THE IMPORTANCE OF SLOW LACTOSE FERMENTERS IN WATER ANALYSIS Thesis {or the Degree of M. S. MECHIGAN STATE COLLEGE Edgar Walton Kivela 194? THESIS \— < V -..4 .' 1" .. ‘1' . 1.5! ‘ 'm .- 9- Cain P’- .V ‘, ‘ . 0 . ’ A .s. ' "_ » . "$31k, ”r. h- - . ,1; 7 v ADJ. w $2.515" . M -5 4. I . ‘7 ., , 1'9: A $646 \ll 3 ‘. I . .'I ”‘1‘ 'vb 23" ’ k .j OM u) ‘rfign .- V .,j ‘ .I—l ‘32-’95 L. . . «7.1g ,fiu . _ 4:. . . :3" . x ‘ .. 1‘-' ' I .-4‘ 0‘. . ' '_ A 4 ' 5‘ . . , .. ‘J'.’ .19}. I‘ n “i; . .. 1" {r} . v [T‘Iy~.’ T} .f ‘ . ‘r:‘-'\{"‘\: t“ 4 . « w I-” . fl 3: -( r. I‘ Q . ‘ < .‘u . '.°§". ,"- ...~,_."L _' . -' ..' 3.x“. '1> 3'4. .‘l - J . .l ‘ H 1'5“sz 3"» ' 1w ...,. . .. ' ‘15“: t " 14... ';‘ '-I- ‘ ‘1 4.... "It” . 1‘ .1 l A “5.1-5.5 flu A: A 7'." . I l‘\:h‘,% .' . 9" V a f V‘, ‘ . ' k" , 1'.“ ‘ "1' ‘iv 1 # . ' .I. '2’ r" ‘ a' ‘.k 'I’l' O *} '_. (12;. - "a ‘21:. v '- ‘ \J ' I . 1 "k” I. p .w'J-R' jf'" I “ r ' ‘ . .. l ' ‘ " I , l h At”: ”Lfiz‘fi'gfafiét‘ .3;{*' iii], if”, ‘ :t' 5-” \ . E THE IMPOHTnNCE OF SLOJ LACTOSE FLRMLNTEHS IN WATER by Edgar Jelton Rivela A TnESIS Submitted to the Graduate School of Michigan State College of Agriculture and Applied Science in partial fulfilment of the Requirements for the degree of MnSTER OF SCIENCE Department of Bacteriology 1942 TH E813 I wish to express my sincere appreciation to Dr. w. L. Mallmann by whose able assis- tance this thesis was made possible. he C? at") £3 ”My «A. Red -w\ l‘iIo‘l‘oiiICnL bxxbndfiuUhn many men have reported the resence of yecillus coli t , .L in the intestinal and urinary tract oi Jen and in e few instances tne; have been noted in the feces of horses and cattle. Gilbert and Lion (1895), apleer to have been the first to describe iarecolon bacilli. Morgan and Ledinghem (1909), encountered such types in human feces end divided them into three groups. On thy basis of cul- tural cheracters, they re;orted them es numerous in cases oi dizrrhee but regarded them as of little pathogenic significance. on the other hand, Gyergy (1920) suggested that the) i,leged a cansiderable kart in dierrnee in both men and calves. Jones, Urcutt, and Little found, during a study of infectious Cierrnea in cows, matile Gram negative rods which failed to attacn lectose. Iheir numbers varied greatly; at times they made up 90 percent of the organ— sms, at others the, Lore present in relatively small numoers, and in neny cases no such colonies tere iound. In one instance they Lere found throuénout the intestine of a cow slaughtered diring an attacn of diarrhea. In earlier worn Jones, orcutt, and Little found many non-lactose fern nters from the intestines oi cows hLViDS diprrhea. in some epidemics all cons had them, in Others very few had them, and in Still later epidemics none were found. Upon examination of healthy cons (50) only one at ,. icel colon ‘ozcilius wee iound snouirw that they are \ \- U l. proba oly not 1res sent in lerJe n1neers in the intestinal tract of normal cows although they may be the 1redomi- natinr types durin3 certain intestinal disorders. L-) 1. Culturally the above three workers found that all strains readily attacaed gllcose, mannitol, maltose, and xylose, with the production 01‘ acid and gas, failed to ferment refiinose, but fermented lactose slowly. In ad- dition all 3eve a ne3ative Vogcs-krosaauer test, 1ositive net: 1yl-ned test, yroduced indole, and failed to liquefy gels tin or rroduce hyd1o3en 3.11pwi . Upon cultivation it was Iound that cetain strains of the aty1ical form could be built up to fast lactose fermenters by ra1id transfer and Others co1ld not. ltwas not possible to r"ain “ROCK them down after they more built up. when put into Durham lactose fermentation tubes, it was Io und that the or 3)anisms grew rapidly but