IIIIIIIIIIIIIII III I I I II ___—_. III A COMPARATIVE STUDY OF CCNFIRMATORY MEDIA USED IN THE DETERMINATION OF THE CC'LI-AEROGENES GROUP IN WATER ANALYSIS Thesis for {he Degree of M. S. Doris E. W'iIson I936 I." . ififi'fi’: .;{v':&.‘ an 3"” I'- b 1m): is .. 3A ‘4 a. 2"... '. -'._.'-"‘ 15"." :4 . “.3 ‘I‘ *‘71‘r‘c’"‘7"_t X' (' z‘ ‘3”. PAR“ ”‘34“: . .' ,3 fit ~37 ,~ ~, .m.-_..;L " 65—9.; awm-yz“ . \VLZL': ‘. < up ‘ \ ”SHAR- S .. , ",l?~"¥ .- . ' ._‘.-.', -,.,1 . ? 1" 1. \s"! 'v‘ 1:! L. I"‘ It. I 3' -5 f » Y’ 9 .3 .3] .5- ,. .3 3| ' :‘2’. ‘1 Sty. . . , ‘3 .R‘ I" V I? ‘7'“. ( "J 'V a tip gin-'3‘? ,2}! .A‘. -. - . «i. f- 7536 :2?“ ‘ ‘. -'~ I .‘\ A ‘J if‘fi. ‘ - a.) 3,? fit“ I“ ' - " “I" .:~ w» ‘ I . 'x'q’ _a,'4' .s _ I 5'.“ ~ 4: 2 fl » . 4 -sg.O:-;IW l. 3" " b ..‘_r‘l:g L, . ~. ~ -. ‘ . u;.r.\.' , .. .~ -. . 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I: P’Ltlb :’ Dorls EI'Wllson 1956 of A; icultural ani Applied Snience ‘J A Cougarabive Stuiy of Confirmatory Teiia 3381 in the getcrmination of tne Joli-AeCOZenes Groug in Later Analysis Doris 3h ZTiIson ~+ gasu Lansing, gichiian ;Q ‘- 4—1;; "I '1 JV- 'TDWn A I.“ .u I. Q I n“- 114 IT I J “in; L u 7 .. -1_ \J .'|‘.J._J '.1 7T A? \) 'IIT’ «\ ALI 1.?A'T J-J‘Ju fl] ‘\ I“. J ‘J V fl 3 a nu! } .w .v s). In” \. ... .2 xi; l‘ f. A :-—- . IT 4_ rs," *_ 11' i"" I, -1..;Ir‘.u...'u .JL)-}' “mun. "j- o ’ - r‘x‘ ’ ine writer misse‘ "\ to thank Dr. C. s. Ybung, and 0) Miss Alice waorthy of the Bureau of Laooratories, Licnigan Departnent of Zealth and Jr. 3. G. Danham of the Difco Laboratoriss for their assistance and copperation in making this stuéy possiule. 0 he writer also wishes to express sggrecistion to Dr. 1;. L. :Lallgnen for his guidance anti assistance in carrying alt her work. Y" \1 LC ) Y' Irv-me“) ‘ . -..- “v‘t k) *5 r-1 1 \. f Jonsideraole OjjOSitiJfl has develowoi in recent years to the staniari msthols of water aha ysis. A aehani for a trocsiure which is sore 3 out, siholer, and gore oractical has been orevalent. Prescott acd “inslow (l), A.J. Jalle (3), Unnham, 3-.- m J me an (3), ani others have reco:nizel Pl raiy, and Jon. the neel for ingrovei xethois for isolation of the coli~acrogenes qr up fros water supplies. Idle n ti :3 :3 3V Bil Water analysis of the American Public Health Associa- tion (4) was published in 1335, it was reCOjnized that there were numerous new procedures being triei. The (D committee selectei brilliant green til lactose broth [4 t (‘1‘ (1‘ h 8 time l-‘° for primary isolation in purified water. . it was thoaght that only media which Hal been proved to be atisfactory should be used for "Staniard Methods". C) VJ ’ 4 -is attituie mas consistent'with that of “inslow and Prescott (1); they statei that the met: should be a standari one so that uniforh resilts could be Obtained by various workers. In 1:35 the use of four media - fuchsia lactose broth, crystal violet lactose broth, brilliant green bile lactose broth, ormste ricinoleate broth - were endorsei as confirmatory media by the Committee on Standards of {ater Analysis (5). A series of sets on sixty “tr 118 0i the coli- serO'eies group were made in these selective “3113 by fifteen cooye¢aoing labor— stories. (6) It was ibdnd that none of these were as selective for the coli-aerofene s group as lactose broth Ho I (D I...‘ and that brilliant green b actose broth, crystal violet broth, anl fornate rioino leate bnoth were too inhibitive as primary isolation weiias It was thought that these media might be used in the c3nfir:nat 011 pro- cedure. It was recommended also that the use of brilliant green bile lactose brofi1 fa? orinary'isolation in water -urification plants be removal to the M oendix of Standard Methods. (5) No study has been.aade of the value of these four media for confirmatory tests on water supplies - raw, purified, or those being purified. Such a comparison VJlth one of the present standard method procedures seemed ex- pedient. Through the cooperation of the jureau of Labor- atories, nic.igan Department of health, confiruiatory tests ‘ were made. a group o H) 202 tater samples were tested using L) the follm~ing ifco dehydrated media: brilliant green bile lactose broth, crystal violet lactose broth, gentian violet bile lactose broth, fornate ricinoleate broth, and eosin methylene blue agar (one of the no confirmatory media pre vi sly recomnneni d). This work has been done in an effort to determine the .racticability and Specificity of these media in determining the presence of the coli-aero- genes group of organisms from water supplies. -7- ,3." ‘73 a , ,_ ”I J... VLL 1"];le The first procedure for bacteriological analysis of water was formulated by a group of bacteriologists who were requested in 1895 by the Committee on Pollution of hater Supplies of the American Public health Association to draw up methods. (4) This report was published in 1898. From this period until 1312 dextrose brothxvas used for the isolation of the coli-aerogenes group from water. In 1912, the procedure was changed so tint tdle broth me- dium was recommended for the standard test. An outstanding contribution to the detection of polluted waters was made by the United States Public Health Service in lvld. A standard method for the contnal of water supplies with definite criteria.regarding the safety of the