L |\ mumumnmmmwmmum Ni “WM 3TUDIES ON COLIFORM BACTERIA ISOLATED FROM ‘MlLK . Thai: for the Den“ of M. S. ‘1} MICHIGAN STATE. COLLEGE ‘ Elizabeth Ballard Burlcigvh 1944 STUDIES ON COLIFORM BACTERIA ISOLATED FROM AILK By Elizabeth Ballard Burlaigh V..- ‘ ~. _. - a. 1.4LJ.~V ++ 1- +Lr ~w 1 ~ T x~~w \ V... )e .1 ' m"" 7 r" .-. M w r-v1-‘ v- v v ‘,_ g L __ LI - I Iv _ .l ‘ 1‘ " r :3 -. “ fi . 1 5" 1 r‘ 1 Y“!('\V"t' uiy ‘— ‘ .~'_ _ klx —.-‘ L ‘5‘ 1" Tflfi‘j'i’“ "I- ‘ +1‘r- "1"" r‘ n- I‘ .H ' A~ J J L L) " - \ . Tfi] q..- t .u HAL .L J4 ‘ -4 -‘ r‘ w‘! n4 an 7 l ‘1 -7 A :\ . - ‘5 - 0 <2. m3. 3&1' CH ‘1 n ‘3 .3 ‘- k,‘.‘.lri .. .heu n ’1: ‘Ch’ 4‘ r‘ II. III. IV. \‘r I o OUTLINE OF CONTENTS Introduction I o o . Study I. effects of 30 C. and 37 C. Incubation on Three Biochemical Differential Tests A. Introdrction B. Procedure C. Results and Discussion D. Summary Study II. The Eijkmqn Test A. Introdnc+ion B. Procedure 3. Resulis and Discussion D. Conclusions Study III. Pastonrizniion Siudies A. Introdtction B. Proccdn“e C. Results 1). Summary is. Conclwd one Afpengix. nedia and fiéagents Literature Cificd. Pun "§29?% Kl INTRODUCTION THE COLIFORM GROUP OF BACTERIA The coliform group is antigenicly heterogeneous and has widely diversified metabolic activities. For these reasons, classifications proposed for the group have been many and complicated. The 5th Edition of Eggggyig flange; 23 Determinative Bacteriology (9), classifies the group in two genera unler the family Enterobactoriaceae. The two genera, Escherichia (Type species: ggghgrighia coli) and Aerobacuer (Type species: Acrobacter ser: cues) are differ- entiated from other nembers cf the Tqmrlu bv inei” ability J - ‘ ‘r l .- ' '.- - . --‘ ‘ . . to inr‘ont iuCLCDC 'lLi ,.s ;.cluct.on . A Lhird “onus, fit .. _. J.-. _,., ..... .‘ -. '-', .- .. . ."‘ . ' . ' :zi122i2;3£’ fins frog re; h; "v.2“rn 'nJ fliilei to includ: .._- I..\ h L L, . - . \. - - ran]: ‘. — 1 -.- - .-.~--._ _, H 1:: u-.. .. .._‘ o l“ " r c".- .-~. w... _. .- n 1 _-_ ,_- JI . - .. - - -‘- - " lia. s oi loin. cool ‘ L v- '_ r ~* ~' ~- 3‘ -I , 1 1‘ L- ' ' l .L u 11'. . ,. . . . . w. . v . . —.D-u J I .. a. v v _.__ . -v .' v" -1 _ J . 1 . . _L_ . _._. -'l - _ .1 .. u . . .. _ '1' ' .I . _...'\....- - 'Vl ‘ LI. .. — . - - . . _ .-- .P r e ‘_ . . . _L c be ‘ - :_" -"1 ”1' 41,, .q'I '- v ' LI I — \ ‘ y. - , .. . . .. r . .g .. c ._ . _ ... v.'v..L—'.lL-o (na. (1;; _._...— _ a — I... . . - _ __ _.. _ a -u I 1_ H.— L A - . ha...— v.4! -\. —. - J —u ..a. " . . . . " ...,_--. .' .- ‘I.. .l.‘. uuv-J. J... -...L. .\. v. e ._._-‘ .n .-..d. .1. J LA .- - . ___._ _-_— ' 'I J. . fl ' ' .‘ _ , . ..- ‘- f“ ‘ * “°"- - ' - i - b :-L' _i;;u;;:; *‘ ““ .- .- ... .- -. .- .— n. .. - .- .. .— _ .. ‘ ash A. n '1- .. Tr ‘. ~ ‘ . . " . 1 .. . ' .. - _ ‘. \. I L. LL-.. - A ~ ,3. _ n _..__ k v I _ I." J. ‘4 Pa'\ a.“ vhf; _._ .. 1... .. _ _ - . . 'ilgi; Sreunq on grains and plants, in the soil and to a varying degree in the intestinal tract. Because of their association with pathogenic bacteria in the intestinal tracts of man and an- imals, the bacteria in this group have assumed public health significance in the detection of fecal contamination or of eater supplies. In dairy products, the H. O :5 sewage pollut presence 0; numbers 0? colifer: bacteria is retarded '3 k—l p "1 ( l U} taminauicn from barns 0” dirty dairy equip- Jo .3 ’1 (”J r) I3 6: irr": (-01 -:_V ‘..,L JC.,.J— 3 (' :3 F ‘0 ": -ut \ t 3 ) ct- ) wq .l O C V \ (-1 C C .. C ( .I L i—h r—d p.) ‘4' m ! -J 1 C. ’"5 El ( I d J "\ (a o 'N k D \I A {\"i CD \d f‘\ L—J V 1 , D A ° f" ! A y‘ ‘I [N r v “ " ih= J.cu; "“ b-c r“ c‘rru r a“ a r i c i-c.y \- y‘ A I" -_: u .'-7. at \‘ - . fi‘f‘ r» _. f 1" .v-‘ r " 3‘ - .1 _. .I . 1 .a .' . O J.‘ o ' _, s — -L x o .z \v v " Iv \. -7 n ..n. v I L) J ’ — l.. r - "’ C‘ ’1 C‘ - ' '1 r1 d '. ~ "I‘ I. "' 1 \ — H h. h ‘ t ‘T r‘ I q _ G LI 1. ‘- cn. -- \ d- .- l . J J ‘ 5' \J v I k - l a H + ‘n '_ (.1 ‘I‘ r. ‘ .1 41;" ‘ .L f- “ ._A 9' - C Is 1‘ > ,_ . l' I 1‘ I ‘_ _‘ fl ’3 f f‘ 4- (A 1 1- 15 f r~ ‘ 1 A r ' .z ‘ p ‘& W Vt _. - ._ 1‘ . C‘ o C a n .. 4 ~ U r (1" _. -~--~-—— --, -. - 3', +1“. -~ , _.t -- -t . '- , ... ,- ,q . "- .,1 . ' x . . A . . - . .u - -_ .r _. \ .. / _-. J V --__-~_ -—-- ——- -_——_—_—--—- —— ——~.‘- ——— _‘—--H- .- 1 'u 1 f' I‘ 1 _ 1‘ fi 1 _1 A '1 f, -_ — w o _ ;-_——_ —— —— ' —— ——- —-- -—_“ -———_ -— -—~—h —_——— -—.p-—- \ l r‘ .. .. 1 * .. Ft 1 o r\ ‘ .. : ‘ - . a " J 4 }fi r~ . Y 1 A) \* q 1 r‘. “ [1 ’\ Y‘ A ‘ P T‘ ’1 ‘ . .i 1" A ’ ‘ '1 a. m A. r 1 + ‘ 4-L ~n~+e~. D . ,\ ~-%nw un+i‘ w 1‘ A ’ A w — ‘ fl Y‘ ' ’% ‘1. P u .‘ r . ' _ v ’ 9 5‘ o _‘ - 1 — K 5 a ‘ n A ‘ 1 : A _‘ ‘ H fl 1!} 1 r m A h .— t n p r k V c 7 r f VA I r r} A1. 0.. a- 1-.+r‘ 1.. '1 ~11, a A‘ (we “ ". . . o ' L .1- a n 5“" \«A 1y} {innr‘ r3 ~ Tn aJ-~1vf 4 r n [\A-o 1“ «a h . o- ” an. r01 ’, -e -‘ m .0 In. .- n - .. .q ‘TI l 1 n . _ - | I '~ ’- ‘ A.) .. \ -K: V AA - 'V - ‘ - "r V J " V —-—-—~I———- n.—— n. 1 4 ' .- - v V u I J v ,4 z tryptose lactose broth3 were used for presumptive tests on all samples. Lauryl sulfate tryptose dextrose broth4 was used on the first two series of samples indexed,.and formats ricinoleate broth5 on the remaining eight series. Eosin- methylene blue agar5 plates were streaked from the positive- presumptive tubes (those tubes showing gas in 48 hours at 37°C.) Wherever possible, typical Eggh. ggli or gag. a332- SEEEE colonies were fished from the plates after 24 or 48 hours incubation at 37°C. and planted into lactose broth for confirmed refermentation tests. Occasionally an atypical colony was fished from a plate into lactose broth. Agar6 slant cultures were made from those lactose tubes which showed gas production within 48 hours at 37°C. About 80 cultures were diluted and plated. New slant cul- tures were made from isolated angle colonies on the dilution plates. Stock slant cultures were stored at room tempera- ture and transfers were made from them as needed. Five hundred cultures that fermented lactose with gas formation within 48 hours at 3700. were isolated. It is with these cultures that the following three studies on methods of detection and identification of coliform organ— isms from water and milk were made during the years 1942-44. 3The formula for tryptose lactose broth is given by Darby, C.W. and hallmann, J.L., J.Am.Water Works Assoc., gl:689, 1939. The addition of lauryl sulfate to the tryptoso lac- toso broth is discussed by these authors in Am.J.Pub.Health, 81:127, 1941. 