did not produce acid reactions. inis experimental evidence shoved that the bacilli utilized the lactose but at the same time 1roduced alkali, for a time at least, in suificie nt quan— tities to mesa the acidity of fermentation. The indicator in the tunes rerneined uncna3n' ed for tvo or m01e days. Acidity ass firs t noticed at the bottom of the tube, and the reaction gradually CILEH ged, accom1snied by a moderate gas roducti1n. Ten days or more mes re 1ired to change the color of the indicator completely. Un lactose agar 1lates these organisms may readily be mistaken Ior peretyphoid bacilli, especially when freshly isolated and CulthLtUd in glucose, ltc sucrose. They also form smegth trsns1arent colonies on agar plates. Immun0103icolly it mes Iound that members of tne grout difier only in degree rather tnen constitutionally. Clint} SID" 1011‘l‘lON according to dtuert, nicnle, end barman, since so n terminology has occurred in 'he liters- M mucn coniusion ture rel.ting to slow-lactose fermenting members of the coliform group, it was su33ested that tne term ”eberrent coliforms" be used to describe ell Gram negative, non— s1oruleting rtds which ferment lactose teanly at 570 0. They also su33ested a tenative SCytPEtion oi the aberrant tyies into four grou1s besed on s study of more then 10,000 coliforms isolated from mater, soil, miln, feces, end other sources. The classification 1roposed is as follows:- nberrsnt toliforms Strains diIiering from ty1icsl coliforns in rcs1ect to tneir fermentttion oi lactose (producingless then 20 percent gas in 46 hours at 570 C.) I. Micro-mercaenic Coliforms - aberrant coliforms pro- ducing gas from lectose slowly or in small amounts at ther 370 U. or 200 C. (D H. II. rseudomicro-nerogenic Uoliforms - aberrant coli- forns having tne enerscter’stice of the true micro- 5. herog3enic streins st 2370 but snowing; normal lactose s 11Lt1n3 ectivity at 200 c. III. repiilee-bormin3 Coliiorms - aberra nt coliiorms SllOVJiI‘JS the ty1e of dissociation evidenced by t1ct. coli m1ttbile, but net c nfined to the 5enus fiscnericnie. IV. anterogenic toliiorms — eberrent coliforms 1roducin3 ecid but no gas from lactose. Tne existence oi e grouf of non-lactose-iermentin3 coliforms is reco3nise , but this group is not suffic- iently well delineated for sstisiectory discussion. I. micro-nero3cnic Uoliiorms - belong to rerotecter, Intermediate, or sscner icliie 5roups. The produce acid in lectose and ass: a bubble to 101 Icent gee in 48 hours et 570 0., some 20 to 100 Lercent gas in 3 to 9 days st 570 0. acid and gas are groduced more slonly at 200 C. The orgenisms 5ron as well in 24 hours in lectose es ty1ical cultures, but at the end of 24 to 56 hours 'ne lectose be “comes stron Qy elneline. Tiis is iollowed first by ecidificetion of tile bioth in the insert, and then (1 to 4 days later) by a general ecid reection and sp1eer- ane of ges bubbles. 0n nos1n~Metnylenewblue s3: ar mazny Lroduce only white colonies, end when is oleted do not ferment lactose any fester than the Parent colony. If the 1lttes tre left in tne incubator, the centers of the colonies becoue blecx, out no cht 3e occurs in the steed of fermentetion of lactose. 