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A study of bacteria of the esherichia-aerobacter group responsible for an alleged feed flavour and stable.. Hudson, Vivienne G. 1932

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A  BACTERIA  OF  STUDY  T H E BS&H1IRIGHIA-ABRCBAC H E RESPOJYSIBLE  m  ALLEGES  FEED  FLAVOUR  GROUP  FOR  AND S T A B L E  ODOUR  or  MILK  by ViTlenne G. Baadson, B» A. and G. Duncan MacKenzie» B . S, A.  A. Thesis Submitted f o r the Degree of MASTER  OF  ARTS  i n the Department of BACTERIOLOGY  and f o r the Degree of MASTER  OF  SCIENCE Of AGRICULTURE  i n the Departments of ANIMAL HUSBANDRY and DAIRYING  With One Table  The U n i v e r s i t y of British. Columbia April  1032  ^  A  STUDY OJ?  BACTERIA  OF  THE  ESCHERICHIA - AEROBACTER GROUP  RESPOHSIBLE AH  ALLEGED  FEED  FLAVOUR  AND  FOR STABLE  ODOUR  IH  MILK  The work reported upon by Sadler, Irwin and Golding  and by Sadler and Irwin  suggested the  c r i t i c a l necessity o f a comprehensive i n v e s t i g a t i o n being made into the r e l a t i o n of b a c t e r i a to the incidence of & soc a l l e d feed f l a v o u r and stable odour i n m i l k . Cast i n a somewhat popular mould, describing i n d e t a i l how the enquiry came to be engaged i n , the former paper ^  has shown that an alleged feed flavour and stable  odour common to milk, i s to be a t t r i b u t e d t o s p e c i f i c s t r a i n s of aerobic gas-producing  b a c t e r i a ; and that t h i s so-called  feed flavour ean be detected by the association therewith of a penetrating nauseating and c h a r a c t e r i s t i c odour*  The  micro-organism found to be responsible f o r the defect was i s o l a t e d from corn s i l a g e , but no evidence i s to be adduced that t h i s organism i s present i n a l l corn s i l a g e as such*  (2) Sadler and Irwin  describe the c u l t u r a l c h a r a c t e r i s t i c s  of the organism and c l a s s i f y i t as an a t y p i c a l s t r a i n of  - 2 -  Aerobacter oxytocum  (2) , . (2) (3) (Migulajf, Bergey et a l •  On entering upon the more comprehensive study with which the present paper i s concerned, i t appeared to he essential to examine representative milk supplies, the various feeds that go to make up the rations of milk-producing cows, and also such other sources or materials as might he a possible habitat f o r organisms capable of producing t h i s c h a r a c t e r i s t i c feed flavour and stable odour i n milk,  More-  over, the p o s s i b i l i t y of implements and equipment to be found on farm premises being c a r r i e r s of the feed flavour producing b a c t e r i a had to be borne i n mind* already studied and reported upon  fhe t y p i c a l  organism  having proved to be a  s t r a i n of Aerobacter oxytocum» i t was decided to follow such procedures and use such methods and media as would make possible the i s o l a t i o n of bacteria of the EscherichiaAerobacter group i n p a r t i c u l a r * I t seemed reasonable to conjecture that providing we could secure data on the r e l a t i o n of b a c t e r i a to feed flavours and stable odours i n milk, some contribution to knowledge would be assured and those things that constitute good management on milk-producing farms might be the more c l e a r l y defined*  - 3 METHODS  AHD  PROCEDURES  I t became necessary to adopt, adapt, apply and employ methods and procedures suitable f a r the securing of samples of the material to be examined and likewise suitable f o r b a c t e r i o l o g i c a l examinations of such material*  The Taking of Samples In the c o l l e c t i n g of a l l samples the usual aseptic precautions were observed throughout* MILKS  Using s t e r i l e pipettes and b o t t l e s , samples  of milk--morning's, evening*s and mixed—were c o l l e c t e d a f t e r the a r r i v a l of the milk supply a t the dairy or a t the r e c e i v i n g station r e s p e c t i v e l y , FEEDSs  With s t e r i l e forceps and spoons, samples o f  feeds were c o l l e c t e d i n s t e r i l e containers* FARM PREMISESt  In order to examine farm premises, equipment  and implements, swabs—such as those i n use i n h o s p i t a l p r a c t i c e — w e r e employed, the swabs having been s t e r i l i z e d previously i n the usual manner*  - 4 Examination of Material MZLKs  Inoculations of 1 c.c*, l / l O c.c., and l/lOO c e .  of each sample of milk to be examined* were made i n duplicate into f l a s k s of s t e r i l e milk*  One s e r i e s was incubated a t  3?°C* and the duplicate a t 30°C*  A f t e r 24 hours' incubation  the f l a s k s of inoculated milk were examined f o r the presence of the c h a r a c t e r i s t i c stable odour*  I t was found that i f  the odour developed a t a l l i t was as pronounced a t 30 C. as at 3? CU, and i n many cases even more pronounced a t the lower temperature* o  thereafter, the routine procedure was to  incubate a t 30 C* f o r 24 hours when seeking t o detect the t y p i c a l odour i n milk*  From a l l f l a s k s i n which the feed  flavour odour could be defined, or i n which the production of gas and f r o t h i n g were evident, plates were made i n varying d i l u t i o n s on neutral red b i l e s a l t agar* o  The p l a t e s  were incubated f o r 48 hours a t 30 G« and were then examined f o r the colonies c h a r a c t e r i s t i c of organisms of the Escherichia-Aerobacter group*  Tubes of litmus milk  were  inoculated from common colonies and a f t e r incubation were examined f o r gas production or f r o t h i n g , and f o r a c i d i t y * From a l l tubes i n which gas production was evident, 1 c.c* of the milk was inoculated i n t o 250 c.c* q u a n t i t i e s of s t e r i l e milk i n f l a s k s ,  and these were l a t e r examined f o r the  c h a r a c t e r i s t i c feed f l a v o u r ©dour and f o r f r o t h i n g * # For d e t a i l s as to media see page 6.  