB denotes growth in tubes as judged by turbidity. X Srowth ? Questionable — no growth -15.. acid Product is n In DeXtrose Froth Under Anaerobic Con T .‘h, ' r 3”-“ 7,. 4...-30 r1 n0 rz O n I: *IICLLX uthn Penii-e‘tkblhiv UV C 97 U L) AP* D‘** Ar D: as Organism Irober 1 .05 - .05 - .30 2 .00 - .05 - .15 5 .05 ? .10 X .10 4.10 ? .05 2 .00 6 .15 X .05 X 50~ 8 .15 X .05 2 00 15 .05 2 .25 ? 00 17 .00 ? 40 7 .50 Group A 21 .00 ? .55 .05 22 .00 ? .15 2 .55 25 .00 ? .00 ? .5 26 .0' ? .00 9 .00 28 .5 X .50 X .05 29 .00 - .70 X .95 50 .00 - .40 X .85 51 .00 - .‘5 2 .6 52 .00 - .00 ~ .15 5 .15 X .5 X .05 12 .70 X .35 X .20 5 .65 X .65 X .45 14 .55 X .55 X .25 Group B 18 .40 X .20 X .25 19 .70 X .70 X .25 20 .65 X .65 X .25 25 .55 X .70 X .65 24 .80 X .66 : .40 55 .80 X .85 X .25 *AP denotes increase in titratabie ac idity. This is t difier ence eX~i ess ed in m1. between the titration fiqu' or the eXp *imental tube and the control tubes when U) H) portions fr.m these were titrated with E/2O KaOH using pher Help} t1a1ein indicator. **DB denotes growth as judged by turbidity. X growth ? questionable -Xo growth 'O'O'O'OH'OHXH «DNH NNHN NMHNNHNN«O-O L) (D TABLE IV Incubation Tenperature 500 C 55 Organism number Final pH Value 1 5.86* 5.8 2 5.94 5.9 6 5.91 5.8 Group A 28 5.41 5.7 29 5.42 4.9 51 5.95 5.0 52 5.80 4.9 12 5.18 5.6 15 5.18 5.5 14 5.55 5.4 24 5.58 5.6 55 5.57 4.8 Control (uninoculated) ".10 0.0 *pH value determined electrometrically -17- .x. O3U‘nl—‘U‘Il-‘rblo Cfil—JOLON Hoypcoo .0 OH the 00. mm. OH. 00. 00. OO. OO. )0 C OO. mo. OO. OO. OO. OO. OO. OO. 05 .moQSp map was moose HmpqofiHeamao exp pom moqdem :oHpsAPHp esp consume mesonHHHe mop .mpHeHoo womaoeona .Hfi 5H wommbamso asHmc mpaswmh mop HephOHeoH :HonnpamHoumHm f ' r“! monz Hora ON\H des manna HOHpmoo map fioam mHmEUm .Hfi m was mondp pruoaHaeawo deem mHmasm .HH m mowpsepHp hp dmoHanopow mm huHsHos doaoomooH o1» mH mHHBfi F o l OO.H ON.H no. 0H. ON. ON. OO. OO. OH. OH. 0n no. OH. OH. Om. om. an. OH. ON. OO. 00. 5H 1 OM. OM. OH. mm. ma. mv. mH. ON. O . m . SH m OSOHO ON. ON. 0 . ON. mm. mm. ON. mm. rn. mn. NH OO. mo. 00. mo. um. mm. OH. ON. a. m . O OH. OH. OO. OO. OO. 00. OO. OO. O . OO. mm OH. ON. OO. OO. OO. OO. OO. OO. OO. OO. Hm mu. OO.H OO. no. on. OM. OO. OO. OH. OH. on mH.H ow.H OO. no. OH. mm. OO. mo. O . OH. om OO. OH. mo. 00. mo. ON. 00. OH. ON. OH. N 00. OH. OO. OO. 00. OH. OO. O . OO. no. mm mo. mm. 00. oo. oo. so. so. oo. 00. so a macho 08. OH. mo. no. OH. a. 00. me. no. so. 06. OH. mo. mo. OH. OH. 00. go. so. no. OO. OO. no. OO. OO. OH. OO. OO. 00. OO. FHMUD¢!OGD OO. OO. OO. OO. mo. mo. OH. OO. OO. OO. OO. O. O. OO. OO. OO. no. no. OO. OO. OO. km . roped- SmHzouHO Ow OH H Ow om OH H 05 ON OH H omoeodm puma new O com a can O oow bespreQSon ooHp83502H mpoam mmomodm 5H nOHpozeoem owed .4. n 4.1 > r-r RTE -18- Sunmarjr ” Ta bles II and III shows that the organisms isolated at 25 o and 500 0 produced acid in dextrose broth under both aerobic and anaeI obic C ,- . r3. ' : O I 1 :2 O ,‘N ,- ,7 C' L10 -‘V 3 3-, .,,. ‘ ,. ..... 0 onaItIc ns at 50 v, 57 o an Ju o. Ianinun aCId production occurred at 500 C and 570 C with the eXcep- tion of two organisms which orefe “ed 550 C for maXimum F— (1 acid production. M1108 the greatest acid production, as judg ed by titratable acidity, was in the aerobic tubes, the author It els just ified in considering these organisns as facultative anaerobes. “ea juice inocu- *- ‘l‘he hydro;en ion coIIcen ‘ " tion of ~, .