4. Tnese or5enisms have been isol;ted from normal and _betholo3ical.lrrnen feces ix1 :uell numeers a1m1 were almost invariably ecconienied b3 coliiorms Leicn groduced normal 5es amounts. the workers had encountered e33reciable numbers 01" all three strains (nero’eggteg, ll’iterznediete, chiu) in gublic mater sup lies, nostl3 ITO? sen les .9- 4. H- LSCLCI‘ of untreated xttcr. Il. kscudomicro-Lcro3cnic toliiorm - cougaretle to merobecter enc Intermediate strains. Cultures were isolated from veter, soil, end milA, tudnone from 600 fecal samples. The) procuce in lectose broth ecid end gas: some a buetle to lO yercent in 48 hours at $70 0., others no gas in 21 da3s st 570 U. Un L.M.b. agar et 570 U. the3 Lroduce colonies from t33icel coliform down to colonies berely Visible to tne e3e. Large colonies; raised, moist, con— th 1-)- fluent mith darn center, or flat, dry, and discreet 1 large blacn center and hivn metallic luster. smell colonies; similar to excegt the smaller the colonies, Fl 0 *5 C'; (a the less intense the tlacn center, the very small ones bein5 entirely thite. It was discovered the when the flats mes left st room tenierature ior 24 to 48 hours, all colonies became lar3c and t3licul. then tests (Guglicete) mere run on cultures at 200 and 570 0., tney found a warned Variation in re U) ilts. At 200 C. all Cultures ,roduced 20 to 100 tercent was in lactose in 24 to 72 hours, were metnyl- Q .1 fled ne3ative ens Vo5es-1rosktuer yositiVe. ht $70 C. the; produced from a bubble u; to 10 percent gas in 24 to 48 hours an‘ vere tositive to some de3ree in both the methyl- ded and V03ee-1rosnuuer tests. en running growth curves;- 570 C. for 18 to 24 hours the 5rthh mes stationery or on the decline; 200 U. for l8 to 24 hours the grout; mas in the legerithmic phase. They found thet cultures put into preheated lactose (37° ‘.) and incubated st 37° produced acid in only 5 out of 32 in 48 hours and no gas. Others either died or did not produce gas or acid in two techs. The cultures transferred into lactose broth at room temyerature and then glaced into the incubator \eir) produced a bubble to 10 percent gas. This showed that the organisms begen growing before the broth mermed to the 370 temperature. lII. Pepillae-forming uoliforms - may belong to nerobecter, Intermediate, or Escherichia, but the sreeter 1 majority epgear to be Lschericn a. In lactose at 370 U. they may produce acid and small amounts of ges in 48 hours. Later as much as 50 to 40 percent gas mey be produced. These or5enisms, by repeated transplanting, were induced to produce 20 percent gas in 18 to 24 hours. Un E.M.B. agar both bleed end white colonies devel- Op. The black colonies resemble tyyicel coliforms end when picked produce 20 percent or more 5es in 18 to 24 hours. Ihe white colonies in 2 to 5 days form small, black, daughter colonies vmthin the parent colony. The white 6. colony, when Lut into lactose, produces acid an; gas slow- 1y. Better than half of the fecal sanyles collected from 100 cas s of gastroenteritis produced this tyye of aberrant coliiorm organism. hone were isolated from the feces of normal individuals. Facts show tnis t33e to te, probably, the most im- portant of aberrant coliforms. brequently they are not detected by standard hethods of Later analysis since many strains do not produce gas or even acid in 48 hours. lV. nnaer05enic Uoliforms - produce acid in lactose in l to 7 days at 570 u. but no gas. They have a close resemblance to aberthella and Egigella. They were isolated fr m soil, water, cereals, normal and pathological feces. several were found in an outbreak of gastroenteritis. Stuart, nicale, and borman stated that their know- ledge 0: these t3;es was so limited that no su55estion \' A: of their relationship to the coliform group or of their sanitary significance could be advanced. it?