4Dextrose was substituted for lactose in the lauryl sulfate tryptose broth. 5Formulae of these media are given in §EEE§EEE TEEEOQE 293 the Examination 0; later and éegage, 8th Id., 1936 (I). ——————————— "" ‘y‘)I"p(ll"L1JGiOU Of l’lLI'tT'i—ent Elgar- 6See appendix for formula and STUDY I EFFECTS or 30°C. and 37°C. INCUBATION ON THREE BIOCHEMICAL DIFFERENTIAL TESTS Introduction The coliform group of bacteria is defined as includ- ing "all aerobic and facultative anaerobic, gram-negative, non-spore-forming bacilli which ferment lactose with gas formation" by both the iiaaéass Tsihsds £2: IE2 Esaaieaiiea 2i V2225 and §§E§EE’ 8th Edition, 1936 (l) and the §is2§a£é T212242 £2: The Esasiaaiisa 2f 221:1 EEQQEEIE’ 8th Edition: 1941 (3). However, as both these §tgnda3d Egthgds limit their presumptive analysis tests to one temperature of in- cubation, 37°C., only those bacilli which ferment lactose with gas formation at 37°C. are detected in the routine examination of water supplies and dairy products for coli— form bacteria. In recent years, data have been reported indicating that not all coliform bacteria produce gas from lactose at 3700., and therefore are not detected in examination of water and dairy products conducted at 3700. only. Hiscox (20) isolated fm 3 milk a member of the coliferm group that fermented lactose with vigorous production of was at tem- peratures up to and includ_ng 300C., but which produced no gas at all or, at most, a scarcely perceptible amount at U.) \ C') 0 0-4 ‘4 m J also cited a similar organism isolated from butter by Grimes and hennerty. Levine, Carpenter and Coblentz (° \\ 8) described 88 of 196 celiforu strains isolated from chlorinated watersi as producing gas slowly, if at all, at 37°C. but very luxuriantly, as a rule, at 30°C. Their detection, they stated, would be facilitated by lower- ing the temperature of incubation for presumptive tests to 30°C. These reports indicating that some coliform bacteria produce gas from lactose more frequently and abundantly at temperatures below 37°C. raise the question whether more coliform bacteria might not produCe positive reactions to biochemical differential and identification tests at lower incubation temperatures. Reports on the effect of incubation temperature on biochemical differential tests made on aberrant coliformsl differ. Hiscox (20) found hb bacillus to give the same citrate, indole and Voges-Proskauer reactions at either 37°C. or 30°C. Stuart, Mickle and Borman (38), on the other hand, state that their biochemical reactions with "aberrant coliforms"l varied with the temperature of in- cubation. Observations of 221 normal coliform strains led Levine (25) to conclude that a 30°C. incubation tempera- ture would yield more positive reactions than 37°C. in Voges-Proskauer tests of such strains. The following study was made on a large number of l ”Aberrant coliforms" as used by Stuart, Mickle and Bor- man (38) includes those coliform bacteria which produce less than 20 per cent fins in lactose in 48 hours at 37°C. u ' .. - 3;..“1 .~ r #1335}; ' ‘ a - e 375‘5‘Mu ‘ .. Q If”. . 0%” .u T) U‘FQIM‘AE‘E‘ typical 37°C.-lactoee-fermenting coliform cultures to de- termine the effect of 30°C. and 37°C. incubation tempera- tures on three biochemical tests, citrate utilization, indole production and the Voges-Proskauer reaction. The Citrate Utilization Test The citrate utilization test was proposed by Koeér (20)(22), who found, in 1923, that Aer. EEEEEEEEE could utilize the citrate radical as a source of carbon and pro- duce visible growth in a chemically defined medium in which citrate supplied the only source of carbon. Each. cell, although multiplying in such a medium, shows no visible growth until after seven to fourteen days (33). The Tvst for Indole Production The production of indole from tryptophane is charac- teristic of EEEB- ggli (36). Ehrlich's oriQinal test for indole production was mediiied by Kovécs (1928), and fur- ther simplified by Ruchhoft, Kallas, Chinn and Coulter (33). Ruchhoft gt 3;. found that this test for differentiating Esgh. ggli was an extremely sensitive one, and tLat the indole once formed in a culture medium was very stable. The Voges-Proskauer Reactionz £25. aggggengs ferments glucose with the formation of acetylmethylcarbinol. In the presence of atnosphcric oxygen ._. caus ic al'uli oxidizes the acetylmethyICarbinol to diacetyl, ZNamed for Voges and Proskuuor who first observed the reac- tion in 1898. which condenses with certain constituents of peptone to give the red coloration characteristic of the Voges-Proskauer reaction (24)(36). Unfortunatelm the red coloration devdnps very slowly, and it was customary to wait 12 to 24 hours, or longer, after addition of the hydroxide, before recor& ng results (24)(33). Howeveru in 1936, Barritt (6) suggested for a more delicate test, the addition of q-naphthol and a change in the concentration of the potassium hydroxide. This modification permits reading of the test in two to four hours after the addition of the alkali. Procedure Three hundred and thirty coliform cultures isolated from milk and cream in the coliform index survey were used in this study. Each of the cultures fermented lactose with gas formation within 48 hours at 37°C. The temperature of the 37°C. incubator was held during this study at 37.000. or 37.5°c. 10.50, the 30°C. incubator at 30°C. 11.0 Air temperature at the top of each incuba- tor wus recorded as the incubator's temperature. Neither incubator had a fan. Koser's citrate medium3 was used for the citrate util- ization test, tryptone broth3 for the indole production test, and dextrose dipotassium phosphate medium3 for the Voges—Proskauer reaction. All three test media were seeded at room temperature from agar3 slant transfers of the stock cultures. After 3 See apeendix for formulae 11d p‘rparation of media. seeding, the tubes were shaken and placed in the 30°C. or the 37°C. incubator. Tubes of Koser's citrate medium showing visible growth (turbidity) in 24, 48 or 96 hours incubation were recorded as positive, but only those tubes which did not show visible growth in 96 hours were recorded as negative. Two or three drops of Kevacs amyl alcohol indole re- agent4 were added to each tryptone broth tube after 48 hours incubation