supplies was drawn up. The use of lactose broth for the confir- mation test was recommended (4). The committee on the Standard Methods of the American Public Health Associa- tion replaced the use of bile medium with lactose broth as this was found to be superior. The present standard procedure "recommends that the coli-aerogenes group be considered as including all Gram negative non—spore-forming bacilli which ferment lactose with gas formation and grow aerobically on solid media". (4) The method for the detection of this group consists of the following procedure: First, there is the presump- tive test - the planting of water in a series of lactose broth fermentation tubes, incubation of these tubes at 37°C. for 48 hours, and reading those positive in which ten per cent or more gas is produced. The second step is the partially confirmed test-transfer from the cultdre of the positive tubes of the presumptive test to Sndo's agar or eosin methylene blue agar plates, or brilliant green bile lactose broth, crystal violet lactose broth, fuchsin lactose broth, and formats ricinoleate broth. The third part is the completed test - transfer thical colonies from the Jndo's agar or eosin methylene blue agar plates or from the tines showing gas in the confirmatory medium to lactose broth and agar slants, those lactose broth tubes showing gas within 48 hours and showing Gram- negative non-Spore-forming organisms from the agar slants are considered a satisfactory completed test. (4) Since the adOption of standard methods, modifications of the prodecures as pertain to the media rather than a departure from standard methods and the use of entirely different media have been suggested. Since Limbourg in 1889 (7) introduced the use of bile salt with peptone and pancreatic extract for an isolation medium, a considerable amount of study has been made upon the use of bile salt media for use in water analysis. XacConkey (8) in lvOO found that nedia.with 0.5 per cent per cent salt ensued no inhibitory effect upon doer- C,“ to thella typhi and Escherichia coli and allied organisms. He also found that at 37°C. most organisms of the air and soil were inhibited. It was found by Jackson 1906 (a) in his work;on the study of the use of bile in the medium that the effective agent in bile was the cholic acid radical. Thus sodium glycocholate could be used as an inhibiting agent in the medium. Ls ox bile consists largely of glycocholate this is a very good source for this inhibitory agent. Jackson made a series of tests on 5000 samples of water at Mt. Freepect Laboratory and he ootained agreement,with the exception of a few samples in which gas was formed in lactose bile medium, and not in the standard method. Some attenuated strains of Esch. coli required three days incubation for growth. He concluded that the previous dif- ficulty with bile mediumiwas the result of using too small an amount of bile which caused an overgrowth of the coli- aerogenes group by other organisms. He thought that this medium would be very practical in filter plants. Sawain (10) followed this study by running tests with lactose bile medium and lactose broth. He found that the lactose bile medium was more reliable especially in testing con- taminated waters. Opposition to the above results develOped in 1915, after Jordan (11) fmimi that there was no relation between the attenuation of the organisms and the antiseptic action of the bile salt. He concluded that young cultures as well as old cultures were inhibited. Cumming (12) substantiated the former's work. In a series of tests made, he failed to detect the presence of half of the colon bacilli present. In both studies 10 per cent ox bile was used. As a result the Standard methods eliminated the use of lactose bile medium. Salter (1919) (13) stated from his stwiy on the action of varying amounts of bile salts that they are very inhibitive and produce a lengthened 1a: period and increase the generation'time throughout the period of growth. however, he found that 0.5 per cent or smaller amounts of bile salt stimulates each. coli while 1 per cent or more inhibits growth. Levine (1323) (14) in an effbrt to determine whether bile inhibits or stimulates the growth of colon.organisms u) U) concluded that it‘vas nec ssar << to have an optimum concen- tration of bile constituents in the medium. he found that sodium taurocholate up to 1.5 per cent accelerates the growth ofgacrmrenes and similar out irregular results were (D obtained with isch. coli. Difco evaporated bile show d marked and distinct effects upon the coli-aerogenes group; the growth of A. aeorienes was maximum in one per cent dried bile while Lech. coli grew best in 2 percent evap- orated bile. A concentration of 5 per cent bile vas not detrimental to this group; however, 10 oer cent was ex- ceedingly inhibitory to the crLanisms. The addition of certain dyes to the medium to inhibit growth of spore-formers and non-members of the coli-aero- genes gnoup was sugjestei. In 1912 Churchman (15) did a classical piece of work.in his determination of the se~ lective bactericidal action of gentian violet. he used aqueous solutions of the dye and fnund two types of reactions. First, ther was the group which when treated with gentian violet failed to grow; second, those which grew after treat- 31L +e .. do .aL {A 3 H L umnithdse tion .3 S O r 9 Liu 3 n1 U - not 8 fif. n a; e e 3 I ‘ 1 ‘5) C O‘L ‘ W . in none '94 1|. '1 C di’iC'rBi’ ‘ . l ' — L sJ . 