“ 0111‘ 1' 1- CE Stuart, Mickle, and borman state that the exact 5nificance of the aberrant coliforms is not H. sanitary s entirely clear. the true microaerogenic coliforms (Group 1) probably have a significance similar to that of com- parable aerobacter, intermediate, ‘nd Lscherichia strains showing tyyical lactose fermentation. whey conceded no 7. significance to the pseudomicro-aeroyenic group(Group II) which is made up entirely of low temperzture-loving members of the aerobecter section together Mlth a few Intermediates isolated from soil, water, and milk, and which they had not isolated from feces in their inves- tigation. Ihey found that the ptpillee forming group (Group ill) could be determined only infrequently in routine coliform estimates in which gas production is used as the criterion for their presence, and the anaerogenic group (Group IV) would not be detected at all. however, they think that these two groups may be highiy significant btcause of their frequent association with gastboenteritis and genito-urinary infections. They state that ”certainly no aberrant coliform giving a positive completed test in accordance with a strict interpretation of standard hethods of water analysis should be disregarded without a further study of its characteristics”. Ziegler states that non-lactose fermenting tnd late lectose fermenting organisms have been isolated from polluted water supplks and from diarrhea patients. The agglutination of, at least, two varieties of such organisns occurred in high dilutions of patients' serum which in- dicates that they are a cause of the infection. During the latt.r «art of a study of an outbreak of diarrhea in I“ A- Springfield, Aiegler found that typical hseh. coli were not isolated from the meter suy;ly, but pathogenic non- J. I lactose fermenting inteStintl types of organisms uere 8. present indicating gollution thich was not detected by standard flethods of tater snel3sis. McCr d3 ste tes tlizt “since the coliforn dens it3 of a filtered,treeted, or filtered-treated water is a measure of the effectiveness tith thich the treatment has elimi- nated disease organisms frum the water, it is reesoneble to attribute equal szniter3 si Qnificsnce to the presence in such water of either t3pical or et3pical coliform organisms, slow lactose ferncnters or rapid. "Slow lactose fermenters are found, althouLh in small prOportion, in fresh feces, in greater proyortion in stored feces, and occasionally in discharges from cases of gastrointestinal disturbance; the gresence of the OF"énlSHS, even in nzturel waters, tnerefore, cannot elwe3s be dismissed as of negligible sanitar signiiicznce. rurt‘ncrmore, since other outanisrns contained in the scmzle may reduce the amount of gas produced in lactose broth b3 t3gicel coliform organisms, the volume of gas produced cennot be accented as a sure indicetion of the t3pe of orge nism present.” Data mere collected b3 accredy on questionnaires to 25 leborator3 workers as to their ideti on the signifi- cance 01 slow lsctose fcrmenters. rifteen vere in favor of including them, six were in fevor but did not, one fzvored L cludi1n5 those from filtered one treated meters but not from natural, end three were not in ft vor 01 including them at all. Herr states that blemsha's “bacillus P" is a good deal line slow lactose fcrmenter since it yroduces onl3 acid in lectose uith smell amounts of gas if en3, and sometimes neither. bacillus r is said to be common in the feces of gestroenteritis patients. pulsnejy and bmith state that the}, feel that they ”must recognize the widCSQreed distribution of such coliform organisms \slon lactose fernenters) in untreat- " o 4" berg, state. ed waters of eppsrentl3 satisfactory seni 1he3 may or me3 not be detected in the routine examinetions. rhis finding snoild invite caution as to intergretetion of their gresence at an3 time. rht resglts from sanitary survc3s should be crreiully explied and such data cor- r .L' s I related tith bLCtCTiOlJQiCLl finyirws. Lt the szuc time to O n he must recognize thet their non-fecal origin has not 3st been groved end all informetion regarding their goten- tiel guthogcnicit3 should be carcfull3 adoumulzted and evalucted." EthRIMhNTnL £03K as shown in the literature, the incidence of slow lactose fermenters in mater, both treated and untreated, a V l,— I Q Li H. -—~r pJ .L O ‘ is high. This naturell3 censtitutes a serious L P. is s U) the bacteriological examination of water. whis the for the purpose of trgin; to find some solution to this problem. several cultures huve been sent to this laborator3 lO. w.hicn were isolated from rater sznirgles tnLt Led 3esseo the gies mittive Lnd 3ertiell3 confirmed tests in the Stendtrd Lethods of meter nnsl3sis es unpotetle, but hed fLiled to confirm. in some instances the ins yroduction ‘ O .4 in the presumgtive test had been Ls W1 h as 90 to 100 percent, but the organisms LL; still failed to confirm.- Upon isolation of some cultures from these veter emples, it was found thet microsc0picell3 they were dram neeetive rods, and when 3ut into lectoee broth, tne3 produced onl3 a bubble oi gLs in 48 hours, but produced from a bubble to 10 32ercer1t 1n thre ee to five de3s. In an3 cases no gas at all use produced in 48 hours. Since the et33icel reection of these orgenisms oCcurred only after the yertielly confirmed test, it seemed logical that the Eosin-Methylene—Blue agar htd a toxic effect on the ”£8 production. bxjeriments were then conducted » ith a pure culture of Escherichia coli to determine if this were true. A susycnsion of “sch. coli -er slant ves placed in sterile distilled tater une trensplents vere tehen daily end subjected to th stenderd procedure of ueter e nel3 sis b3 Iirs t Llecing them in lactose broth, incubating them st 370 for 48 hours, streaking on E.m.h. agar plLtes, incubating ‘hese et 570 for 24 hours, and then piczing colonies from the plLtes end tren 3s; anting then been into lactose broth. needings were taken on both the grimer3 end seco d: r3 lactose broth tubes at 24 and 48 hour periods. it mes found the there 11. CO was no inhibition of as L. production using a pure culture Since the above method did not 3roduce the desired results, some of the Cultures of atypical orgsnisms uhich had been sent to this laborstor3 were then subjected to epgroximLtely the same 3rocedure. night of these cul- tures mere ylsced in lactose broth and incubated for 48 hours. IanSPlents from these tubes were then streaked on both b.m.B. agar and plein nutrient agar. Colonies were then giched from these 3letes at one, two, and three de3s and gleced in lectose broth. headings were taken on these tubes at 24, 48, and 72 hours, end it was found that there was a definite inhibition, with the exception of a few cases, of their gas production (Table Re. I) An attempt mes then made to increese the gas production of thertcen slow chtose fernenters. It was found that their gas 3roduction could be increLsed sli htl3, but not more than 5 percent at the most. This was accomplished by rapid transfer methods in lactose br th of ph 6.9, i.e., transferring ever3 24 hours to a new lactose broth tube. ahen lactose broth of eh 7.8 was used, it mes found xthet there was a definite inhibition of gas production, probably due to the formation of carbonates b3 the reeetion of the medium with the carbonLdioxide produced. Lactose broth with e ph of 7.2 was then orepared end used, and it .I. was found that there was still a slight inhibition of gas production. 12. Culture {‘3 OO-QCDUILbCA DO (D-QOEUIHhCfl 25% 505;, 107;; 2571 307;; 30% 107:, 20% I Eosin-Methylene-Blue Agar no bub 10% 8% bub bub 10% bub Plain Nutrient Agar day 48 72 15% 20» 25% 50% growth 15% 20% 50% 50% 20% 55% Bib 8% 20% 40% 20% 50% 20% 25% 25% 55% 50%, 55% 50% 60% 50» 40% 15% 20% 50% 40% 15. 