at either temperature. A cherry-red ring form- ing after a few minutes at the surface of the mefi.um indi- cated the presence of indole and a positive test. Forth-eight hours incubation at both 30°C. and 3700. was also used for the Voges-Proskauer reaction. After in— cubation, 0.6 ml. ofcrnaphthol reagentff and 0.2 ml. of 40 per cent potassium hydroxide4 were added per ml. of medium to each culture tube. The develOpment of a crimson to ruby- red color within two to four hours indicates in this ”est 5 the presence of acetylmethylcarbinol. Vigorous sharinc speeded the formation of the red color, and tubes could be read as positive (red color), negative (no color) or partial (inbetween shades of orange) within one—half hour. Results of all tests were recorded as follows: t' +-(positive), - (nega 1ve) or 1 (partial). Of the 330 cultures tests 238 grew visibly in Koser's DJ citrate nocium at both 3000. and 3700. Seventy-four cul- _- _ u. _ cw —- _ — .4 .— 0‘ — u — c— — See 3p_ennix for the composition of reagents. tyres showed no visible growth in four days at either tem- perature. Thus, 312 or 94 per cent of the cultures gave the same citrate utilization test at either temperature. The remaining 18 cultures grew better in the citrate medium at the lower temperature, all 18 showing decided growth at 30°C. and only one a slight visible growth at 37°C. See Table 1. A much smaller percentage of the cultures produced indole. Of the 90 cultures which produced indole at either temperature,89 did so at both temperatures and one at 30°C. only. No partial reactions were recorded for this test, and the number of reactions agreeing after incubation at both temperatures was 329, or practically a 100 gar cent agreement. Results are tabulated in Table 1. For the Voges-Proshauer test, 191 cultures produced enough acetylmethylcarbinol to give positive reactions when incubated at either temperature. One hundred and thirty cultures were negative reactors after 48 hours incubafi.on at both 3000. and 37°C. From Table 1. it can be noted that six positive reactors were positive only at 3000. and one only at 3700. However, 321 or 97 per cent of the cultures tested gave the same reactions after both 3000. and 37°C. incubations. In the first 110 cultures studied, 3700. incubation tests were made soon after the isolation of the cultures; 300C. tests were made four to six months later. On the re- maining 220 cultures, the BOOC. and 37°C. tests were made at the same time and were seeded from the cane agar slant transfer of the stock cultures. Tests were repeated on cultures not giving the same results after 30°C. and 37°C. incubation. The final results on all repeated tests were used in tabulating the results of the Citrate, indole and Voges~Proskauer reactions (Table l) and in typing the cul- tures (Table 3). Results of the repeated tests made on the 220 cultures, whose original 30°C. and 37°C. tests were made simultaneously, are tabulated in Table 2. Stuart 9 al., as quoted by the 9th Edition (unpub- lished) of the éiasiezé flatness £22 the Erasiaaiiss 2E dais: groupse, £95. aerogenes, Esch. ggli, and Intermediate, on the basis of the three biochemical tests: citrate utiliza- tion, indole production and the Voges-Proskauer reaction. Stated in the order just mentioned, these cultures giving +++ a +~+ and -—+7 test reactions are classified as Aer. aeg- gggneg by Stuart. Those reacting as-A» are classified as EEEL' 2211 by him, as are these cultures giving negative re- actions in all three tests but which give positive Eijkman testsg. Most of the -~~ type cultures in this study gave negative Eijkman tests in Study II. many of them after 30°C. incubation gave the reactions of typical Intermediates Accordingly,the --- type culture has been considered here _ — _ _ ~ -I- h— .. _ — -. — _ u— .- 6Stuart gt al.(39) in the original publication point out that this grouping is one of convenience and carries no taxonomic recommendations. 74-stends for a positive test, - a negative test. 8 See Study II for a discussion of the Eijkman Test. -11-, as belonging to the Intermediate group. Stuart considers the Intermediate group to be composed of two types,+—— and ++— . In addition to those two types, the —-— type al- - 1 ready discussed and the type-+r not mentioned in the §EEE§' Egg Mgthgds, 9th Edition, have been included in the Inter- mediate group in this study. With the two exceptions stated, the 330 cultures were classified by Stuart's classification as given in the 9th Edition (unpublished) of the §tandagd ' 9 According to their reactions after incubation at the usual 37°C. temperature, the cultures were grouped into the eight possible types arising from the three tests. The types were then arranged into the‘three groups of Stuart's classification. One hundred and ninety-three were classi- fied as Aer. aegggenes, 58 as Eggh. £211 and 79 as Intermed- iate by the 37°C. incubation reactions. After 30°C. incubation, 21 cultures gave different re— actions to one or more tests, and the classification of seven we affected. Three Eggh. 3311 were transferred at this temperature to the Intermediate group. One Intermed— iate was classified as Eggh. ggli, and three Intermediates as LEE- 223232222‘ Classification based on 30°C. incubation typing would have been changedfio the following: fie I": 2252' ggnes, 196; Eggh. golfi,-5t, and Intermediate, 78. See Table 3 for typing and classification of the cultures used in Study I. 9This classification was also followed in classifying the cultures used in Study II and in Study III. Summary Three hundred and thirty typical 37°C.-lactose- fermenting coliform cultures were studied; 312 gave the same citrate utilization test, 329 the same indole test, and 321 the same Voges—Proskauer reaction, whether in- cubated at 30°C. or 37°C. Twenty-one cultures differed in one or more bio- chemical test reactions following 30°C. and 37°C. incuba- tions. Seven of these would have had their classifica- tion affected by the differences. A tendency was exhibited in all three biochemical tests for more cultures to produce positive reactions when incubated at 30°C. than when incubated at 37°C. .Auuoe asapnamv a “Agnes obwuumezv.s “Apnea o>flpamomv + .n. ooH m.o «.mn n.o m.o m.o m.em ammo use on» m ona a n .n Hod Monasz ; H l I u I I + H + I H + I + H H .