4 Al dye u when the H 11% ox .— of 39188 e h :L x: 9 r, n L. n v Md «5 3 ”L H C CD 9 T Lu .1. r. 31. «U 4 gonfort by ldal 5) dd J I1 gfl' L he did ceinteracted y ‘— .’\ V .. b ; l I the coli-aerow 19fl VLO "OI‘A'. 1111’; t of t of V ‘ 1 violet on n attenfl;t Q :nn ) 3 I (A A. 1 \L . [J 4. in * eff o 1,000,0CC r 0 "1|: t fen ) cryst L; n -- P. ,_ _ .L. of s t 1 (lb i O “OH O . - N.) s ‘,19 lnnij ‘f 1V‘ U 1038 'J 4. - 111 1 ".1 most -1ut d all and presumc the in A ,. LA. U curious A. e found vat 0‘ onenomenon. ‘\ creaSmd was bas- s TT tone to t .1I* '» ‘» Q -‘\ hinslow and Dolloff (la) in a comparative study 0: rosa- L U f‘J' lie acid ran an violet, uni brilliant green found that gentian violet in lactose broth was five to fifty tines 4‘ ‘ 7' ‘1. J" ' : "t we ' q,- .. A ' "‘ O D' ‘ 1- . . as UOALC s rOsalic acid nails in lac ese bile orotn it (D innioited in a dilution of 1 to 10(0 as did rosalic acid. Stearn (13) in order to find out whether gentian violet affected the viabilityrof'tater-oorne organisms cultured for five montus dsch. coli i, Lech. coli I "v.5 i , ,_ H _ "W 1 O _ o 1. o (:1 . _ ‘- a. aeroienes 3,and nscn. aciii lactici. one found that with gentian violet lactose peitone broth an increase in growth, motility, and fermentative action resulted. CI ”alle (2) in a study of 100 samples of water found crystal violet in lactose broth superior to lactose broth ‘1' us su;:estei that the amount of the dye used should v... alone. be increased in proportion to the anount of peptone and other organic matter added to the medium. Stark and Ang- ) H ch strains 0 turesfr~ t (L! laxm.(20) found, with pure 01 m , l violet and found that {J an inhibiting eifect from or ,4 I if. U] i D it was necessary to incubate some cultures five days be- fore any gas formation was produced. Crystal violet and brilliant green were both ori- ginally use to inhibit the growth of the coli-aerogenes group in the isolation of the typhoid group. An attempt to use both of these dyes to isolate members of the colon- aerogenes group was made. In Salter's work (13) (131:) on the rate of growth of jsch. coli a study of various anilin dyes and different media we. trade.I-1e found brilliant ! green to be much more inhioitive than crystal violet. a . l " n tneia 1-» marked effect on the increase period and 105- arithmic phase has notei. Xe found that brilliant gree; inhioits growth in l to 600,C00 and that it sliéhtly stim- ulates the organisms in a dilution of l to 10,000,000. Juer and narris (21) in 1330 studied the use of brilliant green (commercial salt of tetraethyl diamino triphenyl carbinol) in broth, agar, and bile. Brilliant green in bile shows a definite selective action preventing the growth of Clostriiium uelchii and Cl. soorogenes in a dilution of l to 10,000; however, without bile in the medium this selective action for Ssch. coli is restrained. In a study referred to above, linslow and Dolloff (18) found that brilliant green was inhibitive in a dilution of l to 100,000 to l to 1,000,000 in lactose broth and l to 50 to 1 to 500 in lactose bile broth - the toxicity of the dye disappears with oils in the medium. Dunham, McCrady, and Jordan (3) in 1925 found that brilliant green bile broth eliminated Spore-formers; however, the medium was a selective inhibitory'aedium which did not detect attenuated forms of the coli-aerogenes group. In order to determine whether brilliant green bile broth was an improved method for detecting the presence of all active coli-aerogenes organisms, 3.3. Jordan (1927) (52) made a study using the medium in the analysis of water of varied degrees of pollution. He found 95 per cent to 97.5 per cent confirmation of coli-aerogenes group. His work led to the approval by a group from.tte American gater Uorks Associa- tion of the use of parallel primary isolation in lactose - 10 - and brilliant green bile lactose broth for routine testing of waters in the process of purification. Salle concluded (2) that brilliant green exerted a bactericidal action upon the coli-typhoid 5n3up of organisms and that it was not as efficient in eliminating Gram positive organisms as crystal violet. After the Laboratory Council of the American Buolic Health Association appointed a committee to study the es- tablishment of standards of toxicity for dyes, 3. h. Kline (23) made a study of the bacteriostatic action of brilliant green in broth (lactose and peptone). Phis phenomenon was emphasized rather than the bactericidal action because the effect of the inhibition.of various dyes upon the growth of organiSms was of importance. is found that certified dyes from various concerns showed distinct differences. The bacteriostatic titers of dyes can be obtained from the Biological Stain Commission. Bronfenbrenner, Scllesinger, and Soletsky in 1920 (24) studied the effect of china blue and rosalic acid on the colon-typhoid group. They stated that due to the small quantities of the dyes used in the medium that the bacteri- cidal actioninas negligible however, there was an antisep- ; tic effect upon some organsms. They found that china blue did not inhibit the majority of organisms. Rosalie