\ . .‘ ,«n f. .. ,"’ . . . r. . ‘Q ,~ \ —. w". ~ . r“, at Ianisns and Incubated anaeiobically «.4 lated with these or at 500 0 was narkedly increased. The final pH value ranged from 5.18 to 5.94 in juice with an initial UK of 6.10. In pea juice with an initial pH of 6.05, inoculated with the same orqenisms but incubated anaeI ob ic ally at do 0, the final pi values ranged from 4. 85 to 5.94. Acid production, aerobically, was noted in one per cent sucrose bioth with decreasing acid production 1—5 1 o nCI H81 * concer tIat I on of sucrose. H- (a: '~.~ +n‘~~,~~,c -, l-Ll UMUin'LJ L 0. Heat hesistance. Historical The resistance of bacterial spores to heat has been stud ed in great detail. In 1920 Biaelcw and Jsty (8) made a comprehensive study of the heat re- °stance of thermophilic flat sour type spores. Ihey stated that tI Ie iIIitial concentIation of the spores and the hydroien ion concentration of the medium influenced Ireatlyr the theIIal death point of the Spores of resistant or;anisms. The effect of the nyarorcn ion concentration in regard to the thermal death point was determined by heating a definite nu ber of spores in food juices of different pi values and it was concluded that, as the pH value of a medium is lowered the time recuired for the condlete dest'uction of the spores is decreased. They studied the effect of the initial concentration of Is in corn Juice with a n1: value of 6.0 at tentera- syor w tures of fron 1000 C to 1300 C. The results of this work led to their conclusion that in a medium of known hydro en ion concentration, at a given teuperature, the larger the number of spores present the lonaer will be the time recuire d to sterilize the medium. ihey report fin diIIg spOIes of thermonhilic 01s Qisms that would survive a tenperature of 1000 0 for 20 hours. Nyant and Tweed (7) in 1923 made a study of the thermal death times of the sr oxes of flat sour type organisms isolated fr‘m cold cached canned peas. In no case did they have an orgf1that would lesist a ten eratuie 01' 1100 C fo1 over 10 minutes. In 1928, J emeron and fiilliams (2) made an interesting study on the therLal death time of the spores of flat sour thermopuiles isolated from sugar. They found that the effective killi13 time for a spore suspension of 100,000 per ml. in plosphate solution of pH 6.97, at a temperature of 1100 C, varied from 5 to 240 minutes. Experimental Pregaration of Stoc< Snoie Suspen sions Suspensions of the organisms containing between 90 and 100 per cent spores without resorting to a heat treatment to kill t’r e veietative cells, xertn1e“a ed in the followin3 manner: Dextrose asar slants were inoculated with tne orfanisms and in sbited for 4% hours at 500 c (organisms numbers 29 to 33 bein; J il‘lcu— . . 