+ + woumoa Hdpoa or” 000 059 000 arm 00” Ohm con Ohm con Obfl Don Obfl com or” con Obfl 000 mwkdpdso soapodom hosmMmonmImouo> OOH a.ms n.o o.sm ammo Hem one ovm .H mm umnssz fl I I H I I + H + I M + I + H H s+ + crummy Haney can eon opn con can con can eon can eon can con can con can eon can con mohdpaso . qofluozvoum oaousu ooa «.mm n.o m.m H.m> psoo Hem on» «5 H pa mom Hansen n I I n I I. + u + I H + I + H u u+ + eoumma Hdpoa can con can con epn eon can eon obn oon ova can can oon can eon can con weuzaaso soapmuaaaen opeuuuo apnea asowaozoowm H manna Ha NH «N aaammom dapoa mama wawmcwgv uenasz ++ I+ +.+ ++ + + +. + ++ mcwvsomom mom: came + _Obn 000 _+ 069 com Ohm 000 + can can I + + Obmoon + + DEG con + Ohm con daze QOfipoeom HoudMuonmauewo> nowposcoum eaoena conpmuaanpa massage amma apnea concomom m candy .uowpoccu HossxmonmImomob use nowpozvonm oaoecfi .coflpduwaflps cashew» "Houuo unakoaaom on» 2% unmanned one can maazmcn peep awowfiennoap esp ea Rowen momhs* a sauce «Hacposm anomHHoo mo nofiusowmflmmwao n canes one Ha H b on on 0 «ma om .Ooon we mensuaso H t b V w v A Iv a t .4 n ell... 33 m I I. II I. II I. II II II I. II II II .I I. I. II I. I. .I I.op I. I. I. am Ha H n o .ooon pm newsman own mm m n mm mm 0H boa om .oosn pm monsuaso I +,+ l I ++ I I.+ I +.. i.I I +I + + + « oaks Haves cadaveSHean Haoo moquoaoa .n04 guano , .nomfi .0009 an and .oosn pa STUDY II THE EIJKMAN TEST Introduction In 1904, Eijkman reported that gsgh. IO 01 blooded animals fermented glucose-peptone bro formation at 46°C. and that that temperature ited or destroyed ether bacteria commonly fen confirmed by four Jnrrecen workers: Chris..1n, 1 Thoma“ err Buli“ {"‘ .I (“~\ ‘- rn" ‘l -“ -'-I»-.. 3‘: .'. . \"VI' . .. . _ ' . n 11. I. irvcs*1"etinus o: the terfits “. - '1: 7....-.l..+..4 “1,. 3:..‘15-“9. "Mn-H...— 1,, up ,, 311?? -'- ‘ ° - — I. . rbe"s".eristir 0’ “a“i ctr-xns .f ?.ch. cc] 4. _._—... .— _— warm-blooded animal feces. Leiter furthe. sumo“... . .-. i from warm- th with gas either inhib- nd in wz+er (33}. isolated from .‘.. ..-I--.,.'...~ 6...\.3.- - I "Luv. .-.-_. '|_ .. . . 1,..L', .. - .a' . -'~l ‘ " -. - .- .4. -l-e c'uce ELI :~€J.L.:.L' Z- '1‘ "9' 's'v ea 1.1—. l’:---- v‘. -'I- n- I — é UJU . .. . -. ' .. _ I. .' '- - . ... ., - .. .'. yl u-A. .- . .'i 1,... 5.£.’I..v-. ..-. . ' . - .I. --'r'.1.vt'.o .. - , -— . - . _ L. _ _. _ I. ' - g -. v u - v \ I . , I _ I ,_ 1 t I ‘ ..- r. 1 . . A ..- . .— . _L . . I -I _ -.I . '_ - 4.4-9“ I II. | v . ‘ - - n_ I , ' .l‘ ‘ l' . . . . n - v i} .. . L '\ .I. g .1... _. ._' - " u -\ 1 - _ _. . E‘ v Ab- .. . ,_ ‘ u I v v. 3 .-.”. . ., . . I -. _ . . -I. . _._... . . L - v . '- o L . .. - - \ .1”. V— ‘ “ _- —-'—_.'_ ' n. . . - I . _. _ \. . . . _ -I . I l ggli. They reduced the glucose concentration from 1.4 per cent to 0.3 per cent and added a phosphate buffer. In the original medium, the pH after fermentation was 4.5, whereas in the modified medium it remained at a pH of 5.6. In their modified medium, Perry and Hajna found that all strains of EEEE; ggli tested produced gas after 24 hours at 46°C., and that they remained viable Ior 96 hours or longer. As a result of Hajna's expcriments in fermentation of a large numter of mcnc- and Lieurcharides by 353E. ggli, HaJna and Perry (16} Luhst itrted lactcec fer the glucose in their first meiificaticn Cf Lijkma '5 medium. Later, they changed the protein From ;e,tohe t: trjptuee :74 in 3?21, Lc"5.:, Listei; u-. Yang'. 1;), 4'0- --Lc‘ lu'C. as .rc ct‘imum +:mp'rut re "'- the n-6nIrn ts; . re - ferti g parallel in1.L;¢i'L., 'itn terpzra.tre; in the mod- .um I. :50. ., we. I...:-I.. :. it c.. “..e- I... v.1“; good growth c- Escherichia sir in: in baa. aigin n ne;iu“ and standard lactose broth at LI; lrncr DEL? r:.-.;. at ‘25 higher Eeap:ra.”ru, :uh ;as fl T.ruL11:I {hr 4591;31:2ia 1v’--.A? ;. --.'..—vt¢ .-C: VV- .. £1“ -” 1;- irhih; s _ 'a”.. La L.°I. n‘!‘.“.:II": _I"e.,--U-'.io. I- i". in .'f.‘ .._'_ 4.1. I'L'JLJEI was he‘s i“: 3-3Lrvi'; gear by lile;n it 3;. 3;-2G the I'l'].{‘ip. uiuiilI '2.’ ...“: ul‘i—liffie:t:—JI 3.2%.: .I. u'!'-'b'Cu(J \In). "..'I . -.-.,'r.-l.-¢;.'.. .'. ..L ‘ ““c .‘. E' “.."-, 'II:_, “IT:— e:t:‘i_tei +" fr? ' L ‘. . -. ‘. Q 2;. "‘. I on r‘.- __ -_ I" 1 I. I n .. :- ~I .‘.”;L .-. J" .4. 3...- ill I. .L‘." -:‘-L.....- -:I- van“\-_:; - A Er-l‘ ’abu.._..-l:\;t' a: 1— -— ‘ v""&“ m‘ - V 'VI1“- “' .. . c . . _ . _ . . . .. . . . . . u .n _ r . . . . . . . .. ... . . . . . — ... . stressed the importance of a carefully controlled, uniform temperature in the medium and the need for using a water bath rather than an air incubator. (7) Minkevich, Alexandrov and Soboleva (30), using Bulir's mannitol medium, in 1936 recommended 43Q;to 43.500. incuba- tion for the Eijkman test. They determined that 46°C. did not hinder gas production of Escherichia cultures when in massive inoculations but that it repressed their development when in small numbers In 1939, Hajna and Perry (19) experimented with loner incubation temperatures for the Eijkman test. In their med ium they found that many Aerobactor and Intermediate strains produced gas at 44°C., but very few did so at 46°C. On the other hand, of 1,374 Eggh. ggli strains tested, only five did not produce gas from lactose at 46°C. These workers favor the use of an incubator with a temperature in the medium of 45.50am 46°C. Using a water bath and Perry and Hajna's medium, Stuart 21 2;. (39) found that among a large number of coliform strains, Aer. agggggngg and Intermediates seldom produced L‘- gas at 45.500. and that sch. ggli seldom failed to do so. To date the Eijkman test HCS had a checkered career in the hands of many investigatorsl. Some condemn it entirely; — — .. _ ... _ .— .