acid 0.005 per cent was found to be inhibitory upon Gram posi- tive organisms. The combination of the two dyes gave th same results as rosalic acid alone. fiinleW'and Dolloff (17) found rosalic acid to be inhibitive in lactose broth in a dilution of l to 1000 and in lactose bile broth in l to 10,000. Schreiner (25) in the Kansas State Laboratory in 1921 developed a meiium to use for confirmation which consisted of the addition of basic fuchsin to lactose broth. A com- prehensive study of fuchsin lactose broth was made by Ritter (26) covering a four year period with 33,552 positive tubes confirmed or 94.53 per cent. She found that for ground waters the medium was equal to lactose broth and for surface waters it was superior. The nedium showed a marked selective action in chlorinated waters. Stark and England (27) made a medium using sodium ricinoleate and sodium formate for the inhibitory and selective agents for the isolation of the coli-aerogenes group. They found that sodium ricinoleate inhibited the growth of bacteria which caised false positive presumptive tests and that sodium formate accelerated the growth of the Jscherichia-Aerobacter group. The total number of bacteria was increased partly because of the buffering effect of the sodium formats; the hydrogen-ion concentra- tion of the nedium was pH 6 after six days incubation. They obtained satisfactory results with the forxnte ri- cinoleate medium. Noble and White (28) using the Butter- field-Hoskins quantitative—' I *8 - " A. 3 . __, ~_ - H r v I I, .. ‘, ‘ _ . ’ ‘. I .3 :1, - j , 43 :ognr 13nere 24ers Olly'4ui alloures i lied hal _lw>llcei no 4 i‘i i‘1 was in the liouid confir ::a torv media. In ?aole go. these reactions are tabulated. 1 . 'v "x .I .p Ot' -L 3;;‘1'613nL911t ted—1‘11] 3‘2; hI‘S. 48 hI‘S. Lactose broth, presumgti e tests - + ' ooo Josin methglene oluc ago” growt. not 44 ty.ica Lactose brotu coqoleted tests 44 :ornate ricinoleaoe broth 34 44 :Crystal violet lactose oroth 104 éé irilliant green bile (2;) lactose broth 109 44 Fuchs in lactose broth lab 44 3entian viole t lactose o’le oroth 157 44 If there was no prowth trollcel on the eosin rn thylene blue aiaj'a;fter :4 hours, time cultlres were incloatei for 48 thPS. If no growth appeared at this tine, these cultlres were recorded as proausin; no growth on eosin methylene blue agar. Bhere were 40 cultures which produced no growth on eosin methylene blue agar and forned no {as in brilliant green bile i2“) lactose broth, crystal 5 violet lactose broth, fuchsin lactose broth, iormate ricino- leate broth, and gentian violet bile lactose broth. Two cultures produce no growth on eosin methylene blue agar but produced gas in all of 'the five liylid confirmatory l media. These cultures were found to be members of the coli-aerogenes group. The above reactions are found in Table Ho. III ubes Gave Iegative Test on Josin thylene dlue agar Positive Presumptive T :3 .‘J. th Bositive Broth Hegative 0 ts Confirmatory Tests 3 Confirmatory Te 48 hrs. 48 hrs. Formats ricinoleatc broth 40 2 Crystal violet lactose 4o 2 broth Brilliant green bile 4O 2 (2p) lactose broth Fuchsin lactose broth 40 2 Gentian violet bile 4O 2 lactose broth Twenty four cultures prodlced colonies on eosin methy- lene blue agar whichflwould be onsidsred typical of either L V ass cultures failed to fer- I ‘ l . |‘ -. :1 . osch. coli or a. aeromenes. -h J I msnt lactose broth andxvere not members of the coli-aero- genes group. There'was no gas formed in the five broth media by these cultures. This group of cultures was negatively confirmed. The results as recorded are listed in Table No. IV. In 39 cultures discrepant results were obtained. These were not further studied so that the value of these tests is questionable; however, they are tabulated in Table V. In this group of cultures there are three types of variations found. First, there were 22 cultures which produced positive completed tests from eosin methy- lene blue agar but were ne ative in brilliant green bile , . c; ‘_J a (2L) lactose broth, crystal violet lactose broth, fuchsin lactose broth, formats ricinoleate broth, and gentian violet bile lactose brotl. Second, there were a cultures which showed typical growth on eosin methylene blue agar; these did not produce gas in lactose broth when transferred from the agar. The five broth media planted with these 9 cultures showed no gas production. Third, there were 8 cultures which did not ,roduce typical growth on eosin methylene olue agar, did not produce gas in lactose broth, but produced gas in the five broth confirmatory media used. The cultures in the broth media were not transferred to lactose broth so that they could not be considered members of the coli-aerogsnss group. as m I Table To. IV Examination of 24 positive cultures on Blue [LEBI' Josin Lethy ene Negative Negative 24 hrs. 48 hrs. Lactose broth, completed tests 24 24 Formats ricinoleate broth 24 24 Crystal violet lactose broth 24 24 Erilliant green bile (2w) lactose 2 24 broth Fuchsin lactose broth 34 24 Gentian violet bile lactose broth 24 24 Discrepant Reellts in Confirhatory idedia heretive Positive 45 hrs. 24 hrs 48 hrs Losin methylene blue agar 22 Lactose broth, completed tests 22 Brilliant green bile (2;) lactose 33 Dr OE h Crystal violet lactose broth 33 