3-0 Dated at ob C). These slants were then placed in jars and incubated for a~period of from 4 to 8 days under reduced p1essu1e (a311o11uatelv 22 incnes o1 va cu1u). Suspensions of these syores were made by carefully w1s sLi13 the slants with sterile saline (pE--d.9) and transferring to sterile tubes. These spoie suspensions -21- were then exagined microsco: ically to detertine the percenta3e of spo1es and standardized by dilution with sterile saline until one ml. of the suspension contained aptroximately one million spores as deter— mined by the standard plate count, allowances being made for the vegetative cells. This was considered as the stock spore suspension and held in the ice box for future use. Heat Lesistance netegiklutions. By experimentation it vm-s found tli at a temperature of 1000 C could be controlled by immersin3 a 200 ml. Erlenmeye1 flask containing saline in an oil bath held at between 1050 C and 1100 C. 10 determine the t1me17a l resistance of the spores, 200 ml.rlen:1eyer flask s contai n3 99 ml. of sterile saline were immersed in an oil bath held at between 1050 G and 1100 C until they started to boil. (Te:perature--lOOO C). They were then inoculated with 1 ml. of the stock spore sustension and thoroujhly mixed. (This mixing must be done by keeping the flasks imm reed in the oil, other wise there is a rapid drop in te:1we1ML:e.) At definite time inter- vals, en1 ichuent tunes containing 5 ml. or dextrose tryptone broth were inoculated with ene al. of he 0 1 o o 3 . O ssore suspens1on M1ich was be1n3 held at 100 c, -22- The sterility or this Spore suspension was deternined by incubating the enrichment tubes for four days and determining the acid olouuct1c1 as indicated by bromcresol Purple indicator. IRS first tube show1n. F;- p:- (D H (D C U) (‘1’ (D P. i... 1.1 (1‘. O I i In (7’ H (D «4 H no acid t1ouuct on 't;;* cons shows the heat resistance ci these syores as deternined by the above method. As a check upon the efficiency of this method for deter. Lain: the ste1ilit;' of the s: ore sussehsions, dextrose tryntone agar plates wer made using one ml. samgles from the first enrichment tubes which showed no acid production after four days incu- bation. Uoon ere; lination after a 48 hour incubat on I period all 01 these plates were lo and to be sterile. Fur her heat resistance studies were made on 10 of d' Fv‘ } J (D 0') Cf 0 S: (O O H (D m g (.0 ( I 0‘ :3 m H. O H U) wh n ubsequently su pended in pea juice uh 6.10. hrlenmeyer flasks containing 99 m . of sterile pea juice were imne reed in an oil 1 bath, the temperature being regulated to keen the con- tents of the flask at 1000 0. They were then i ocu- lated nith one ml. of the stock syore susgension, the thermal death time being deternined by the previously described method. Table VII gives he thermal death times for these spores when susgended in pea juice pH 6.10 as compared to their thermal death times when suscended in sa lin W( {0.9). An attempt was made to determine quantitatively the thermal resistance of these spores. For this work -25- I'll (xiii if! flasks containing 99 ml. of sterile saline were held J'r’j‘ at 000 C in an oil bath and illoculated wit n one ml. of the standard spore suspension and thorouchly mixed. At delimite time intervals tubes containing nine ml. of sterile saline were inoculated with one ml. of he spore suspension from t} ese fla sks. Dilution plates were then made in an attempt to deter ine the number of viable spores greseLt at the end of the various tine intervals. t was dete; :Lined th-t a 1a id late of killing was obtained durin: tLe fist inteiva ls of 1~ea tirg with a small percentage of heat resistant sycres surviving. Inconsistent results were obtained when the number of viable spores wast determined bv plating Lethods. It M38 often noted that when few viable Sjores renained it was