when the  - 5defect could be detected, the culture was transferred te # agar and s e t aside t e await further study* FESDSt  Small quantities of the feed samples secured were  inoculated into f l a s k s or s t e r i l e milk f o r examination* I f the smell of the p a r t i c u l a r feed as such prevailed a f t e r incubation t r a n s f e r s ( l c.e.) into f l a s k s of s t e r i l e milk were made from the milk containing the o r i g i n a l gross inoculation*  On the detecting: of the c h a r a c t e r i s t i c odour  neutral r e d b i l e s a l t agar plates were made, the subsequent procedures being as already described* SWABSs  The swabs were transferred from t h e i r test tube  containers into tubes of litmus milk*  From a l l tubes that  showed production of gas, 1 c.e* inoculations were made into f l a s k s of milk.  The same procedure described above was  followed f o r i s o l a t i n g the organisms*  Employing these methods of examination, s t r a i n s of aerobic gas-producing  b a c t e r i a , each of which produced to a  greater or l e s s e r degree the c h a r a c t e r i s t i c feed f l a v o u r and stable odour i n milk were i s o l a t e d from*  milk, the  tongues of d a i r y cows, baled a l f a l f a hay i n the feed room, scrapings from dairy stable mangers, a l f a l f a from a manger, grain mixtures from a feed truck, feed room, and granary bins, pieces of cut mangles from the s l i c e r , soaked beet  - 6 pulp from the feed room, water from a drinking howl attached to stanchions, a hay truck i n d a i r y bam,  a beet pulp shovel,  mangel f o r k s , mangel cutters i n feed rooms, milk sheet b l o t t e r s i n m i l k room attached to dairy bam,  the f l o o r i n a feed room,  the f l o o r where g r a i n i s mixed, straw bedding i n dairy s t a l l s , the f l o o r s behind s t a l l s , a milk s t o o l , a grooming brush i n d a i r y stable, dairy barn gutters, the w a l l i n a d a i r y barn, the r a f t e r s i n a barn, stanchions, a d i t c h , mud from a paddock, and stable manure*  These described sources and the c h a r a c t e r i s t i c s  of the b a c t e r i a i s o l a t e d therefrom are set f o r t h i n Table I*  THE  Cm^TURAL  STUDY  Media Employed Agar, gelatine and the various d i f f e r e n t i a l media used i n the study were prepared from the dessicated media of the Digestive ferments Company of D e t r o i t , Michigan, and were s t e r i l i z e d f o r 20 minutes at 14 pounds pressure* Slueose Agar: B i f c o nutrient agar i n which 1$ glucose was dissolved before s t e r i l i z i n g * Milk! For the detecting of smell and odour, skim milk i n f l a s k s (250 c.c* quantity) steamed f o r 45 minutes on one, two, or three successive days* Litmus Milk* Approximately 8 c.c* skim m i l k — w i t h added azolitmin s o l u t i o n — f i l l e d into test tubes and s t e r i l i z e d f o r 20 minutes at 12 pounds pressure* Carbohydrates f o r Fermentation Studies. With the exception of glycerine and adonite, a l l carbohydrates employed were obtained from the Digestive Ferments Company*  - 7 The method o f preparing the sugar media and the procedure followed i n the fermentation determinations are here excerpted from the former paper (2)t •For the determination of the acid and gas formed from the various substances used, the shake agar method was employed* With the exception of adonite on account of i t s cost, and of a e s c u l i t e , because of the d i f f i c u l t y of using & greater concentration, whieh were added to the extent of Q*5#, the carbohydrates were added t© nutrient agar to the extent of 2%* Brom-cresolpurple was used as an indicator* Portions of about 8 cc* of the various sugar media were placed i n tubes 6 i n * long and 0.6 i n . i n diameter, and these placed i n an autoclave f o r 20 min* under 13 l b * pressure* A 17-hr. old nutrient broth culture grown at 3 7 ° C , was used f o r inoculating* In order to get r e s u l t s as c l o s e l y comparable as possible, a s i m i l a r amount of the b r o t h — t h e quantity held by a loop Z mm. i n diameter—was added to each melted sugar agar* Bach agar tube was immediately rotated i n such a manner that the medium was not d i s t r i b u t e d around i t s w a l l s , and i t was placed i n cold water* A f t e r s e t t i n g , the inoculated tubes were placed i n a water-bath so as to reach as r a p i d l y as possible the approximate temperature of incubation; they were then transferred to the incubator kept a t 37 C* The tubes were examined a f t e r an incubation of four hours and at regular i n t e r v a l s to 10 and 12 h r . incubation* A l l inoculated media were again examined a f t e r 24 h r . Each tube being removed from the incubator to be examined was held i n a water-bath at a suitable temperature.* The following carbohydrates and higher alcohols were useds dextrose, lactose, sucrose, d u l c i t e , s a l i c i n , glycerine, mannite, sorbite, adonite, xylose, arabinose, rhamnose, laevulose, mannose, galactose, maltose, r a f f i n o s e , dextrin, i n u l i n , soluble starch, aesculin*  - 8 -  B a c t e r i a Submitted to Study One hundred and four organisms that had d e f i n i t e l y produced the c h a r a c t e r i s t i c feed flavour and stable odour i n milk were r e p u r i f i e d and retained f o r study. the  Thereafter,  procedures adopted and the methods employed followed  c l o s e l y those described i n the paper already c i t e d  v  The r e s u l t s of the d e t a i l e d c u l t u r a l study are summarized i n Table I*  AH ANALYSIS Olf THE DATA IH TABLE I Of the one hundred and f o u r organisms that produce the  t y p i c a l feed flavour and odour i n milk, one s t r a i n  culture Ho* 212, a short Gram negative rod f a i l s to produce gas i n any of the carbohydrates employed*  Another s t r a i n  Ho* 20, appearing under the microscope as long slender rods, produces acid and gas from lactose but f a i l s to ferment dextrose to gas*  Throughout the present discussion, no  further mention w i l l be made of cultures Ho* 20 and 212* One hundred and two s t r a i n s that ferment lactose to aeid and g a s — t h e presumptive test f o r b a c t e r i a of the Escherichia-Aerobacter (coli-aerogenes) group—appear under the  microscope as very short Gram negative rods*  - 9 The Bacteria That Are P o s i t i v e to the Voges-Proskauer Test F i f t y - e i g h t of the lactose fermenting s t r a i n s are p o s i t i v e to the Voges-Proskauer test f i v e are a l k a l i n e to Methyl Red.  w  and of these, t h i r t y -  Table I .  