24 bub bub 0% 0% 10% bub 0% 10% bub bub bub 0% 2 da3s 48 bub 15% 10% bub bub 20;; 8% 8% 57:. bub 72 870 8'76 bub bub 25% 20723 8% 8% 24 bub bub bub 0W bub 20:36 bub bub bub 8% bub bub bub bub bub 5 da3s 48 1035 8% bub bub 15% 50522 bub 20% 8% 25% 50% 20% 10% ; 4078 10% 5030 Since the attemgt to 3roduce t33icel hsch. coli 0 1 from the slou lactose fermenters b3 regid trensfer methods ‘ .4 failed, the reverse vas tried, and several methods mere ‘ = to tr3 to attenuete e pure culture kt33ieel) of been. {‘1 use coli so thut it would teue on same of the greperties of the slow luctose fermenters thtt had been isolated. The procedure used was as follows:- a pure t3gicel culture of hseh. coli was grown in nutrient broth for 2' hours, and I“. then put on glain nutrient a ur slants. inese, in turn, were then incubated for 24 hours so that a good gronth would be obteined, end then the cultures were scraped off into flasss containing different dilutions of brilliant green, 0033er sulphate, sodium ursenete, and lithium chloride. This 3rocedure yielded a good suS3ension of organisms in each of the flasks. The fleshs were then placed at ver3ing temperatures end tran83lsnts texen from thin at intervels:- brilliLnt green in l, 2, 5, 4, 5, 6, 8, 10, l2, 1:, end 20 days; lithium chloride and sodium srsenute in l, 2, 5, 4, 6, 7 du3s. Transplants from these flasks were yleced in lzctose broth and then run through the standard procedure for water enel3sis. it was found thet the coy3er sulphate was too toxic and killed the organisms in one to two de3s, but up until the time that the organisms were destr03ed the3 did not show any sup- prcssion of gas production. It use also found thut a ten- *3 ereture of 420 U. was too high for the organisms, tie flasks becoming sterile in one to two dz3s. l4. The above exteriment proved that, by the methods used, slow lactose fermenters could not be troduced from a pure tgticel culture Of L‘JSCl’l. coli. ilOX-u'evgr, Hershey and bron- fcnbrenner retort thut they were able to build up the gas production of slow lactose fermentcrs t3 "rspid transfer methodS” and were also eblc to regress it again by per- tielly destroying the lactase b3 the use of sodium suc- Cinote. Shortly after comtleting the above experiments, a water sam,le was received in our leboretory for analysis which produced gas in all live primary lactose broth tubes and also in ell IiVG tubes of the trygtose-lsctose-lauryl- .‘ sulfate broth \T.L.S.) of hellnenn vhicn us run in U) gs tr llcl with the stencrrd isotose DrOtn, upon streufiing F—o from the tubes to E.M.b. agar, however, it was found that from the lactose tubes tno tkyical ACPOo &€P0£SHQ§: one t gicsl fisch. coli, end two styyicel organisms were ob- (.5 P“ 4 .5 teined; and from the T.L.S. broth three t3uicel Lech. coli and two ety;ical organisms were obtained. Colonies were yicged from ell 10 plates and seeded into lectose broth. ill colonies produced gee exceyt two thich were picned from Lletcs streaked from the T.L.S. broth. These pro- duced smell bubbles of gas in 24 hours, and 10 end 15 percent gas in 48 hours. Later ten atggicel colonies were ticked from etch plate showing the slow lactose fernenters erd ylzntcd into lactose broth. Of the 20 colonies tn:t yere flCHCd, 15. 9 of them produced only a bubble of as in 24 hours, and e; in 48 hours one produced 20 percent gas, one lb p.rccnt, and the rest 10 percent or less. All 20 of these lactose tubes were then strcsned on s.M.b. agar snfi incubated for 24 hours. uu,licste colonies were then piched from each tlste, an from the 40 colonies ticked, 10 produced 8 percen gas or less in 48 hours. Leenwhile other water samples had been received at the lsborstory for analysis that contained many slow lac~ tose fermenters. hour colonies were pieked from each glate shoeing atytiCtl colonies, and of the four, two more Ilaced in lactose broth and the other two