Fuchsin lactose broth 23 Formats ricinoleats broth 22 Gentian violet bile lactose broth 23 Josin methylene blue agar 9 Lactose broth, completed tests 9 Brilliant green bile (2V) lactose 9 broth Crystal violet lactose broth 9 Fuchsin lactose broth 9 Formats ricinoleate broth 9 Gentian violet bile lactose broth 9 Sosin methylene blue agar 8 lactose broth, completed tests 8 Krilliant green bile (3n) lactose 8 broth Crystal violet lactose broth , 8 Fuchsin lactose broth 8 Gentian violet bile lactose broth 8 Tormate ricinoleate broth 8 Table 30. VI Classification of Josioive Confirmatory Bests Obtained from One or J‘rs irotn gedia Jsin; gosin Lethy ens jlue Agar a the oasis _. Iumoer Total fiercentage hidium neactions not positive not confirmed confirmed Lact se broth _ Bgesumptive test - - 210 Josin methylene clue agar 121 Lactose brofii, completed test 121 Fornate ricinoleate broth 104 31 73 Crystal violet lactose broth 79 9 70 Fuchsia lactose broth so 27 59 Brilliant Green bile 54 9 45 (2a) lactose broth Gentian violet bile 29 4 25 lactose broth Bosin methylene blue agar 41 Lactose broth, 41 completed test Formats ricinoleate 32 3 29 ' broth Crystal violet 24 7 l7 lactose broth Fuchsin lactose broth 21 8 7 13 Brilliant green bile 8 8 (22)lactose broth Gentian violet oile ll 2 9 lactose broth Sosin methylene blue 58 agar Lactose broth 38 completed test Formats ricinoleate 20 5 21 broth Crystal violet 2O 3 l7 lactose broth 7‘ 1 ‘9 o . o ".-- r'\ i~ 22 8 l4: lucnsin lactose orotl Brilliant green bile ll 1 lO (2y)1actose broth Gentian violet lactose 4 1 3 bile broth Table JI Classification of :ositive Confirmatory lasts Cb‘ained fro:n Qne of Lore Broth uedla Icing obsin methylene blue agar as the Basis. Number Total :ercen'aée Hedium Reactions not positive ngt confirmed conf‘irmed Sosin methylene no blue agar growth Fuchsin lactose broth 11 4 7 7 Crystal violet 2 5 lactose broth :ornats ricinolee to 8 5 5 broth Brilliant green bile 5 5 (2H)1actose broth Gsntian violet tile 4 5 l lactose-broth Totals Eosin methylene olue 121 94 c.3ar Fo rmate ricinoleate broth _ 126 89 20.58, Crystal violet lactose 109 106 16.15, broth , ruchsin la ose broth 95 120 53°57N Brilliant green bile 69 146 12.82p 2nlactose broth , Gentian violet bile 58 177 20.85p lactose broth '71 .he remaining 215 cultures from the 685 lactose broth presumptive tests shared clnsiderable variation in the five different broth confirmatory media and on the eosin methylene blue agar. Using the eosin methylene blue agar as a basis for classification these esults have been re- corded. Table No. VI shows this classification. There were 121 cultures*which showed typical colonies of the coli-aerogenes gnaup on eosin methylene blue agar and produced gas when transferredto lactose broth. From this group of cultures 54 cultures produced gas in brilliant green bile (2N) lactose broth; 8 of these cul- tures were not found to be bombers of the coli-aevogenes group. In crystal violet lactose broth 79 of these 121 cultures produced gas; 9 of these cultures were not coli- aerogenes organisms. Lignty six of the 121 cultures formed gas in fuchsin lactose broth; 27 of these were not identified as memoers of the coli-aerogenes grou to In formats ricinoleate broth 104 cultures of the 121 produced gas; however, 51 of these cultures were not found to be coli-aerogezes organisns. only 29 cultures of the 121 cultlres formed gas in gentian violet bile lactose broth; five of these 29 cultures were not mem- bers of the coli—aerogenes group. In the next group, there are 41 cultures which did not prod‘ce typical colonies on eosin he hylene blue agar and formed no_gas in the completei lactose broth test; however, from this group of cultures produced in some of the variols broth media. Shifty -29... t o cultures formed jas in fa‘date ricinoleate broth. ._ 1 . , I 1’. . .‘ : _ "r , . , o W, '_ _‘ _ '. rdifl uflreg cello 11130 COLL-QBF'usefldd 01‘: 13.31;.- {1.3 'v."3r‘e .1013 41 cultures producs‘ gas; seven of these 24 cultures were not lien tif ed as nemoers of the coli-aer genes :rouo. In fuchsin lactose broth 21 of the 41 culture \— u :as. From eight 0 these cultures sensors of Ft; for me 1 the coli- -aerogenes group were found. sigh t cultures from the 41 cultures produced gas in brilliant green C‘ H (S of y bile (at) lactose u; all of these were found to be coli-aeroie nes orzanisns. In gentian violet bile lac- tose broth eleven of the 41 cultures produced gas; two culture’ were iot id ntif ed as members of the coli- aero- 3enes group. The third group is compos3: of 58 cultures which produced typical colonies on s sin methylere plus a3ar and did not fermentivhen trausferrel to lactose broth. Tron hese cultures a variety of rea CtiOilS was ootained in the broth confirmatory iedia. Twenty six or these cultures produced gas in fornate ricinoleate broth and coli, é. aero:eocs, or Zitrooacter (interneilatcs) -‘,- 'V, 1‘ (‘1) \., '2- _ , ‘. , ,“.- .r ose broth 22 CL tuese o8 cultures prouuced \L‘ . r- ' A “ ‘, . q cg. .: L , .4 n :‘ t _ 9 _ 3' u- , ; elgflt oi tuese;.ere;nt “HJJLFQ oi tie coll-aero:.ne3 . . o ‘. , 1". ‘tL- ,. ., ..-‘. — .- -. ? M...» went} 0: tne )3 cuu,urcs ~CV11331 :ao in crystal 0 . 4- x .. J— ‘ .V ,3 f 1“. ' r r) ‘-. 1 _ .