Thus the  c o r r e l a t i o n of the r e s u l t s of the Voges-Proskauer and Methyl Red reactions i s established  f o r t h i r t y - f i v e of the f i f t y -  eight organisms d e f i n i t e l y p o s i t i v e to the Voges-Proskauer test.  Of the remaining twenty-three cultures that are  p o s i t i v e to the Voges-Proskauer t e s t , a c o r r e l a t i o n i s not to be found.  Four s t r a i n s , Ho* 206, 137, 139, 198, are  £ (2) * I t has been observed by Sadler and Irwin that t h e i r Aerobacter oxytocum (atypical) s t r a i n gave a p o s i t i v e reaction to the Voges-Proskauer test a f t e r 24 hours incubation, and f a i l e d to do so a f t e r 96 hours incubation* In the present study f i f t y - t h r e e of the f i f t y - e i g h t organisms gave a p o s i t i v e VggesProskauer t e s t a f t e r 24 hours incubation at 30 G. and 37°C* respectively. Five s t r a i n s , Ho* 206, 251, 32, 52 and 116 were positive to the test i n 48 hours. Judged by the Voges-Proskauer t e s t , no acetyl-methylearbinol could be detected a f t e r 96 hours incubation i n f i f t e e n of these s t r a i n s — c u l t u r e s Ho. 24, 249, 261, 275, 282, 109, 110, 112, 113, 114, 115, 122, 34, 67, 116.  (4)  In view of the observations of Burham and Levine et a l v5) think i t of i n t e r e s t to report that even a f t e r an incubation period of 8 hours we got a p o s i t i v e Voges-Proskauer reaction with cultures Ho* 24, 249, 261, 265, 275, 276, 277, 283, 49, 198, 109, 110, 112, 113, 114, 115, 122, 66, 68, 34, 67, 119, 121, 166, 167, 168, 169, 170, 188, 229, 47, 197, 209a, 209b, 120. w  e  10 d e f i n i t e l y acid to Methyl Red, while nineteen are v a r i a b l e to the Methyl Red reaction a f t e r periods of 48 and 72 hours incubation of 30°C. and 37°C. respectively*  As precautions  were taken to insure p u r i t y of the cultures studied, we  can  o f f e r no explanation of t h i s v a r i a t i o n t o the Methyl Red reaction of s t r a i n s that unmistakeably produce a e e t y l methyl-carbinol•  The eultures that produce aeetyl-methyl-  earbinol and are variable to the Methyl Red r e a c t i o n are Ho. 161, 162, 163, 164, 51, 66, 127, 213, 251, 53, 67, 113, 119, 121, 229, 52, 116, 120.  117,  Discrepancies such as  these to which we have drawn attention, have already been observed by Johnson and Levine  v  '•  Proceeding with the analysis of the c h a r a c t e r i s t i c s of the organisms that produee acetyl-methyl-carbincl, i t i s seen from Table I that thirty-two s t r a i n s f a i l to l i q u e f y gelatine and twenty-six are gelatine l i q u f f i e r s * are motile and forty-three are non-motilet only form indols  fifteen  seven s t r a i n s  a l l of the f i f t y - e i g h t reduce n i t r a t e s to  n i t r i t e s : and the production of gas i n litmus milk i n a l l eultures was pronounced. For the fermentation studies, twenty-one carbohydrates were employed.  As can be seen from Table I, an attempt  has been made to give a comparative idea of the extent to which the individual organisms attack the carbohydrates? furthermore, an i n d i c a t i o n i s to be seen of the preference shown by p a r t i c u l a r organisms f o r s p e c i f i c sugars--  11 observations "being made a f t e r incubation f o r 4, 8, 10, 24, 48 and 72 hours r e s p e c t i v e l y .  In a l l cases, when the  organisms produced a c i d and gas, l i t t l e f u r t h e r development i n gas production took place a f t e r the 24 hour incubation period; whilst the reaction of a l l cultures to the carbo-  (3) hydrates used by Bergey et a l of  i n the d i f f e r e n t i a t i o n  species w i t h i n the genus Aerobacter was s p e c i f i c a t t h i s  time.  Continuing the incubation period f o r a f u r t h e r 24  hours served to accentuate the d i f f e r e n t i a t i n g value of c e r t a i n of the sugars and higher alcohols*  For instance,  the gas production from xylose by cultures No* 161, 162, 163, 164, 127, 213, 121 and 32 was more pronounced: likewise, more gas formation was evident i n the maltose agar tubes inoculated with cultures No. 49, 137, 139, 127, 34, 67 and 72, and the attack on the glueoside aesculin was more d e f i n i t e by cultures No. 127, 117, 118, 119, 121, 229, 72 and 19*  fhe production of acid and gas from arabinose by culture  No*. 120 and from dextrin by cultures No* 24, 249, and 275 was accentuated by continuing the incubation f o r 72 hours* The Bacteria That Are Negative to the Yoges-Froskauer Test Of the lactose fermenting organisms there are f o r t y - f o u r strains that are acid to Methyl Red and do not produce acetyl-methyl-ear hi no1*  A l l f a i l to l i q u e f y gelatine*  t h i r t y axe motile and fourteen are non motile:  f o r t y produce  - 12 i n d o l and a l l reduce n i t r a t e s to n i t r i t e s *  Except cultures  Ho. 144, 123* 146, and 99, a l l produced gas i n litmus milk* Usually the gas production was not as pronounced as i t was i n the  case of the organisms p o s i t i v e to the Vbges-Ixoakauer  test*  In the fermentation studies employing the twentyone carbohydrates* observations were made i n a manner i d e n t i c a l with that described above*  Except i n the production  of gas from d u l c i t e and s a l i c i n by culture Ho* 40, from d u l c i t e by culture Ho* 44, and from s a l i c i n by cultures Ho* 66 and 58, the presence o f acid and gas when produced a t a l l , was d e f i n i t e a f t e r 24 hours incubation} and the d i f f e r e n t i a t i o n of species w i t h i n the genus Escherichia, a f t e r Bergey e t a l (3) , was s p e c i f i c a t t h i s time.  The 43 hours incubation  period served to accentuate the action of c e r t a i n of the eultures on s p e o i f i o carbohydrates: f o r instance, the gas production from d u l c i t e by cultures Ho* 57, 63, 146 and 222, from s a l i c i n by cultures Ho* 45, 46, 54, 123, 4 and 85, and from maltose by cultures No. 50, 21, 22 and 222 was more pronounced.  The production of aeid and gas from d u l c i t e by  culture Ho. 263, from s a l i c i n by culture Ho* 64, from glycerine by cultures Ho* 240 and 269 and from aesculin by cultures Ho* 45, 54, 43, 175, 35, 146, and 41, was accentuated by continuing the incubation period f o r 72 hours.  