in T.L.8. broth. Th gas production use recorded at 24 and 48 hours, and it 8 percent gas or less was froduced in 48 hours, a slant culture was made from the tube and nept. in this manner 108 slow lactose fesnenters were collected from 7 water sungles. These organisms were then identified according to the classification of coliform eigenisms found in water as stated in Tepley and milson. to tests were run on gelatin, nor wts the bijkmun test run. A summary of the results is as follows:- ___12' S 0.2;? Licjiisflli Variety l - t cultures Variety lI - 3 cultures Intermedietes Variety I - 60 cultures (5 others coniormed but did not give m.R. test) 16. Variety II - none eerobacter aerogenes Variety I - 29 cultures (5 others conformed but gave h.R. test) Variety iI - 2 cultures Une culture could not be identified according to the rave only a Lositive Voges- ) 3.) classification since it Proskauer test, the rest of the tests being negative. in running these tests, the barritt modification of the VOQCséProsheuer test was used and run on a 40 hour culture of buffered dextrose broth. the citrate test was reed in 60 hours, and the indole and methyl-red tests were run on 64 h ur cultures. in running the routine water azalyses, it was noticed that the average gas yroduction of standard double strength lactose was 21.6 percent per tube, uhile the average gas production of I.L.S. broth was 26.5 percent yer tube. it was also noted that of the 55 ,lates streaked from lactose broth, only 15 of then gave the tyiical metallic sheen; while of those stressed from the T.L.S. broth, 25 gave th~ tgtical metallic sheen. Lnother thing of interest which was noted has the where the growth was heavy on the a.h.b. plates there oc- cur7ed the tyfiical metLllic sheen, but there the organisms were more Spread out and well isolated colonies were iorm- ed, the typical sheen was not yrescnt. Ugon Licking these well isolated colonies, it was found that the; were often the slow lactose fermenters, while, on the other hand, 17. those with tne sheen gave ty'icel lactose fermentation. .5 UOKCLUSIONS From the above exieriments and-obscrvrtions it vould be logical to conclude that the incidence of slow lactose fcrmcnters in meter samglts of both treated and untreated water is suite high. however, even though yrzsent, these organisms do not Show up in the stfindtrd method of water analysis, but, as shown in the literature cited, it is quite probable that their ,resence cen mean fecel con- tamination. thus they should have all the sir of tggicel esch. coli with its regard to water analysis. these etbyicelorganisms may be missed in the LL?- tielly confirmed test then the n.M.B. agar Lletes are streaked from Lrimery lactose tubes rather then from tn 'L.L.S. broth due to the absence of the tygicel metallic sheen. if, houcver, the tyrical reaction is obtained on the heavily seeded iartof tre plate, it is euite yossitle that the well isolated colonies are of the atygical ver- iety. Since tne standard Hetnods of meter analysis states that well isolated colonies shall be picsed for the completely confirmed test, these well isolated colonies, et*.icel, will probably produce little or no gas in 2'11" 0 1- «.1 48 Hours. in addition, these organisms will have been sub- jected to the toxic effects of the n.M.B. eger, and thus .mill have tneir gas Lroduction further depressed, Uonse- qucntly, the chance of detecting the gresence of these slow 18. lactose fermenters is ,uite smell, but the; should be detected since they are an indicator of gollution. I" ‘ _ 3"“ *‘m‘vnIfl $7 1' Q J Jun...) l Urn...) q From the ebove oats gresented, it would seem thtt tne following ;rocedure for tnc becteriolodical examine- r-‘ . v C 2’ lVGIl- '- Q tion of water m; 