‘ Violet lactose broth all an thls grouu o: J? cultures 1 " D~ :, ‘L. i.! I "u . ' ‘e‘ . “r‘ .1" tnree nele not HudflbllLCu as coll-aero3enes OPfiEHLSbS. 30 8 . .n n S O .1 . l e 1 7.J t 3 a .1 w. l «i e .U 3 O S . h 0 V C e nu. a S u. S h 3 1 4 2 a .L a f .1 -t r. e a .d o e c c t. 3 n l 2 1 .. V ..... O f c .U 1. 1 n... 1 6 .r... S . .. M .1 O n o 1 .1 .1 m... {u u n1 C C e in .C 1v 1 n. . 1.6 . e. .r n 1 3 S 3 3 .L d .1“ P to . . 1 t 0 C .o to P .L a. .s P S .3 ...l. .i O .6 .1. d C Q 1 a... 1 ..... o”. u .1 C .3 n“ .1 1.. o S 4.6 . ... U S to T F e L n. 1 .5 3“ ., L C We. 1 J 1 as n . l... n r I n... 1n .D d .1 C H J t o... 1 ma. 3 U f d 3 8 C m . ., 3 .. o 0 e .r... j 8 O 3 3 O a .1 V 1 C 3 . t F 1 ...., d 3 C ....; f l S n. f O o... 3 6 a .1 ".1 h .l - 4... Q 1 .: .L A e n . n. T. 8 .1. .0 O 1 O ._. -1 P 1 n... a... . A O C nu. .9 C o_i 1 +o O .1. . O .1 U . ., m V + o O 3 .1 Q .i 3 3 Lb e 1 +.. a.” +6 1.” Ti 8 ca .4... m .- . to o to t 3 a n... .C B 1 .3 l o P .H n C .1. n. 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O 8 .P. r 1 .h a . ..... -o .. . 8 d n... . . i fl fl Q .m. .. t e d O o-.. h .n. i . O .3 fl 3 t t 3 2 cu m... e . L O n c e. H O 5 .41 O C 0 GI M .l. 4.... 1 S 1 lo .L .1 P 8 V. H 1 Pl C .. .i 4.1 .1... r.“ i. .. . 1...“ .H 3 9.1. .4... .. fl 1 o L n h .1 n. 3 .nl n). . .. a. u C a o 3 n .1 4.1 3 r1 .4... L .C iro Q m . .. 1.. S .u . .h t .o f .P n - a 1 1. e a. e. 3 3 .i f S in... .. J 8 O 1. 46 8 O 3 P 8 .. . 1 u to ....,... ...l 1 3 3 1 no .1 O 1 A T 3 .u w. .n. 3 O S 0 .1 O 3. O 1 3 ...i n 0 n ..l. n. .1 3 . . S .2... O .6 r r f .1 O C .0 o... u .r... .1 a J C s . o r t V . e h e D r n 3 ..l O 7.: O t h a. 8 1 t - a . E .L a 8 O a. .l . to O f e 8 w... e .8 m. S t .. L P Q 0 . n mi n1 3% a mi T V O 3 .o a. a. c... e c . a .. is f .1 .1 e u . , ... in e S e .1 C C 9. .1 9 .3 an .1 C C .1 u... n n. ”ca 3 C C. c. . a L6 8 t _ n. 4... .. . .0 t 1 a O S r L r O n ..l . . .i no 3 t :1. no 9... J l O O l O C a 2:4 0 r .J Di .1 S. S n o... 1 h 1 r. C m- .i S n. u e t 1 t p. O 3 11 P... C .4. i A.“ .0 o .3 .0 Lb .To .5 3 o n1 1.. o 2 0 TL mi n. . . n. O C O a, .1. . e h 1 h D 3 . u h C u 0 9 .1. a... .1 t 3 in... 1 . ., To 3 +o .1. 8 .1. C. t .5 e r l e e To A 1.1 S u. 0 3 C 0 r 1 O B 1 .41 8 .1 on an 3 9 e ol 0 S .1 9 f u o i r d O 5 r h d e a .0 O t C 1 m 0 t .. .. V as 0 d O ......_ t C 1 .0 t C -. 31 3 .n e . . 3 n t _ S . i r 3 S 3 a i O O . O a .1 o... S 41 e R .i B t n F r S . .1 .0 .... .11 w . W a 3 0 e 6 .v .1 9 h d .. . O .n. ..i .1 .11 C C .u. .1 ...... h n.” n1 n1 n . .11 t u 1-.. t 8 n1 n n 1H,. G 9. ”Q .U Q h a .1] 4U ”11 C 3 3 1 u a... O C P. ..1 .1 :L 3 a .3. C 11 C «1 1b .1 n1 - C .1. a .w W .1 .1 1 . .1 O 8 1r. .1 n . .. O 3 O .11 .1 _ S l .1. m1 n. n. n1 n1 “1 3.. 11v .1... 1. n1 0 11 .. \ .C e .1. .4. .m. .1 n. a, ., n. .1. .u a. .. n. 1. .11 ». ... .o .u .1. n. .mu .m ..1 7.”. U ) .11 ..r n S n.1,. C .M ..i “1 .11 c .H 1b .1 n1 C .5 e #1.. 3 01 1b .1 .3 r .n 1 0 . L O a1 .01 fl 8 .fl .1 S C . 2 . a 3 . J J 7 1. 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Any . a. l 51, «.11 .n.“ HM. n... o w u _. ._ 3 u o 1 n . i .. 1 m... u 0 .111 1 I1 1.. u L14 .. v 4.11 1.1a . .~ 1W.“ 0 O a: t . _ .WL ..Hu . u .111 1 AU. 1 r‘ t C 3 -J L g n . q 1 U C . s n. .3 t S t 3 t l C F .L E O 0.. .3 3 . .. O 11 l O a. an. 1. O H 3 S n1 3 .. . .1. .3 l 7.1 l +u .r1 .11. n1 4b h d .1. n1“ 4.... o 21 6 n1 3 .3 Pi .11 .i J O t 8 15 U . 2 . .. C t n J . 1 91. C .L x. .. .. . 1. .L n H O U \I n... F 3 .r“ .C .3 .1 W1 1.1 ,1... n. 3 t rd. .11 o, O .1 3 . .1 S 3 P 3 1. I. 1 J .1 W S :2 .i .U :1 1 .3 O J 3 .1 .11 C Y 1 u 3 q 3 .3 .... u S O O 3 O t O s .1 O n. C l . t ..._ ... - . O .1\ 8 r . O a. P \l .. . e 3 n1. 0 O .1. J T. P .d n 11 .11 .3 n1 .1 Q. 3 10 .J. -U .4 3 n: .11 O U n. O a. .1 1.1 Lu .1. o .u 3 e .1 3 .1 3 C J n n O S W. 5 3 t n .1 o .3 1 .1. a. .1 LL .1 n 1 O c1 3 a” 1\ S 3 .7 3 11 O .1“ +1 .C 1.1 o1 M F 3 V l .1. .11 U m. .. u 11 1b S +u .fl _ .1 9. .11.. 1 u S o... n... 3 O .7... U .11 n , 1U 3 n1 3 3 .l1 t O C :1 G d . ! mu 1.1. n n1 .1 .1. n C q 3 .1. 1.1. .11 1.1 P H S .11 M n .L .1 1 T E .1. P 3 9. .1 J 8 11 .1 .i. .i 3 .L a. e e .1. n... 9. a... O .. 1 a S k S a. .- . 3 .3 a 1 V 1 J 1.. O 9 u C V 91 V" 1. .1 H1. 0... . . ..i H O 4... .. I. h d O .i O Q U n .11 P .1 . “i .. .. .i C .1 R. a a...” 9,. 8 t .«L A.“ flv r LIN P .L .1 1 n t cl Av 01 e 11 O my t ”1 .mb .1 . . 8 .. . -3 S t 3 t e 3 C .i r u .1 .1 a J 3. .U ._ P1 . . n I B .i 3 .1. V 9 J r O t +u e r 1b 1.1 m 1H .13 e V t 3 0 v. 3 .i r l P O l C e r O .1 h 0 .1. O 1... .1 .1 ,1. a n t n). F .1 O O .2... .0 3 g D. + V e T .0 P O a L .. .. .1 a, 1. O o. i .L 8 .i 91 S 3 f .1 C .U f .I .1 9 .1 .1 e f + . V t V t .a O 3 S .1. f 2. e .3 l h 5 e e l 3 O a. .1. 9 n1 3 e 1 u e a a O m L a .1 e t O V .v 11 .... a .1 n m .i a h r e n W r .1 9 e e «1. .1 .. .1 .1 .1 n. P .3 o... r 3 .1. 41 O .. z 9.. .8 J n O .c n S .1 C t T Q. 8 .i P .1 O t .11 m .u a 1. . h t O f n f. .1 O h e B .1. .1 Q U o 1 O 1 u a: .. .11 :1 1C 0 +u .11 .1 an... 3 .3 1L W n . S S e m. H P fl .3 .1 S t n r O V t e t t C O O 3 O O n . . l O r e n P P a... 9. 