13  THE ClASSIEICATIOH OF THE BACTERIA  In attempting the c l a s s i f i c a t i o n of the b a c t e r i a , we have considered the c h a r a c t e r i s t i c s as summarized i n Table I, and throughout, have kept before us, the q u a l i f i c a t i o n s to which we have drawn attention i n the foregoing c r i t i c a l analysis of the reactions of the b a c t e r i a * In d e f i n i n g the genus and the species of each of the (3) organisms, we have followed i n the main, Bergey et a l • In the a n a l y s i s of the data on Table I, we drew attention to two cultures, Ho. 212 and 20 and f o r the reasons given there, we r e f r a i n e d from including the organisms i n the discussion*  two  In the absence of other s t r a i n s  showing i d e n t i c a l c h a r a c t e r i s t i c s , we propose to o f f e r no suggestion as to the c l a s s i f i c a t i o n of these two organisms* Consequently, i n the c l a s s i f i c a t i o n of the b a c t e r i a studied, we are here concerned with one hundred and two organisms* The motile or non-motile rods that are dram negative, that form gas from dextrose and lactose, and  that  produce acetyl-methyl-carbinol, are placed within the genus (3) Aerobacter, Beijerinek, Bergey et a l • On the sum of the c h a r a c t e r i s t i c s determined, Table I, cultures Ho. 161, 162, 163, 164, 165 and 206  are  (3) c l a s s i f i e d as Aerobacter oxytocum (ifigula) Bergey et a l  ,  - 14 even though culture Ho. 206 f a i l s to produce i n d o l , and forms no gas i n i n u l i n .  Cultures Ho* 24, 249, 261, 265, and  275 a r e i d e n t i c a l , the one w i t h the other, and with the  (3) a t y p i c a l s t r a i n of Aerobacter oxytocia*  (2)  by Sadler and Irwin  reported upon  . Cultures Ho» 276, 277, 282, and  233 are also c l a s s i f i e d as a t y p i c a l strains of Aerobacter oxytocum  • Each of the cultures No* 49, 51, 137, 139 and 198  i s c l a s s i f i e d a s Aerobacter aerogenes, (Kruse) Beijerinek, and cultures Ho. 109, 110, 112, 113, 114, 115, and 122 which f a i l to attack adonite, are to be considered as being a t y p i c a l s t r a i n s of the same species* I f we consider only the c h a r a c t e r i s t i c s used by Bergey et a l ^  f o r the major d i f f e r e n t i a t i o n of species  w i t h i n the genus Aerobacter, cultures Ho. 66 and 63 appear (3) to be related t o Aerobacter aerogenes  ; culture Ho. 127  i n some measure to Aerobacter ievans (Wolffin) Bergey et a l and cultures Ho* 213 and 251 to Aerobacter aerogenes  ^ *  In the ease of each of these s t r a i n s , however, very s p e c i f i c v a r i a t i o n s from the respective types are to be seen—Bergey (3) et a l  and Table I of t h i s papers  hence, considering the  c h a r a c t e r i s t i c s as a whole, we place these cultures within the genus Aerobacter but r e f r a i n from attempting a more precise c l a s s i f i c a t i o n .  15 As can be seen front Table I , many of our s t r a i n s are motile or non-motile Gram negative rods, produce a e e t y l metiiyl-carbinol, ferment sucrose to a c i d and gas, and l i q u e f y (3) gelatine r a p i d l y or slowly*  According to Bergey et a l  and i n the l i g h t of the data on Table 1 certain of these cultures, to wit, l o * 72, 19, 47, 71, 197, 209a, 209b, 32, 52, 116, 120 and 220 might be considered as strains of (3) Aerobacter bombycis, Bergey et a l • Yet, on the other hand, based on the sum of the c h a r a c t e r i s t i c s , these cultures are to be recognized as variants of Aerobacter cloacae* C7) (3) a f t e r Jordan  ,  :  and the f a i l u r e of the strains to  agree the ©ne with the other i n the a b i l i t y to attack c e r t a i n of the carbohydrates, notwithstanding, we suggest that cultures So* 72, 19, 47, 71, 197, 209a, 209b, 32, 52,  116,  120 and 220 be c l a s s i f i e d as variants of Aerobacter cloacae« (Jordan) ^  Bergey et a l ^ * . 3  Cultures Ho* 34, 53, 67,  117,  118, 119, 121, 166, 167, 168, 169, 170, 188, and 229 are the mom-motile s t r a i n s of the group of organisms whose main c h a r a c t e r i s t i c s are summarized above*  These s t r a i n s f a i l to  agree the one with the other i n the a b i l i t y to ferment certain of the carbohydrates employed i n the study, and even though the o r i g i n a l strains of Jordan a l l proved to be motile (7) ' we f e e l j u s t i f i e d , considering the sum of the characteri s t i c s , i n placing, t e n t a t i v e l y , cultures Ho* 34, 53, 67, 117, 118, 119, 121, 166, 167, 168, 169, 170, 188, and 229 as  - 16 • (?) non-motile variants of Aerobacter cloacae (Jordan) Bergey et a l  ( 3 )  ,  .  The Gram negative motile or non-motile rods, that produce gas from dextrose and lactose and do not produce acetyl--methyl-carbinol from dextrose are placed within the genus Escherichia, G a s t e l l a n i and Chambers, Bergey et a l Of cultures No* 240, No*  45, 46, 54, 56, 53, 64, 144  ^ ^. 3  and  54, 144, and 240 f a i l to attack r a f f i n o s e , none  produce gas from dextrin and as can be seen from Table I, i d e n t i c a l reactions i n a l l the carbohydrates employed i n the study cannot be observed*  Yet, i n considering the  c h a r a c t e r i s t i c s as a whole, Table I, each of these cultures i s to be c l a s s i f i e d as E s c h e r i c h i a c o l i (Escherich) (3) G a s t e l l a n i and Chambers, Bergey et a l  *  On the major c h a r a c t e r i s t i c s , culture No* 263 i s c l a s s i f i e d as Escherichia enterica (Gastellani and Chambers) Weldin, Bergey et a l 'i and, cultures No* 12 and 123, which are i d e n t i c a l with eultures No. 263 except that they are very a c t i v e i n adonite, are c l a s s i f i e d as adonite (3) v  fermenting s t r a i n s of E s c h e r i c h i a e n t e r i c a  •  Cultures No. 40, 43, 44, 57, 181, 192, 194,  233,  236, 245, 175, 267 and 269 form i n d o l , and c e r t a i n of them, Table X, f a i l to attack d u l c i t e . Even so, on the c h a r a c t e r i s t i c s as a whole, these organisms must be looked (3) upon as s t r a i n s of E s c h e r i c h i a formica and we place  - 17 them as i n d o l producing variants of E s c h e r i c h i a formica * Bergey e t a l (3)• (Omelianskl) In so f a r as the l