— — —-. — _ — — In this introduction, only a few of the many investigations resorted in the literature of the Eijkman test have been mentioned. For a detailed review of the literature on this test, see Leiter (23) for work previous to 1929 and Batty- Smith‘s comprehensive survey (7) of the work from 1929 to 1942. others claim it to be the most valuable single test for the detection of typical Eggh. ggli. Apparently the results obtained are influenced to a great extent by the technique with which_the test is performed (7)(35). One most import- ant factor in technique is temperature of incubation, and the Optimum temperature seems to vary with the medium em- ployed. Moreover, it is influenced by the length of the lag period between the medium‘s planting and its rise to in- cubation temperature (so)(35). The 9th Edition (unpublished) of the §t§nd§£d pgtpggg incubation temperature of 45.500. and Perry and Hajna's modified tryptose-lactose medium for the performance of the Eijkman test. It was the purpose of this study to determine how the Eijkman test performed as recommended by the §tanda§d gath- eds, 9th Edition, would correlate with the type and classi— fication of a large number of coliform strains. Type and classification were based on citrate utilization, indole production and the Voges-Prcskauer reaction. Procedure The procedure followed was that recommended by the 9th Edition (unpublished) of ihe iieeiizi Esiheis :2: the seed- ination of water did §gwgg§ (2). The cultures used in the study were isolated from milk and caeam in the coliforn index survey and had been growing on artificial media for two weeks to several months. All . . . o . produced gas from lactose within 48 hours at 37 C. PreVious - 17 _ to testing, fresh agar2 slant transfers were made of the cultures. The transfers were incubated 24 hours at 37°C. be- fore plantings were made from them into the Eijkman medium. Perry and Hajna's tryptose—lactose modification of Eijkman's original medium was used.2 It was tubed in 10 ml. quantities in Durham fermentation tubes. The tubes of Eijkman medium were seeded at room tem- perature, shaken,and immediately placed in a 45°C. water bath. The temperature of the bath was held between 45.00 and 45.500. Throughout the test the water level was maintained above the level of the medium in the tubes At first,an attempt was made to incubate the cultures in a 45°C. air incubator, but with very irregular results. The reactions reported in this study are those of water- bath incubated cultures only. Gas production, the criterion of a positive test, was determined by displacement of the medium in the inserts of the fermentation tubes. Any amount of displacement (from a bubble to 100 per cent) was considered as positive. Read— ings were made after 24, 48 and 96 hours. These cultures not producing gas in 96 hours at this temperature were re- corded as negative. Results and Discussion Over a period of several months, ”ijkman tests were made on 320 coliform cultures. Sixty-one of the cultures fermented lactose with gas formation at 45°C. The remaining 28cc appendix for formulae and preparation of media. 259 either failed to grow in the modified Eijkman medium at this temperature or, if growing, did not produce gas. Only 19 per cent of these coliform cultures, isolated from raw and pasteurized milk and cream, produced positive Eijkman tests. Typed according to their reactions to the citrate ut- ilization, ind>le production and Voges-Proskauer tests, 65 cultures (20 per cent) were classified as flesh. ggli; 186 (58 per cent) were classified as Aer. 233352333 and 69 (22 per cent) as IntermediateB. Only 51, or 78 per cent, of the 65 Eggh. ggli cultures produced positive tests. Fourteen cultures in this group were negative to the Eijkma; test. On seven of these four- teen, the test was repeated, but with the same negative re- sults. Among the 186 435 agrorgnes cultures, five produced gas at the Eijkuan temperature. All Jive were citrate pos- itive, indole negative, Vogos-Proskauor positive type cul- tures and were isolated at one time from two milk samples v fle possibility d- of the same dairy. They therefore present of originating from a single source. '4. In the Intermediate eroup, t is interesting to note that three of the five noeitive Eijkman cultures were the only three indole producing cultures in the group. One culture negative to all three biochemical tests4 and one — —. .... _ .... _ _ — _ ..- _ .- See St'dy I for a discussion of the three tests 1nu the modified Sthrt gt 3;. method of classification. 4This culture would have been classificd by St art gt El' (2)(39) as an Jsph. gall. l9 -tive to the citrate test only, produced the other two ositive Eijkman tests. The Eidkman reactions of the 320 cultures are tabul- ated in Table 4. With the 255 A25. agggggggg and Intermediate cultures included in the study, the Eijkman test presented excellent correlation. However, among the Eggh. ggli only 78 per cent gave positive Eijkman tests. Williams, Weaver and Scherago (42), working with the Eijkman test in 1933, experienced certain changes in the fermentation characteristics of pure strains of £523. ggli after several transfers on artificial media. To some ex- tent, the property of gas production we .s lost. At that time, these workers s'ggested over-cult iv ati1on on artifidz=l media as a possible explanation of the poor results obtained by many workers with the Eijkman test. Although Leiter (33) had only four years pPeViOLIS 1y stated that the Eijkman re- action was a fairly constant Characteristic of pure strains of Eggh. Egli, the remarks of Williams, Weaver an; Scherrro “u -. merit consideration in a study in which 14 of 65 gsgh. gel; cultures did not produce gas from lactose at 45°C. Conclmnions The Eijkman test performed as ‘ocomnendod by the 9th Edition (unpublished) of the gjfindagd EEEEQQE £23 tflo EEEE‘ ination of Water and Segra“g (2) seldom produces positive reactions Tron cultures in thc Aor._agrgggng§ or Intermed- iate groups. On tht other hand it does not prOuuce pos- itive reaction: from all 533;. ggli cultures. .novao pun» an .mvmop nosdxmonmnmomo> use oaovsa .opdkpao on» op muowpoeom * am an“ ma on «a m oea an o>opmmez caexnflm mondpaso oa an a a m a an n o>opflmom nesxnom mouspaso ooa can ma a m be no m nea mm eopmoa monspaso w: ++| u++ «1+ |+ l +..1 +|.+ ++L.