ixpossible to obtain a |""\ ir ite t (D H de nd in th rate of killinf. Qualitative methods gave results that indicated a definite and orderly trend in th 8 rate or killiné nnen the sa"; method of heating and sampling was used. TABLE VI Thermal Death Time at 1000 0 Scores susgended in saline (pH 0.9) T! minutes required to destroy Spores at lOOO 0* Organism Iunber Inefficient Efficient l 80 100 2 80 100 3 60 8 4 10 20 6 10 20 8 5 60 5 10 20 17 20 30 ~roup A 21 5 10 22 30 40 25 10 20 20 10 20 28 10 20 29 180 240 30 180 240 31 350 ? ** 3 360 ? ** 5 10 2 12 50 60 13 50 60 14 60 70 Group B 18 5 10 19 10 20 20 20 3O 23 60 70 24 5 10 33 180 240 *Concentration of spores 10,000 per ml. **0rganisms El and 32 were the organisms received from the Rational Canners Association. They report that a concentration of 100,000 spores per ml. of these organ- isms, SUSQGHCGQ in corn juice witn a pH value of 0.1 would resist a texperature of 1000 C tor 17 hours. . .lvrlianhl. Pl; Organisn Sunber l 8 12 13 14 15 22 25 25 17 *Concentration of hermal Death Tine 50 10 J I?" uU 60 10 I! Ob 010‘: 5 rec red to destr in pea Juice 1.111 C .10 “ficient 60 20 00 60 70 20 TABLE VII Ine icien n )4 \1 so so so 10 so 60 10 20 .I'L’ f..- 9ft I ‘_D '31-, I w., I " emm.” K umnary Tile tleiuul deatn tine at 1000 0 f“: the snores of J-‘ ~'. 7“. ‘ : ._. -‘ '3 “ 1 r I '— ‘ '1‘ i <' c‘ r71“! W' ‘V‘ 'V‘ ‘ ‘ ‘ tne Cfdelbwb lbOlutsu me do v sud UV 0, WLBL SL5- ; nded in saline 3E 0.€0, varied ire: 10 minutes to 100 minutes. Thirteen of the 22 spore suspensions incltied in this enjeiitent nor aeotrOfeC within 30 Linutes. Four additional spore squensions were 1‘ ‘ ‘ I: o uwv 0‘”. ".’\ .‘ ‘Jn I... 1“, ~.,.,-‘. .v‘M o. aestroye- Witnin 0v ninuues wnile tne rcwglnlufl live .... .: v x ..‘. an -1 ., su' v' sions reoui: ed between 0 and ob -.i.-utes for isolated at so 0 was 240 m flutes. (D [.4 (—5. tT (I) c+ (D H 6 H m (J L (+ t 1 c+ H. b I d There was a decre r‘ O h) 1000 0 for the spores of these organisms when suspended 6.10 as ccnoared to the thermal death c+ E0 wlen sus side in saline ph 0.00. give of the ten orwan's 318 includ in this wovk were d stroyed within 20 minutes when susaended in pea juice as conferel to two when susyended in saline. Three additional organ- isms were destroyed within 60 ninut es wr en susyended in pea juice as.com:ared to five erranisns when sus— yended in saline. The renair 1R” two of the susrensions in pea juice were destroyed within 70 minutes. rwo or the regaininé thi‘e e susnensions in saline were destroyed 0 A within 70 minutes, he third refiuiring between 7. an .n 100 minutes for comt‘11ete des ruction. *0. J.‘ a- \J o“ l .. Tro— 88 :me a V I1 .1 2.. n O .l l +U cl AWU +.U .1 ca .. M W +0 u + o L. _.. l +0 m u +0 1.. .. u L C “a +0 Z We ., Lu Q t a l l e .. , .L .. a t L “I no :1 0... try ...L 11— m, Ag 3 .1 r1. .0 .l .w l .0 l i a ”L .1 .l .. n L. J S C e m _. S U. . 11 S .1 O S L h 0 fi)‘ (V n .b S ... . e. S R an a u. a .l +0 .5 .1 a .._ T h _ T .n u C Q n .0 .Tu 4..u rU .4). .1 u L TL. 1. e C a. V .n I 9. O 3 O x. u .3. S .6 v.’ . w H, ~ 0 u flaw S 0 VJ .“ U 0 Q ..1 10 A...“ S 70 -To C, +0 r n .13 1.1 Rd W .l 8 l C. .1 .3 .1 U C 5v Cy m . 7/ n. u. 1 v1“ .1.