i t e r a t u r e permits of comparison, the c u l t u r a l c h a r a c t e r i s t i c s of our s t r a i n s No. 31 and 63, we suggest that each he c l a s s i f i e d as Escherichia v e s i c u l i f onaam (3) (Henrici) Bergey e t a l • Cultures Ho* 4, 16, 17, 35, and 145 vary i n some (3) measure from E s c h e r i c h i a commanior  i n t h e i r action on  some of the higher sugars and they appear to he s p e c i f i c i n that they are weak i n the fermentation of sucrose and s a l i e i n , Table X* Whether or not t h i s l a t t e r c h a r a c t e r i s t i c i s a d i s t i n c t v a r i a t i o n i n "degree* from Escherichia (3) cowman! or  cannot be said*  not overlooking t h i s  q u a l i f i c a t i o n we c l a s s i f y each o f cultures Ho* 4, 16, 17, 85, and 145 as E s c h e r i c h i a communior (Durham) Bergey et a l  •  v  Cultures Ho* 130, 146, and 219 f a i l to produce gas from s a l i c i n and consequently i n one of the p r i n c i p a l sugars used (3) in differentiation a d i s t i n c t v a r i a t i o n from the type (3) E s c h e r i c h i a communior  i s shown*  Even so, we consider we  s h a l l be subscribing to the p r i n c i p l e s that should guide i n c l a s s i f y i n g i f on the sum of the c h a r a c t e r i s t i c s , we define cultures Ho* 130, 146 and 219 as non-saliein fermenting (3) s t r a i n s of E s c h e r i c h i a communior (Durham) Bergey e t a l  •  Of the non-motile forms included i n the genus Escherichia, cultures Ho* 50, 21, and 22 ferment adonite to  18 a c i d and gas and produce i n Simmon*a C i t r a t e Agar, the blue color which i s s i g n i f i c a n t of the genus Aerobacter*  Culture  Ho. 50 forms indol whilst cultures Ho. 21 and 22 do  not.  These and l e s s c r i t i c a l v a r i a t i o n s notwithstanding,  the strains  without doubt are c l o s e l y r e l a t e d to the t y p i c a l E s c h e r i c h i a (3) neapolitana. Table 1, Bergey et a l t but i n view of the v a r i a t i o n s we c l a s s i f y cultures Ho. 50, 21 and 22 as a t y p i c a l (3) s t r a i n s of Escherichia neapolitana (Emmerich) Bergey et a l As non s a l i c i n fermenting s t r a i n s of Escherichia neapolitana (3) Bergey et a l , we place cultures Ho. 28, 41, 39, 99, 222, on the sum  of t h e i r c h a r a c t e r i s t i c s . Cultures No. 13 and 255,  i n view of the  c h a r a c t e r i s t i c s shown i n Table I, are d i f f i c u l t to d e f i n e . Consequently, we leave them within the genus E s c h e r i c h i a but r e f r a i n from suggesting a more precise and d e t a i l e d classification.  THE PRODUCTION BY THE ORGANISMS STUBIEB OF  THE  CHARACTERISTIC FEED FLAVOUR AND  STABLE ODOUR IH MILK  These observations were made on f l a s k s of milk inoculated with the respective bacteria*  Rather than attempt  to give a description of the c h a r a c t e r i s t i c feed flavour and  # See page 6*  •  stable odour, we excerpt d i r e c t from the paper of Sadler, (1) Irwin and f o l d i n g t "The f l a v o r produced i n steamed or s t e r i l i z e d m i l k . . . . i s very d i f f i c u l t to define. The taste i s disagreeable and d e f i n i t e l y unclean. There i s an astringent e f f e c t , and a s l i g h t t i n g l i n g sensation on the tongue and on the p a l a t e . I t i s not necessary, however, to taste the milk to be sure that the defect i s there. The odor i s an u n f a i l i n g guide. On taking the cotton wool plug out of the neck of the f l a s k the characteri s t i c odor can be noted a t once. I f , however, the f l a s k be shaken vigorously f o r a moment, the odor i s accentuated, i s more pungent and penetrating. The shaking; produces a very heavy foam, and the milk culture f i z z e s almost as a syphon of soda water. When the odor c h a r a c t e r i s t i c of a defect i s present, one i s reminded of a combination of s m e l l s — a combination of the footed close a i r that may be encountered on going into a badly v e n t i l a t e d heavily populated cowshed e a r l y of a morning; the mixture of smells that the remains of various feeds given to the c a t t l e the night before can produce! and the s t a l e , s l i g h t l y sour penetrating smell that memory associates with the clothes of the o l d time cowman, to whom the actual milking of the cows was not always the most important part of an arduous day's work. Withal, i n the odor i s a pungent, nauseating q u a l i t y not e a s i l y to be mistaken......." Except f o r eultures Fo. 53, 72, 47, and 71, a l l of the organisms we have placed within the genus Aerobacter, produced, when f i r s t i s o l a t e d , a feed flavour characteri s t i c a l l y i d e n t i c a l with the d e s c r i p t i o n already given ^ . In the ease of cultures l o . 53, 72, 47 and 71, the flavour and odour were d e f i n i t e but not nearly so intense.  When  f i r s t isolated, twenty-three of the f o r t y - f o u r organisms, that we have c l a s s i f i e d as within the genus Escherichia,  - 20 produced the odour and flavour described as c h a r a c t e r i s t i c While s t i l l <tuite d e f i n i t e , the i n t e n s i t y of the flavour and odour was not quite so pronounced i n milk inoculated respectively with the other twenty-one s t r a i n s placed within the genus Escherichia--cultures Ho* 45, 46, 54, 56, 58, 64, 144, 40, 43, 44, 5"?, 269, 61, 63, 85, 130, 146, 219, 41, 39 and 99* A f t e r the organisms had been i n a r t i f i c i a l media for  some s i x months, i t was found that except cultures Ho*  66 and 68, a l l those of the genus Aerobacter produced a t r u l y c h a r a c t e r i s t i c feed flavour and stable odour to a pronounced degree*  Hot only i s i t of p a r t i c u l a r i n t e r e s t to f i n d that  a f t e r t h i s period i n pure culture, the a b i l i t y t o produce the c h a r a c t e r i s t i c feed flavour and stable odour was thus f a r a constant c h a r a c t e r i s t i c o f the majority of these organisms, but i t i s to be observed that the four s t r a i n s which, on i s o l a t i o n had produced the defect i n some measure, now gave the f l a v o u r and odour of an i n t e n s i t y comparable (l) with the f l a v o u r and odour o r i g i n a l l y described * A f t