+ mama ammo 9mm Haves ovdficoaheunu “Hoe .nomm «anemones nopoepouod soameHMHmmeao moussaso anoeflaoo om» Mo meoopoeem essence v wands STUDY. III PASTEURIZATION STUDIES Introduction .Early work on the resistance of Eggh. sell to heat indicated that this bacterium would be killed in much less than 30 minutes at 62.5°C., a time and temperature commonly employed in pasteurization of milk and cream. Val Geuns reported that it was killed in one minute at 62.5°C. (4), Kitasato and Weisser, at 60°C. in fifteen and ten minutes respectively (34), and Chantemesso and Widal, in five min— utes at 60%.to 61°C. or in fifteen minutes at 590C. (34). However, in 1906, DeJong and DeGriff described heat— resistant strains of Eggh. cell which were not killed in 30 minutes by temgeratures below 70%;to 72°C. (4). Shippen (34» in 1915, isolated coliforu bacteria from raw and pasteurized milk samples. After determining the thermal death points on some of the cultures, he concluded that the thermal death points of these bacteria varied in different strains. Ayers and Johnson (4) at about the same time heated EEEE' ggli cultures, massively inoculated into milk, for 30 minutes at increasing tenpcratures. Twelve cultures (7 per cent) survived 62.8°C., but only one survived 65.60". In 1924 (5), these workers reported further pasteurization studies in which thvy determined that the majority of Eggh. 321i were killed at temperatures lower than 57.200. when held for 30 minutes but that a few resistant cells were destroyed in 30 minutes only by temperatures above 65.600. Finkelstein (16), in 1919, stated that pasteurization by the holding process destroyed practically all coliform bacteria in milk. Yet he reported an average survival of 42 per ml. As early as 1903, Ringeling had attempteddo use the presence of coliform bacteria in pasteurized milk as an indication of imprOper pasteurization or subsequent contam- ination (4). With the report of Ayers and Johnson (4) in 1914-15, showing that a few coliform strains had the ability to survive pasteurization time and temperature, the group came into disrepute as an index of proper pasteurization and subsequent handling of dairy products. However, in 1927, Swenarton (40) suggested that a test for coliform bac- teria in pasteurized milk could be used to goodadvantage in checking up on dairy plant performance. Beavens (8), in 1930, examined 100 samples of pasteur— ized milk, taken from the vat after holding at approximately 62.800. for 30 minutes. In 32 per cent of the samples, coliform organisms had survived the pasteurization. Yet, in 1932, thrady and Langevin (29) found that the coliform group was practically absent from one m1. quantit— ies in the pasteurizing vat after holding but that they often reappeared in the milk because of contamination in the cooling or bottling process. Fabian and Coultor (15), performing laboratory pas— teurization in 1930, found no colifor; cultures surviving . o in skim milk for 30 Minutes at 62.8 C. Stark and Patterson (37), in 1936, inoculated 505 pure Escherichia and Aerobacter cultures into milk in densities of about 100,000,000 bacteria per m1. A11 505 were destroyed in the milk by 52.800. for 30 minutes. These workers stated that their results with the 505 cultures tended to show that the presence of coliform bacteria in prOperly pasteurized milk is mainly due to recontamination. That same year, Chilson, Yale and Eglinton (13), after working with 271 samples of raw and pasteurized milk, felt that the test for the coliform group should supplement the agar plate count for the detection of contamination of pas- teurized milk. They claimed that properly pasteurized milk that had not been recontaminated should have no coliform organisms in one ml. quantities” The 8th Edition of thc éissésaé gatheés £23 £22 £532- 1231122 22 221E; Ereézgis (3) reflected, in 1941, the re- ports of these later investigators. Coliform organisms, it stated, were practically eliminated from milk and cream by preper commercial pasteurization. The presence of these organisms in one ml. samples of pasteurized milk was consid- ered cause for suspecting improper pasteuri ation or subse— quent contamination. In the pasteurization investigations previous to and including 1930, isolations of coliform bacteria from the pasteurizing vat immediately after holding were reported, and strains of coliform bacteria with the ability to sur- vive pasteurization time and temperature were described. If coliform bacteria do survive pasteurization in quan- tities that could be detected in laboratory tests, the value of their presence would be limited as an index of proper pasteurization, and they could not be taken as evidence of contamination of the milk after pasteurization. In the coliform index survey of the bottled milk and cream of fourteen dairies in the Lansing milkshed, a large number of coliform cultures had been isolated, in most in- stances from pasteurized samples. This study was undertaken to find out if such coliform cultures could survive pasteur- ization at 62.500. for 30 minutes and,if so, in what degree they might possess that ability. Procedure The cultures used in this study had all been isolated in the celiform index survey. Three hundred and ninety_one cultures were included in the study. One hundred and ninety- nine had been isolated from milk, and 192 from cream. Three hundred and seventy-four came from pasteurized samples, and only 17 from raw milk and cream. All the cultures fermented lactose with gas formation at 37°C. within 48 hours. Typed on the basis of their reactions to the citrate utilization, indole production and Voges-Proskauer tests, the cultures . were classified as follows: $93. agrgggngs, 237 (61 per cent); Intermediate, 91 (23 per cent); gsgh. 221i, 63 (16 per cent)} After isolation, the cultures were stored at room 1570 Study I for a discuSSion of who three tests and the meJI ? of classification Iollowed. (temperature on nutrient agar2 for periods ranging from two weeks to several months. Previous to laboratory pasteur- ization, each culture was carried through a series of three '24-hour nutrient broth2 transfers at 37°C. From the last of these broth transfers, the cultures were planted into autoclaved skim milk for pasteurization. Pasteurized skim milk was obtained from the college dairy the day it was separated. It was autoclaved that same day at 12 pounds pressure for 15 minutes. Occasionally the milk was autoclaved at 15 pounds for 10 minutes. In no case did the milk show extensive caramelization or the presence of coliform bacteria. The milk was tubed asepti— cally in five m1. quantities in large (20 mm. diameter) sterile cotton—plugged culture tubes and brought to temper- ature in a constant-temperature water bath. A deep water bath in continual agitation was used in the pasteurization work. The thermostatically controlled temperature varied t0.5°C. At all times the water level was above thqhevel of the milk in the tubes. One ml. or one-half ml. of a 24-hour broth culture was pipetted into ihc heated milk tube. The tube was immedi— .L ately replaced in .e 62.500. £0.50 water bath and held at that temperature for 33 minutes plus or minus one minute. ized milk were removed. One ml. was placed in a lactose brothgfermentation tube, and an agar2 pour plate was made of the other. The lactose tubes were read a cer 24, 48 and p see a:pendix for lCPfluch and greuarqtion 0; media. 