8 e r .1 3 .1. .fl 8 .n a P .3 w” m... 3. .1 t n C B c 1 .1 C .J W l O a .0 1L m d t m 0 l .D 8 least .3 I .1 tar \- ‘1’, LI r" \l c‘ W 7. ’ ‘3 'x3- r17» :- .' ‘L‘ra-v ' Ln orler to fultub) ueoarqtaul bAvbe V:“L?C 10118 1: the broth melia, the ooaitlve reactions of each have oeen tone broth 0 tabulated in Dable TII. Jaoh of the 215 la ~;iisagreement in one or more of w *3 CD 0" c D ' .3 cf H. <: (D ct- C” ' D (I) (I) »w—- '_J 9 5". C H 't u the broth teiiam has oeen lietel ehomir; the cougarisoa of each broth meiium with the other four meiia Ffli 80; r-" n methylene clue agar. Table VII Reactions of formats ricinoleate broth Positive Con 1 tag; Bagth in Comparison with Tubes 1n disagree- me a sad Sample 1.4.3, Ref. F.r. 3.5. C.v. F. G.v. No. Agar 24 48 24 48 24 48 24 48 24 48 24 88 24 48 95 .. + + + {_+_- + - + + _4: - - 94 + + + + - I - - - — - - - - 95 + + + _- + — - - -0 - - - - ,- .. - .. - + - - - + - + .. + 2’: + + + - + - - - .. - + - : 38 + ’ + - + I. II - -0 "' + - - 99 + + + + + —. + 1. + .. i - — 100 i ' ' ' + " ‘ ’ 3 ' + ’ ' 101 + + + + + .. - - + - + - i .. - .. 1'. + .. - - _4: .. _-_n-_ - .. .. - .. 1 + - - - i - i - - + + - - .. + - .. - - .. - - .. i8? :- + + + - + + + -3 - - I - + 9 No growth .. + .. .. - _. _. + _ + + .. - .. + - + .. - - .. .. .. 104 + - 1 - - - E - 1 - 1 - - . + - 1 - + - - I- + - - - 1 .. + - _ - + - + 2. .. - - - .1 1'8; .. .. - + + + + '1 + - .. - + 108 ' - - _+_ + - - 1 + - 1 - 1 - - - 1 + — - 1 1 M 1 f 1 + .. + + + .. - .. + .. + - .. 110 + 1 + 1 _ _ _ 1 '_ _ + + + + - g + .- o - .i ii: ::se::za:--:§ 113 I I T- I 5 I I I E I I '- I + - :6 - + .. + .. .t - - - - + - q: - + .. I - + - -. - -. 114 + + + + 4' II- -0 'I' 3 - _t - _‘t + + + + + .. + .. + + - -. 115 - - - - + - : L. : - I .. + '- - - I- I u- — I' - - + 117 _ , _ - Z, _ .. .. -. - + - .. 118 + _ - i + - + - - - i a - 109 + + + + + + + - - - + .. + 116 + + + i + - - _+_- ‘+ - + - + 120 + + + - + - .. .. + - + - + 1 - 1 — 1 + - 1 - -. - - p- - i .. + + - + - + 121 + + +’ .. + + + .. + .. - — + Table VII ! Reactions of formate ngeiiLgs ed ricinoleate broth Positive Confirm- atory Broth in Comoarison with Tubes in disagreement with l ~40~'-o- o , ,1 I Jingle‘ ~ ‘ ' Tr o ! _:_:“Q_r ‘ief. For. ‘3‘ ‘J‘v° 3' ”1"" ‘24 4:3 ‘3'; 4e :3 £3 *3: J... .3- 313 .34 1:3 is. 3. ‘ _ fl 7 1 1.22 + + + + - + + a + l .. - - _. 123 4’ i + + 4' .- + — + I _ _ _ + 125 + - + - + - - + I - + .. + + .. + - + - - .. _ .. -, _ _ 126 + + + + + + - + - - + + 137 + - 1+ - + - - .. - - : .. + - + - + .. - - - - - - 12C. - - — - 4- .. _ + _ + _ _ - - - - + + - -+ - .+ - - - - .. - + - .. + - + .. .. - - - — -. - - - - -. - - - - - - -. + - .. - .. - .. 139 - - - - + .. - + .. + - - 131. - — — + + _ _. + _ - _ + 152 NO growth - + - - - + - + _ - 133 No growth - + - 4. .. .. + 4. _ 4. 130 + 1 + + + - - - - - .. - - 1.34: No growth - + .. + .. + .. + _. _ 1.36 + - - .. + I _4: - .. - - - - 137 + - - - + - + _ - - + - + 159 + + + - -+ - :. - .. .. 1 - - i : i _ + - .. - - - : - - 142 + - - - -+ - — -. - - .. - - + - .. .. + - - - - - + .. .. 144 No growth + + .. - - + - _ - - 145 No growth - + .. + - + .. + - _ n n _ + _ + _ + _ _ _ + + — + - + .. + .. + .. + - - + — I - + - + .. + - + - _ 146 + - I - i - - - + .. i - .. 147 + + + + + .. + - .. .. _+_ - + 148 4‘ 1 _t - + - + - + - + .. .. 143 - - - - + i + 1 + : .. - - 150 + - + - + - + - + - — - 1 .151 + - + + + 1 1 1 + ".i - - + - + - + .. .. + + - + .. .. + - + .. + _ - _— _ _ + _ _ 152 4' - + - + - — - + - E - - + - + + + - .. - + .. + _, + + - + - + - - .. + - + - .. + - + - + - + - + - + - - 155 + - + - + — + -. + -. + - - - .. - + + .. + + + + + .. .. - - - 3: + - -. _I_ + g + - - — + .. + .. + - - i + + - .. ; - - 104 v+ + : + + _t 1 3.. 1 - - l - - I i 1 l ; L IL 4 Table VII Reactions of formute- ricinoleate broth Positive Confirm» atory Broth in Comparison with Tubes in disagreement with 5 media usecL ' ’ Sanole :S.B.3. D r - 7 I 1 . 1 1 $0. [it-:91“ 4Lefo id“. 3090 dove :- ’JOVO N: 43 L; 11., .34 4&8 3‘1 4‘3 334 «1:; 3e: 4:73 1.11 ‘13 + - + - + - - - + —. + - + .. + .. + + + - - .. - .. + + - :4:- .. + : - - + - + - .— 157 ' ' - ' + ' - ' - ' ' ' ' - _ — - + .. - .- _ — _ - - 158 + + + + + + + + + - - .. + ’ 159 _ _ _ _ _. _ _ _ _ _ _. __ _ T + - .. .. + .. - - + .. + - + 160 _ , - _ _ + _ _ _ _ _ _ _ : + - - - I - .. - - - - - - + + + + I - - - + .. + - - .0 + : -. + + - - - + .. + .. - 19'“ + — -. + + - - - + - - ' ' 103 I I _ - + _ _ - : - + - .. “>4 + I + + + -. + .. + .. + .. - + . + - + .. + .. + -. 3» - .. | ,. + .a. I + + - + - + _+_: + - - i 105 + - I - + - - .. - - + - .. V ‘ + ' .. + .. + - - - - .. 1- - .. 166 + -. _. .. + _ + - + - I - -. ' + - .. - + - + .. + .. + - - ' 167 _ _ _ + ., _ _ .. _ _ .. _ _ 183 - + - + - + - -. - + - .. - - - + - '.' - + .. - - 1' - _ - - + l + + - + _ - - + - .. .- - 1'71 + : : - I - _ - r - - - - 175 - - .. + + .- -. + + - - - -. + + + + + .. - - - - -. - - ‘ + CI - + + .. -. - -. - -. - _ 17“} + - + + + + + + + _ —. - - 17b 4- - .. + + + + - + - -. - - + + + + + .. - + I + + - .. + - - + + - - + + + + .. - L 17$ + + + 2: + + + I + I + - - 179 + + + + + + + + + + + .. - + - + - + - -. - + + + - - 180 + - + .. + - .. - - - - - - ? + - + — + - - -o -o - - -o -o 1 i181. + - + _ + .. -. - + - + - - ‘ i182 + -. + - + - .. .. - - + .. - 1 183 + .. - .. + .. .. - + .. - - .. F + - + .. + - - - -. - -. - - l :184 + - 3; _ + - _ - + - + .. - i . 1185 Li .3 i-.'