e r a s i m i l a r period i n a r t i f i c i a l media, t h i r t y three of the organisms of the genus Escherichia gave a pronounced and c h a r a c t e r i s t i c feed flavour and stable o d o u r — cultures Ho. 45, 46, 54, 56, 144, 240, 12, 263, 40, 5?, 181, 192, 194, 233, 236, 245, 175, 267, 269, 4, 16, 17, 85, 145, 130, 50, 21, 22, 28, 41, 39, 99 and 255. The remaining eleven  - 21 organisms placed i n t h i s genus produced the defect i n l e s s e r measure*  I t w i l l not he overlooked that* when making  observations on a defect such as t h i s with which we are concerned* i t i s extremely d i f f i c u l t to define i n t e n s i t y of smell i n a manner more precise than say s l i g h t , marked or pronounced*  Even so* quite d e f i n i t e l y our study permits us  to report that c e r t a i n c u l t u r e s were, over a period of time, consistent i n that they produced the c h a r a c t e r i s t i c feed flavour and stable odour to a greater or l e s s e r degree respectively throughout* On the whole, the b a c t e r i a c l a s s i f i e d as within the genus Aerobacter, produced a more pronounced feed flavour and stable odour than d i d the organisms f i n d i n g themselves within the genus Escherichia*  POSSIBLE CHABTHELS OF IKPEGTIOH  As a ready means whereby one may see something of the r e l a t i o n s h i p of known strains to the sources from which respectively they have been i s o l a t e d , a series of p i c t o r i a l designs has been prepared*  These p i c t o r i a l designs give one  an idea as to possible channels through which the i n f e c t i o n of milk by organisms responsible f o r the alleged feed flavour and stable odour may proceed*  As an example three such  pictures or diagrams are given here—Diagrams A, B, and C.  - 22 Considering Diagram A, i t w i l l be seen that the (2) (3) a t y p i c a l Aerobacter oxytocum has been recovered from „ t l ) (2) , „ v  corn s i l a g e ,  t f c  t  manger scrapings, milk, the tongue of a  eow, a l f a l f a from a manger, straw used f o r bedding, and from grain mixtures*  According to Diagram B, Aerobacter aerogenes  has been recovered from a mangel f o r k , milk, a mangel cutter, and from a milk s t o o l *  According to Diagram C, E s c h e r i c h i a  c o l i has been recovered from a milk sheet b l o t t e r , a beet pulp shovel, a hay truck, manger scrapings and from the r a f t e r s i n a barn*  - 23 -  - 25 -  THE APPLICATION Off THE FIHDIIfSS  Reviewing the work as a whole, i t would seem that the f i n d i n g s r e l a t e themselves to the p r a c t i c e of milk production on the farm, to the methods i n vogue i n the routine inspection of dairy farms, to the management and control of milk, and to the public health aspect of milk production* The study has shown d e f i n i t e l y that t h i s c o - c a l l e d feed flavour and stable odour i n milk can be caused by s p e c i f i c s t r a i n s of b a c t e r i a within the Escherichia-Aerobacter group, and that these s t r a i n s may be obtained from numerous and varied sources*  This study has demonstrated possible  channels through which milk might become infected* Consequently, i f the defect i s to be avoided, i t i s necessary to employ the most approved methods and practices i n the production, management and control of milk*  Whilst  the using of any p a r t i c u l a r feed f o r milk producing cows i s not condemned, i t w i l l be observed that the greatest  care  must be exercised i n management so that no b a c t e r i a l contamination from the feed i s possible* This investigation has shown that the presence or absence i n milk of the b a c t e r i a of the Escherichia-Aerobacter group may be taken as a measure of the conditions and the  - 2? q u a l i t y of management p r e v a i l i n g on milk producing farms and i n milk d i s t r i o u t i n g depots.  Possibly therefore, i n the  l i g h t of the r e s u l t s of t h i s study, the whole question of milk c o n t r o l , of farm inspection and of the grading of d a i r y farms f o r milk production might to advantage he reviewed*  SIJMMABY  The r e l a t i o n of b a c t e r i a of the I s c h e r i c h i a Aerobacter group to an alleged feed flavour and stable odour i n milk has been investigated* Bacteria have been i s o l a t e d from milk, feeds and farm premises* One hundred and four organisms that were found to cause the c h a r a c t e r i s t i c feed flavour and stable odour i n milk have been subjected to a detailed c u l t u r a l study* The respective sources of the b a c t e r i a recovered and the complete r e s u l t s of the d e t a i l e d c u l t u r a l study are summarized i n Table I* A c r i t i c a l analysis of the data i n Table X i s presented* Based on the sum of the c h a r a c t e r i s t i c s of the b a c t e r i a under study, one hundred and two of the one hundred  -  28 -  and four hare been c l a s s i f i e d *  F i f t y - e i g h t s t r a i n s are  placed within the genus Aerobacter; f o r a l l but f i v e the species has been defined and the c l a s s i f i e d organisms include s t r a i n s of Aerobacter oxytoeum* Aerobacter oxytoeum a t y p i c a l * Aerobacter aerogenes* Aerobacter aerogenes a t y p i c a l , and variants of Aerobacter cloacae*  as can be seen from Table I*  Forty-four of the organisms are placed i n the genus Escherichia and for forty-two of the organisms the species has been defined and the c l a s s i f i e d organisms include s t r a i n s of E s c h e r i c h i a c o l i * Escherichia enterica* adonite fermenting s t r a i n s of Escherichia enterica* indol producing variants of Escherichia formica, E s c h e r i c h i a vesiculiformane*  Escherichia  eommunior, non s a l i c i n fermenting strains o f Escherichia common!or, Escherichia neapolitana a t y p i c a l , and non s a l i c i n fermenting s t r a i n s of Escherichia neapolitana—Table I* Hot only have observations  been made on the  i n t e n s i t y of the flavour and odour produced i n milk by the b a c t e r i a when f r e s h l y i s o l a t e d , but also on the i n t e n s i t y a f t e r the same organisms had been held i n a r t i f i c i a l media for  some s i x months*  Certain cultures are consistent i n  that they