96 hours incubation at 37°C. The agar plates were counted after 48 hours at 37°C. Survival was indicated by colonial growth on the agar plates and gas formation in the lactose tubes. Eight cultures were pasteurized in decreasing densities at 62.5°C., three at 69°C. to 70°C., and four for increas- ing time intervals. In these experiments, a series of dilu- tions were made from the 24-hour broth culture. One ml. of each dilution, including the original broth culture, was planted in five ml. of milk for pasteurization. Results To eliminate the less resistant cultures from the group of 391, all cultures were planted in excessive quantities (1 ml. or 0.5 ml. of a 24-hour broth culture per 5 ml. milk) for the initial laboratory pasteurization. One hundred and seven cultures were eliminated by this process; 284 (73 per cent) survived the pasteurization under these abnormal conditions. Correlating the survivors as to type, it was found that 76 per cent of the Aer. EEEQESBEE had survived, 55 per cent of the Intermediates, and 86 per cent of the aggh. 3311. These percentages would indicate that gsgh ggli is slightly more resistant to pasteurization than the other two groups. Eijkman tests had been made on 306 of the 391 cultures. It is interesting to note that among the survivors were 52 of the 55 positive Eijkman cultures in the group. (Table 5) No correlation was f0 nd among the survivors with their original source. Cultures isolated from flilk showed about :- . from raw or pasteurized .-nsity of coliform organisms "ect the resistance of cultures The cultures which were used tion'studies had all survived the. teurization. Originally they were and cream of seven different dairi from pasteurized samples. Because the densities of the laboratory pasteurization were gre . T . samples. Neithe '"13 in the sample seem to isolated from it. for further pasteuriza- 'nitial laboratory pas- isolated from the milk es. All but one came bacteria in the initial atly in excess of those occurring normally ized in decreasing 140,000,000 to 275 For each ization. ing pasteurization in raw milk, eight cultures were pasteur- densities. The densities varied from bacteria per m1. of milk before pasteur- culture, the number of bacteria surVIv— increased with the number present. when the logarithms of the numbers of bacteria per ml. surviving were plotted agains t the logarithms of the initial counts, they fell along straight line curves. Bigelow (10) finding that thermal death time curves for spore—forming bacteria were logarithmic and parallel, suggested that for also be logarithmic and approach parallelism. non-spors-forming bacteria the" might The pasteur- ization survival curves of the two cultures which are plotted on Graph 1 corroborate his suggestion. In Table 6 are the bacterial count tabulated, for each of the eight cultures, per ml. of the greatest dilution showing survival and the count of the next higher dilution in which 1. 12,800 to 5" in which all were killed was from 275 to 1,200 ' 'H:ria per m1. All plantings above 2,800 per ml. showed a. vival, and all below 1,200 per ml. were killed by the pasteurization. By raising the temperature to 69%_to 70°C., but other- wise using the same pasteurization procedure, it was found that three coliform cultures could survive 30 minutes at this higher temperature, but only if present in greater den- sities than were required for survival at 62.500. From Table 7 it can be seen that plantings from 45,000 to 550,000 bacteria per ml. showed survival at the 69%;to 70°C. pas— teurization, but that in one planting of 53,000 all were killed. At 63.500. plantings from 2,800 to 15,500 survived 30 minutes. The resistance of four cultures to increased holding time was also ascertained. Cultures were held at first for 30, 45 'and 60 minutes at 62.500. It was soon apparent that 15-minute increments were too small to noticeably affect the Survival of the coliforms. Whereupon, two cultures were held for 3 hours at 62.5°C., withdrawing samples at half- hour intervals after the first hour. In both cultures, re- sistant coliforms were found that would survive 62.500. for three hours or longer. with one culture, a planting of 5,200,000 bacteria per m1. survived for three hours, one of 520,000 for two hours, and one of 52,000 for one hour. (Table 8) I:1;*; 7 I ‘w»” ties. At the same time, the celiform ’ samples were determined. Coliform bacteria from the samples with coliform indices of 100,000* survived the gasteurization, as did one of the two samples with an index of 10,000. Neither of the two samples with indices of ten or less showed survival. (Table 9) The results with the raw milk samples agree with the density range for survival arrived at with the cultures grown on artificial media, although indicating that the artificially grown cultures might be slightly more resistant than those in raw milk. Summary Pasteurization studies were made on 391 coliform cul- tures isolated from raw and pasteurized milk and cream. Even when present in quantities greatly in excess of those found normally in raw milk, 107 of the cultures failedho survive 62.500. for 30 minutes; 284 survived. For the eight cultures studied, the number of bacteria surviving pasteurization increased with the number present. Celiform organisms survived in plantings ranging from 2,800 to 15,500 bacteria per ml. but not in plantings ranging from 275 to 1200 per ml. Three cultures survived 69%,to 70°C. for 30 minutes, but only in densities greater than were required to survive the 62.5°C. pasteurization. *3 . raw milk producers' 30 minutes a‘ 62.5°c. Conclusions Many coliform cultures isolated from milk and cream can survive pasteurization at 62.5°0. for 30 minutes if pres- ent in quantities greatly in excess of those occurring in milk and cream under ordinary conditions. Some cultures are capable of resisting increased pasteurization tempera- tures and times when present in such excessive amounts. A few coliform strains do exist which can survive pasteuriza- tion when in densities that are found in high-count raw milk samples. Acknowledgment I wish to thank Dr. W. L. Mallmann for his suggestions and guidance in this work. .venfiHSoammm monsuaso one me am so eves was who? meme» smaMnHfi+ no om n eosaas meanness nmm Has an ospms> mmsspaso +eon Ham on eossazeamsm accesses eases I .