“ «1. I T. .1 +U .q B -M ”4. .5 "J . C m. . L e .L r... I a 1 1. .5. d O C +0 u .3 ted med ‘1 T, I, 4;- 1 LU o 1 -L 'J 3‘.” \AU( A lIlOCu- al 011 ta -IC 4. U f“ .1171 l STLS i13j11s ‘7‘. bk . -; v 4.1 1.. in ‘1 ,1... ‘- {: VV -A ‘x4'&'. J. lue L C.) V ’1 I f. 0:. v v tion of 1v - .1 ‘fi'AJ *v O C A :3. U entlv ,, ‘. V -. L; 1-1 1 v. '\ t" J UA‘ 110 rv h (f. - V '3) .1 J" 1 L \_ w -fi+3» J ref f 63 4'1 1w-t 1s V tn 0 1 *neu 0 nos 6 concentr \J...L ~37 \I L. :16 ‘- V CL S. It 0‘ 11 st’ “4.", 1 U‘A 112' O -»1 fix U K o *eriod 10a 7 .1. - .. ) STOPS k‘l b I”. V‘ ‘1‘ 11 C Li a “8111611 10H \ 6.3.0. pit re. t 1000 aerol I E 1-" J. ;t L. Q a’ h fo 8 {31.15}, .r ‘J A '53 ’1 1 iLStrcse t lcub . I l n'r‘ . AAA\.\ fl 1 .'.\.4 'ic.‘ t t ‘7 1 \fl J. :éer w 1. er ml. 0 Y! A L l p stu (N LA 0‘s In I I?“ U 4.4 .1- .0 .1. time varied from 10 minutes to 100 10,000 'J , Pam-n V v ed 0 .‘ 7') *- Va illinj time k ive ) 0.10 «'Y 1.21.1 (. form r ‘ A-.. results con' and r I n 101 “(2.) \J +L U-4L. ~‘n v C.‘ " . U..l‘.r’lo&. ‘ \ - v J ousl V1 4.1.]; UA‘V _") L V \ Y'l J AL: \J 0.1.0 1 +. ,1‘ +w eu'”) 05.15011, death tires 0; the or: \ q . -.... - . - .1. 1.84.36-36.01 .. LL :1 a». s u. .4.', :01 OT 71. F. . 151,1 0 ~-v-'r- .1..L._L T'F' ‘J— , Tables f by no U] 0 IV. 3131513111111 dL'I.;.L1-1liY Re iilm d beet sugar contains facultutive anaer— obic ejore roriinfi orfsuis.s that have the abil- ity to produce scid WithLt fies iron 01110~ 111111 1 1t 500 c. Len; of these on? 1151s, unde 1 anaerobic conditifins, i will groduce acid without 313 in E the pH value of the medium to 5.18 which can be idered as a flat sour condition. OOH U) . ~ ,‘ . 1. 1.: . 11. ' . ,‘ 3-. 4.‘ -. ,.,- ° a... .. . .- , 1011 piosnccion o: these or shishs was p1eetly 0'! nuns J 1... ted by a sucrose conccent11tion 01 between H. A) O end 40 per cent. There was a definite relationshio noted bet ween th 1e Ojtinun tehpereture for acid production and the t1e1L1l dee u: time for these orcenisms. The higher the Optimum temperature for acid production, the wrester was their thermal death time. As t1- e hyd1o3en ion concentration of a medium is in01ess ed, the time required for comflete dcs- truction of the stores is decreased. The heat resistance of the Spores of the orga1isms isolated at 230 c and 500 c is such that the? d {:11 should not be considere oi sianiiicence in the pres cnt 00.1110111 can11n piecess es. However, i—'- b F. U) U) H. O I the heat resi istance OI thesr Oicsr 5°k\$k\r~ukq nific cal tly 11311’tc-be a 5*;Joilc3e hazard in he home canning practices. V K_ V BIBLIOGRAPHY McClung, L. S. 1935. ”Studies on Anaerobic Bacteria", Jour. Bact., 22, 173. Cameron, E. J. and Williams, C. C. 1928. ”The ThermOphilio Flora of Sugar in its Relation to Canning“, Centr. Bakt. Parasitenk. II Abt., 6, 28. Cameron, E. J. and Bigelow, W. D. 1931. “Elimination of ThermOphilic Bacteria from Sugar“, Indus. and Engin. Chem., g1, 1330. National Canners Association 1930. “Bacterial Standards for Sugar“, Canning Trade, March 17, 1930. Cameron, E. J. and Esty, J. R. 1926. “The Examination of Spoiled Canned Foods”, J. Infect. Dis., 32, 89. Esty, J. R. and Stevenson, A. E. 1925. ”The Examination of Spoiled Canned Foods“, J. Infect. Dis. 6, 486. Wyant, Z. N. and Tneed, R. L. 1923. ”Flat Sours', Tech. Bul. No. 59. Mich. Agr. Exp. Sta. Bigelow, E. D. and Esty, J. R. 1920. "The Thermal Death Point in Relation to Time of Typical ThermOphilio Organisms”, J. Infect. Dis., 21, 602. -31- Iii Ili. . ‘ Il‘r ‘l‘llllll..il:.rli .3 mm Mm USE mm fix/W“