produce—to a greater or l e s s e r degree—the c h a r a c t e r i s t i c feed flavour and stable odour throughout*  On  the whole, the b a c t e r i a c l a s s i f i e d as within the genus Aerobacter, produce a more pronounced feed flavour and stable  - 29 odour i n milk than do the organisms f i n d i n g themselves within the genus Escherichia* fhe possible channels through which i n f e c t i o n ©f the milk by the b a c t e r i a may  take place are indicated  pietorially* Attention i s drawn i n the a p p l i c a t i o n of the findings to the practice of milk production, to the routine inspection of d a i r y farms, the management and control of milk, and to the public health aspect of milk  production*  ACKirOfLEDGEMlNfS  To Professor H. K * King and Professor W i l f r i d Sadler f o r t h e i r guidance, encouragement, and advice; to the E a t i o n a l Research Council of Canada for the funds that have made t h i s i n v e s t i g a t i o n possible? and to the University of B r i t i s h Columbia f o r the laboratory f a c i l i t i e s enjoyed, we give our most appreciative thanks*  - 30 REFERENCES  1*  Sadler, W i l f r i d , Irwin, K* Lenora, and S-olding, N. S., 1929, December. An Alleged "Feed" Flavour i n M i l k Caused by S p e c i f i c Bacteria* fhe M i l k Dealer,  2.  Sadler, W i l f r i d , and Irwin, It. Lenora, 1930* Feed Flavour or Stable Odour i n Milk Caused by an Atypical S t r a i n of Aerobacter oxytocum* Can* Jour* of Res* 3, 200-204*  3*  Bergey, B. R., 1930* Manual of Determinative Bacteriology, 3rd e d i t i o n . Williams and Wilkins, Baltimore.  4*  Durham, 1901* (cited by Levine, Weldin and Johnson) Jour. I n f . Diseases* 21, 1.  5.  Levine, Kax, Weldin, J . C , and Johnson, B e r y l R., 1917* fhe Voges-Proskauer and Correlated Reactions of c o l i - l i k e B a c t e r i a . Jour. Inf. Diseases* 21, 1*  6*  Johnson, B. R., and Levine, Max, 1917* Characteristics of c o l i - l i k e microorganisms from the S o i l * Jour* Bact* I I , 4*  7.  Jordan, 2. 0. 1890. Part I I .  Rept* Mass* State Bd. Health,  I  TABLE CULTUREX \NUMBER  SOURCE  FARM NUMBER  OF  BACTERIA  *  MP  SPECIFIC SOURCE\ OF BACTERIA  \GELATME WOT/l/m  DFXTR/N. GALACFOSA M/IL TOJF. WAFF/A/OSF. 3AL fc/N. Vri rcFje/A/A/faw/rA 5OJ?3/TE. \AooA//r£ [Xn OSE. WABINOSE J?f/AW05E\lAEVM05E.\/$4M05E:.  \p£XT/ea5t\ LACTONE.  /A/DOL  INUUN.  STARCH  AE5CUL/N.  Of  ClA55/F/CA T/ON.  M/LK.  W/TRATES. Z4 HOURS  MR. POS.  \l?EDUCT/Of*\ LITMUS  NITRATES REDUCED  AeFobecter  ULTURE\ \NUMBEG.  oxytocum f&i  A G  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  ie/2.  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  I €2  163  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  163  i&4  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  164  165  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  A G  AG  AG  AG  AG  AG  AG  AG  AG  AG  165  AG  AG  Ac  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  A©  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  A G  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  24-9  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  A G  AG  AG  261  3Z3  IG1  m  HOG  MILK  STRAW  "24  ?  BEDDING  NON-LlQ.NOHMJTILE F O R M E D .  NOT FORMED  ACID I  MANGER SCRAPINGS MILK  2.61  206  Aerobacter  oxytocum  24  atypica/  IAS  TONGUE OF COW  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AS  265  -2.-75  .FALFA FROM M A N G E R  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  A G  AG  AG  AG  275  276  STRAW  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AS  276  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  277  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AO  282  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  A G  AG  AG  AG  AG  AG  AG  283  AG  AG  AG  AG  AG  AG  AG  Ac  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  A3  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  A G  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AS  137  AG  AG  AG  AG  AG  AG  AG  A G  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  J39  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AS  J98  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AS  I IO  AG  Ac  AG  AG  AtS  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AS  I/O  112.  A G  AG  AG  AG  AG  AG  AG  A G  AG  A G  AG  AG  AG  AG  AG  AG  A G  AG  AG  AG  112  113  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  A6  1/3  114  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  1/4  115  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  A<3  1/5  12.2.  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  122  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  Ac  AG  AG  AG  AG  AG  AG  AG  AG  A G  AG  AG  AG  A  AG  AG  AG  AG  MANGER SCRAPINGS  AG  AG  2.51  B R U S H IN B A R N  AG  34  M I X E D G R A I N IN TRUCK  FORMED  53  PIECE O F C U T M A N G E L  BEDDING  2.77 GRAIN MIXTURE  -2.83 MANGEL FORK  43 si  MfLK  »37  MANGEL CUTTER  ACID  133 138  xn  MILK  STOOL  M I X E D G R A I N IN B I N  I O S  66  MANGEL  ALK.  FORK  68  ALK.  AG  AG  AG  A G  AG  AG  AG  A G  AG  AG  AG  AG  AG  AG  AG  AG  NOT FORMED  AG  AG  AG  FORMED  AG  AG  AG  HOT FORMED  AG  A G  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  1€>7  12.-7  MOTILE  MILK  -2.13  m  AG  Ac  AS  \Aerobacter aerogenes  I09  atypical  \Acrobacfer  66  AC3  68 /27  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  A G  A G  AG  AG  AG  AG  AG  AG  AG  AG  AG  53  AG  AG  AG  A G  AG  AG  AG  67  AG  AG  AG  AG  AG  AG  Ac  AG  AG  AG  213 25/  Aerobacter cloacae (1)  34-  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  /I7  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  1/8  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  1/9  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  166  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  167  168  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  /6&  I&9  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AvG  AG  AG  /69  no  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  /70  F L O O R IN F E E D R O O M  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  188  MUD ON ROAD  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  6  229  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  Ac  AG  en  ALK. I  LlQ.  