+, name eaaanam ease neaunam spas nonessessoo .nouuo saga ca mpmop Hesdxmonmumom°> end oaovnfl .epsuufio esp op mdoaaosemi. am sea. as as mm m an a as w sedans mouspazo up vmm on ma m n no «m oma Ha nea om mansw> menswaso 00H How am mm m m an no pom NH Had we umufinseumsm mthpaso va08 Ill ++| I++ I..+ n+1 Haven”. +1! +u+ i.+._.+ 0&8 .unoo mawpoa epsaveaumusH «Hoe menewonee .nm< macaw Hem ;: .Aemm mafipdomemmeHo use omhs new: :oHpsHeHAoo aewpwpnasd obwmmecwm QH pcemenm semwyad>fi>nsm :oHpsNHhsmpmem n OHQGH m a. 9 z 0 J a a 0 n4. 9 a I. B d 8 ...... nu .. . ..., Table 6 Pasteurization Survival 62.500. — so Minutes Largest No.3acteria Smallest No.Bac- Culturet Type Eijkman per ml. Showing teria per ml. Test No Survival Showing Survival 545 + + 3 — 325 3,530 380 - +- + —— 10,500 426 + '+ _ 1,000 8,200 435 +-*+ - 275 2,800 1002 —-+~ + 650 10,000 1006 ~ +— + 750 5,200 1013 + —+ — 525 15,500 1014 + -- — 1,200 12,500 Range 275 - 1,200 2,800 - 15,500 Table 7 Survival of Coliform Bacteria in Milk 6 to 70°C. - 30 Minutes * ., Largest No.Bacteria Smallest No.Bac- Culture Type Eigkman per ml. Showing No teria per ml. , Test Survival Shwwing Survival 4:55 + + +* - 53.000 550,000 647‘ +"* — 40,000 400,000 725 "+” ‘ 4,500 45,000 *Reactions to citrate, indole and Voges-Proskauer tests, in that order. ‘ *All cultures were dilution plated except 647 and 723. ‘Culture 647 was isolated from raw milk. Table 8 Survival of Coliform Bacteria for Varying Lengths of in mm: at 62.5%. Time. Culturef Type Eijkman No.3acter1a Survival for 2 Test per ml. 1 1% 2 3 hrs. 435 +++* — 170,000,000 + + + + + 17,000,000 + + ’ + + 1,700,000 ' + ' ‘ + 170,000 — (0 — not run 1013 +-+ — 52,000,000 + + + -+ + 5,200,000 r + + + + 520,000 + + + not run 52,000 + not run **2Explained under Table 7 K” Survival detected in lactose broth, but not on agar plate. Table 9 Paeteurization Survival of Coliform Bacteria in Raw Milk - Producers' Samples. Samples Coliform Coliform Bacteria Sur- Bacteria viving Pasteurization per ml. 1 ml. 1“ 5 ml. 1 10,000 - - 2 10,000 (+) + 3 10 - - 4 100 .000 + - .. 5 less than 10 - - 6 100,000 + + + Q0 was not able to confirm the presence of coliform bacteria. APPENDIX MEDIA AND REAGENTS Media and reagents were prepared according to the sissSers flatness £2: the Esseieeiigs 2L Isis: 222 Egress» 8th Edition, 1936 (l), and 9th Edition (unpublished) (2) with the exception that sodium chloride (0.5%) was added to the nutrient agar, tryptone broth, nutrient broth and lactose broth. - Formulae of Media Hei£i221_ieer Agar 15 gm. Sodium chloride 5 gm. Beef extract (Bacto) 3 gm. Peptone (Bacto) 5 gm. Distilled water 1000 ml. Koser's Citrate Medium The dehydrated medium, Bacto citrate medium, was used for most citrate utilization tests. Occasionally the med- ium was prepared according to the following formula: Sodium ammonium phosphate (microcosmic salt 1.5 gm. Potassium dihydrogen phosphate 1.0 gm. MagneSium sulfate 0.2 gm. Sodium citrate (crystal) 3.0 gm. Distilled water 1000 ml. Errnieas_§£222 Tryptone (Bacto) 10 gm. Sodium chloride 5 gm. Distilled water 1000 ml. Dextrose Dipotassium Phosphate hefi.um Proteose-peptone (Bacto) 5 gm. Dextrose, C.P. 5 gm. Dipotassium phosphate 5 gm. Disiilled water 1000 ml. & -—I—— -—- 5 Beef extract (Bacto) 3 Peptone (Bacto) 5 5 0 'VLLactose, C. P. gm. Distilled water 100 ml. | Bassist: Kovéca Amyl Alcohol Indole Reagent p—dimethyl-amino benzaldehyde 5 gm. Amyl alcohol 75 ml. Hydrochloric acid (conc.) 25 ml. §:!anhthgl_fiaasaai 5, solution of d-naphthol in absolute ethyl alcohol. _(95% ethyl alcohol was used rather than absolute). 40 Per cent Potassium Hydroxide 40 aqueous solution of potassium hydroxide. 10. ll. LITERATURE CITED American Public Health Association and American Water Works Association. Standard Methods for the Examin- ation of Water and Sewage, 8th Ed:210. American Public Health ASSOciation, 1936. American Public Health Association and American Water Works Association. Standard Methods for the Examin- ation of Water and Sewage, 9th Ed. (unpublished) American Public Health Association and Association of Official Agricultural Chemists. Standard Methods for the Examination of Dairy Products, 8th Ed:75. American Public Health Association, 1941. Ayers, S. Henry and Johnson, W.T. Jr. Ability of colon bacilli to survive pasteurization. J. Agr. Res., §:401, 1914-15. Ayers, S. Henry and Johnson, w.T. Jr. Studies of pasteurization. XI. The "majority" and "absolute“thor- mal deathpoints of bacteria in relation to pasteuriza- tion. J.Bact., 9:279, 1924. Barritt, Maxwell M. The intensification of the Voges-Proskauer reaction by the addition of d-naphthol. J.Path and Bact., figzdél, 1936. Batty-Smith, C.G. The Eijkman test for faecal coli in the bacteriological examination or water supplies. J.Hyg., £2355, 1942. Beavens, E. Arthur. The Escherichia-Aerobacter group as an index to proper pasteurization. J.Dairy Sci., l§z94, 1930. Bergey, David H., Breed, Robert S., Murray, E.G.D. and Hitchens, A. Parker. Bergey's manual of Determina- tive Bacteriology, 5th Ed:388. The Williams & Wil- kins Co., Baltimore, 1939. Bigelow, W.D. The logarithmic nature of thermal death time curves. J.Infect.Dis., gg:528, 1921. Breed, Robert S. and Norton, John F. Nomenclature fer the colon group. Am.J.Pub.Hoalth, 31:560, 1937. Brown, J.W. and Skinner, 0.3. Is the Eijkman test an aid in the detection of focal pollution of water? J.Bact., ggzlsg, 1930. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. Chilson, W.H., Yale, M.W. and Eglinton, R. Detect- ing rccontamination of pasteurized milk by bacterio- logical methods. ' J.Dairy Sci., 12:337, 1936. Difco Laboratories, Inc., Detroit, Michigan. Man- ual of Dehydrated Culture Media and Reagents, 7th Ed., revised251, 1943. Fabian, F.W. and Coulter, E.W. Significance of colon-aerogenes group in ice cream. J.Dairy Sci., 13:273, 1930. Finkelstein, R. Occurrence of the colon-aerogenes group of organisms in raw and in pasteurized milk, and its significance. J.Dairy Sci., 2:460, 1919. Hajna, A.A. 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