4t be added to the gethod siretdy Presumptive Test - Double strenétn trbthSC-lLCtOSO— lauryl-sulfete broth should be used since it gives greeter ges production, and the tgpical Lsch. coli :5 O F‘o Q; C‘ b (D 0 reaction on b.M.B. agar has e higher 1 o-l L" tertielly uonfinned rest - if the tygiCtl metallic sheen is gresent in 24 hours on the b.fi.B. agar plates, this could be eccegted es the final test since bormen, nobinton, and Stuart stetc thtt only 0.05 percent of the colonies giving the metallic sheen fail to confirm. This would also do ewe; with the yossiblc picning of et39ical colonies thich miqht have been well isolated. uonfirmed Test - If this test is run, it is advisable to tick colonies that show the tnicel sheen even thou;h they may not be tell isolated. oULIZLniiY In this paper the liters ure concerning the discovery, classification, end ngortunce of slow lectose fermenters was reviewed. rho experimental work tas then related. 19. Ine toxicity of sosin—iethylene-blue agar toward gee tro- duction was tested for by streaking glates with a gure tyiiCLl culture of LSCh. coli, but it mes iounfi that there was no "as inhib’tion. ztggicel cultures were then used in _ k.) b-. the sane manner, 5nd it res found thet their gas produc- V. ‘C tion was decreased b3 the L.Q.o. egg . *5 it use then attempted to increase tie fermentation of the atégieal CJthLII’C—JS b3, ragid transfer metnorls, but it was found that it could be increescd only Slléhtly. it was also attemfteu to decreese the fermentation *3 0 PC“ 0" "i C‘? H (D (D O H m .7: S: t: O 0 $3 H Cf“ e *3 (D O H: [T U) C 5 O O H Ip—lo M U S tr C'f' Fx r-J P. U) M d O O b meter semglcs were then received in the laboratory 7? J. a. unich contained sloe lectose fermenters. brom 7 eat sem;les, 108 cultures of slow lactose fernenters were isoleted. Ihese were then identified end found to be mostly of the intermediate variety. suggestions were given as to the grocedure which might be used in the beetetiologicel examination of water to aid in the detection Of slow lactose fernxnters thet Mléhb be gresent. PC 0 O CC] (it o ('5‘! 7. 9. lo. 11. .9 1" 1""; 1 7‘ ‘_ u '17 o \ piuLlUULLnL 1-1 ”014.131; :;'d HILQUII rirxei les 01 Lecteriolo;; and immunity - e. lth 71 " " ' 7 I , "‘ L. .‘ . \ " I‘ 1". ’ ':~" 1).]. U1.1L.L , all-“zillllmdiu , 511154.411, t-i’lCl «HUD-L iU-Ltlh 7': .- V . .I “ , .:,.- - , - n,‘ :x- w, ,-- .1 1.. .. LLJAJ n “elationsnips oi “e UOlllorm CAM nCluLEd J. Lact. 45: 557- 571; May 1942 .Lnsnbl, .1J3., bdohblidjlsgnbfi, J} Dissociation and Lactsse activity in slow Lactose Iernentirg bacteria. of intestinal p i in, J. Bact. 31: 455-464; 1936 orQLni ns Involve d in tne 101 lutien oi hater irom Lon; stored feces, user. J. fut. hetlth 29: 44 5- 450; “yril 1938 E'UlJlaI"le , 1L 0 D o ’ SJ: IIIII: , La 0}.“ 0 Slow Lactose bermentors in Water .nalgsis, '0 £111er Jo 3711b. “6“. 131.129: 266-” 70; lcvb Lie CLLADI , L; . n . Slow Lectose leruerter in Late nmer. J. Pub.:lt11 29: LG -2 AHIlBSlS, :..a [JILGLLii , 1" all 0 S’L1UJ—.L.L , C. P. o , :HIUiLl—JL , P. L o , LOAL' 1 it , b 01.0 Sum)cstcd urouiin) 01 Slow L;- ctose rermen ntir'~ Coliiorm Urf;‘-rrlsrms, Amer. J. rub. Ieelth 50: 499-307; May 1940 Gblfl‘d , l . "n o , llLULLLLLi-In1u', 11 o Eosin-hethylene-Llue :gtr for Ra; Escherichia coli, emer. d. lub. health Qt: 920-923;'hu3. 1955 .id Direct Count of fiUJMnK,L.h., aosiurot, L.D., bIUAdT, 0.“. A Study of standard Lethods for the Detection of Coliform Urrenisms in Raw and lreated utters, anP. J. lub. nealth 51: 55'7- ~567; June 1941 JUN : 3.5., OIbUfT: 135-; LITLLE, n.B. “tyylcal (310w) Lactose Pernenting 3. coli, 7" J. Dact. 20: 267-279; March 1952 I‘l‘fl‘.‘ . ‘1‘ . . . I h. | q. 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