NOMMJTILE  IIXED GRAIN IN T R U C K  \\1  TONGUE OF C O W  118  1*2.1 1ee  m  MILK  188 229  12*  ALK.  MOTILE  72.  MANGER SCRAPINGS  13  M A N G E L FORK  AG  AG  AG  47  T O N G U E O F Cow  AG  AG  AG  71  MANGER S C R A P I N G S  AG  AG  AG  STOOL  AG  AG  AG  MIXED GRAIN  AG  AG  AG  AG  AG  AG  AG  A G  AG  13-7  i n  203;  MILK  ALK.  -203 b. WATER F R O M DRINKING B O W L  37L  AG  AG  51  AG  AG  AG  Aerobacter aerogenes 4.9  AG AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  A G  AG  AG  AG  AG  AG AG  AG  AG  A  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  /2f  Aerobacter  cloacae (2)  72  AG  IS  AG  47  AG  71  AG  197  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  2093.  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  209b.  AG  AG  AG  AG  A G  AG  AG  AG  AG  AG  AG  AG  AG  AG  32  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  52.  Pi ECE OF C U T M A N G E L  AG  AG  AG  AG  AG  AG  AG  116  TONGUE OF COW  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  1/6  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  120  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  Z20  AG  AG  AG  AG  AG  AG  AG  AG  20  I2.0 220  TU  2.0  W A L L IN B A R N  ALK.  N0N-M0T|.d  MANGEL FORK B L O T T E R FOR N E G NON-LIQ. MILK S H E E T *ao|  45  MOTILE  FORMED  46  AG  AG  AG  AG  AG  AG  A G  A G  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  52  NOT REDUCED NITRATES! REDUCED  AG AG  AG  Escherichia  co//  45  AS  4-6  AG  S4  AG  5£  54  BEET PULPSHOVEL  AG  A G  A G  A G  AG  AG  AG  A G  A G  A G  AG  A G  AG  AG  56  B L O T T E R FOR MILK SHEET  AG  A G  AG  A G  AG  AG  AG  A G  AG  A G  AG  AG  AG  AG  A G  58  HAY  AG  AG  A G  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  A G  AG  S3  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  A G  A G  Ac  AG  64-  AG  AG  AG  AG  AG  AG  AG  AG  AG  TRUCK  <S4  AG  A G  AG  A G  AG  144  MANGER S C R A P I N G S  AG  AG  AG  AG  AG  AG  AG  AG  AG  2.40  RAFTERS IN B A R N  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  J-23  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  2.63  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  A  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  A  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  12  MCN-MOTILE  MILK  4 0  B A L E D ALFALFA H A Y  43  BEET PULP SHOVEL  MOTiLE  44 B L O T T E R FOR MILK S H E E T  AG  144AG  AG  240  Escherichia  Enlerica0J  12 123  A6  Escherichia  Enterica  AG  Escherichia  formica®  263  40  AG  AG  43 44  A  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  57  A  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  /8/  AG  -AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  192  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  Ac  A S  194  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  A  AG  233  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  A  AG  236  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  245  MUD  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  !7S  STANCHIONS  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  267  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  269  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  16  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  17  AG  AG  AG  AG  AG  Ac  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  17  85  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  85  145  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  A G  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  57 181  ILT  19-2.  FLOOR BEHIND S T A L L S MANURE  134 233  TV  23<©  GUTTER  Z45 \1S  FLOOR BEHIND S T A L L S  M U D NEAR B A R N  m  Z67 ZG9  MANURE F L O O R WHERE G R A I N IS M I X E D  61  NONMOTILE  C33 MOTILE  MILK  130  M U D IN PADDOCK  t46 213  MILK TU  50  DITCH ON ROAD NON-MOTILE  GUTTER  2.1  NOT FORMED  MANGEL CUTTER  28  WET BEET  FORMED  PULP  AG  AG  AG  AG AG  A G  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  vEscberichia vesiculi-  AG  -64-  T&rrrmtTisf  €3  Escherichia communior /6  AG  14-5 AS  Escherichia  AG  communior &)  /30 /46  AS  2/9  Escherichia neapol/Tana  AG  AG  AG  AG  AG  AG  AG  21  AG  AG  AG  AG  AG  22  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  -  so  atypical  Escherichia neapoHtana&)\  2&  41  T O N G U E OF C O W  AG  A G  39  B A L E D A L F A L F A HAY  AG  AG  99  MILK  AG  AG  AG  AG  AG  AG  AG  AG  A G  AG  AG  AG  AG  AG  AG  AG  99  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  222  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  AG  2.-2.2. MOTILE  255  1ST m  NOT FORMED  NON-MOTILE  WOOPE N STANCHIONS  AG  A A G  AG  AG  AG  AG  AG  AG  NOT REDUCED  NUMBET<  I II .  T H E UNIVERSITY A  FARM  ON  m "TV  V "Sir  FAT^M POINT  VANCOUVER LULU  ISLAND '  ISLAND  N i l GO IN/IE: IN AT  GREY B . C .  ISLAND  MISSION HARRISON  L A D NE.R  N / I I L L S  • A L L  C U L T U R E S  AG  A C I D  A N D  A G AS A  A C I D A C I D A C I D  A N D G A S A N D G A S . N O G A S .  —  N o A C I D  G A S  A N D  G R A M  N E G A T I V E  N O  P R O D U C T I O N  A D I S C U S S I O N  4/  AG  39  O N  T H E  255  AG  2/2  P R O D U C T I O N  " T H E B A C T E R I A OF" T H E A L L E G E D F E E D FLAVOR ANID S T A B L E ODOUR IN N/llL-K IS T O B E F O U N D I NT H E T E X T PAG ES:  PROFUSE ,  El r>n p l o y i n g S i m m o n ' s Citrate A g a r all organisms wi+hin t h e g e n u s A e r o b a c t e r b r o u g h t a b o u t a b l u e c o l o r throughout the m e d i u m . T h r e e strains, cultures 50,21, a n d 22. w i t h i n t h e g e n u s E s c h e r i c h i a a l s o b r o u g h t a b o u t a blue color throughout t h e m e d i u m .  G A S . fi) C L A S S I K I E D A S N O N - M O T I L E  VARIANTS  AEROBACTER  OF  CLOACAE.  (*) « " VARIANTS OF AEROBACTER (3) W I T H Q U A L I F I C A T I O N S : S E F T E X T .  CLOACAE  /3  AG  B V  RODS.  - M E O I A S*=>L.IT IKI L A Y E R S , AN1D B L O W N U P T U B E B Y © A S . — G A S  *  AG  [Escherichia  MANGER S C R A P I N G S  *  FARM  AG  

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