- 1 :'-' 3;? t. '- 1 z " ; Table VII Reactions of formate ricinoleate broth Positive Confirij ative Broth in Comparison with Tubes in disagreement with] 5 media usedlj . I 1 I . ‘ I ~40 Jo j0| . fl \ ‘. l 301 L. Agarig Ref. r. ‘ 4 I 24 481724 48 I 24 48 34 48 .24 48 24848‘ 24 48 ! + ' - ' Smnole - fl . ng. I Ijov. 2. I [IOVO ___4——— 186 ‘ + I I I I II 11 187 I II-l-II 188 +1 ++-+-++ + I I it a. I I 0 II I II n +n I i189 .190 I II I + +I + +4-+ ++-+ + 0 II I II I II I + I I I I I I I I I ++-+ +| + +I + +4-+ + +++f|l _131 1192 1&3 I + ++-+I + +I + +4-+ + I I I O I II+I I + I I a +-+r+| +Il+ +I I + ++-+ I ‘195 .136 I I +I I n I f I I u | 1137 ,1‘38 I + + +-*| + +-r+-+ +1 I 3199 200 .200 '201 I + +t l +|+l + I + + ++-+ + +-++-+ + +-+u I + +1 I I + I I +| +I + II I II + +-+u 0 II II+| I II I II 0 u I O I o e +4-+ ++-+ ++-+ +.r+ +-++-+-r+ l I I I ‘l I I: I II + II I +1 I II I II I t +1 + +L+I + +l+| +-++ +I '202 84 No growth - . l _L . l l .J_ _j Note: E.M.B.; eosin methylene blue agar; L.b.,1ectose broth; G.v., gentian violet bile lactose broth; B.g., brilliant green bile lactose broth; C.v., crystal violet lactose broth; F., fuchsin lactose broth; F.r., formate ricinolea1e broth. Table VII Reactions of crystal violet lactose broth positive con- firmatory broth in compariSon with tubes in disagreement with 5 media used. . l_ I .— n a . I I 09.111918 Im.fll.3. F I (V I i! '1 I r1 I (N I IIOI ‘ Ibrar l-(ef. ‘u.V. 30:3. I :0 for. :JOV. I v9 A“ , V y; _r {—21 (VI—f 7r WW T r—l—f —‘; " y 7- a ;L4 43 iifléa 24 T3 L; 45 L4 4 L4 4 L4 43 93 -' + + -+ - i - -+ _: + i -+ - - 95 + + + - -fi - .. - - - + - - 96 - - - .. + - - .. + - + - i 98 + + + .. -. - - .. .1 - + - - 99 + + + + + -. + - + + -I - - 100 + - - .. + - - .. I - -+ - - 101 I + + .. I - - .. + + + - + - - - .. + - - .. + - -+ I - - - - + - - - I + .I I - 102 + - - - I - - - : I .. - - 104 + - + .. I .. - - -+ - + - - 105 + + I .. I - -+ - I .. + - - + I. -I - I - -+ - : .. + - - + 'I + .. I .. I .. - - 'I - - 107 - : - -+ + + + .. _ + + - + 108 - - - I 4- .. - - + 4» + - + - - _ 7: + - - .. 'I I .+ - I 110 + - -+ ': +7 - .. - I I: + - : + + -+ - + - .. - + - -+ - + 111 - - - -+ + - _ .. - + + + I - - .. 'I I - .. - - I: I '2 Z - - - g g .. .. - .. .. - _ _ 112 - - - - + - .. - .. - + - - - - .. - i - - - .. - i _ - 113 + - + - + - + - - - + - - + + + .. ‘I - Z .. -. - + - -. 114 + + + .. + - .H - + -+ + _ + I + + 4- - + - -+§ - I -+ + - :. 115 - - - — E - I 1.. II - i _ + 117 - - - — -. - - - + — -. - .. .116 + + -+ .I + - .. - + -+ + - + 120 + ‘I' + - + - .. .. + .. 4. - + + - I .. + - 4 .. -. - -+ - -. + - + .. I - 4- - + - ‘+ - - 121 + + I 4- + + 4- - : 4- + _ - 122 + + -+ ': I -: II. - - .. + - - I123 + + -I; - + - -+ - - -+ + - + ;125 . + : -+I - -+ - .. - -+ .‘ -+ - + 126 I + + + I — + -. 4- - _. t+ + .1 4. .128 i - - - _ -+ - - - -+ I- + - -. ‘ . - i - - - + - .. - + I - + - - . I z I ' : :: i. : :: : i..:: i. : :' §129 I - I' .. - + _ - - + ;_ I _ - 1131 ' - I- - - -+ - - |.. - + -+ - + No growth L1“ + . - ’11 - + 4;‘ +411 - - I132 Table VII Reactions of crystal violet lactose broth positive con-l firmatory broth in comparison with tubes in disagreemen1 _JLthLmedia used. . I I I I ' Canola s3.fl.3. I, I . _ I III ‘ ”(a :{efO COVO I 30%;. F. For. Gov. 110. tut-4:11. 2:; 4a 24 r: I 24 4a 24 4:: :54 4’3 :4 44+ 24‘ 4? § I 154 No growth I .. + - + .. + - +I .. - 140 No growth 4-. + - - _ + .. - ' .. .I. 142 + - .. .. + .. - .. _. .. - .. _ + - - - + - - - - - - - - 148 I : i ' I ' 4 i f“ ' " 1.36 — _ - - - - - - + - + + + + + .. + + + - - lol — — - — 152 + - + " + - — — + .. + _ + + - + - 4- - - .. + + + .. + + .. + _. + .. - .. + - + - - 153 + — + -. + -0 + —. + - + - .- 154 + I- + + + d. 4- I- - a. + - ." P'r“ - — D C + - - - + - — - - loo 158 I + 4- + + + + - - + +2 - + 100 + + + - + - .. _ + + + .. .. 1‘ ,9 + - -0 - + - - - + - + - - I ,. + 3- + - + - — .- + - - - - 103 I _ I 11134 + .‘I‘ + I - + -. + - + + + - - I + I-. + I - + - + "O + - + - '- 2 ‘4‘ + 5-. + - + .. + + + + + .. .. llOb + I-. -. ‘ - + - + - + - + - -0 b + I I — + r — .- *l- n - - - g- - Table VII Reactions of crystal violet lactose broth positive conI firmatory broth in comparison with tubes in disagree- ’ ecL I ~ I . , I I I . , ‘7 39§913| fouoD-I hef. J.v. I 3.5. ?. I E.r. G.v. -0. Luger I I I 2.341 Lt’TfiI Zr; 43 (rt ‘15 Z" figmo‘f 4:0 “.3;er LIL"? {d I I it 1.7]. + .. .. I - + - - I - - - + I - .. + _ .. - + - - - .. _ - - - I173 .. .. .. I + + I- -.I - — + + .. .... I + .. i I .. -. I- — I... .. - .. I .. .. 174 + - + + + + + I- —. + + T - .. 175 + .. -. + + + + I- + .. .. I - .. I + .. -. I + + + + '5- + - - I - - 176 + - - I .. + + + ;- -. + + - .. + - - + I I- .. '+ + + + . .. - '+ .. .. + + - - l+ + + + I - .. 178 + + + I + 2+ + T- + I + i .. - 17:; + + + + + + + I+ + + + . .. - 4n '. + - + - -.I+ + - + I .. - 190 ,+ I- + .. - a .. I- .. - + -. -. 181 + I- + - d- - an. - + - + I - .. 183 + - - .- + - - I— — - 4- I J — I+ - i - -. - - I— -. - + I - - 184 + - _t - + - — I- + - + I .. - .+ - i -. + _ - - + - + - .. 185 + - - .. + .. .. .. + .. + I .. - + + + .. 4- - -. - :- .. + I - - 187 + + + - + .. -. - + .. + - .. 190 ‘+ + + - + - - _ + _ 4. _ _ 2+ + + .. + s + - + .. + - .. - ‘ 191 + + + .. + - - -. .5. a. + I - + 1'93 .. + - - .. + - - .. + - + - .. '- + .. - - I .. - .. + .. + .. .. I- + .. - - + .. - - + - .. - .. 194 =+ .I + - + .. _ - + - .. - _4: 195 I + + + + + + + + + + + - .. 1216 + ‘+ + .. + .. -. - -. + + - - + 4- + - + . + .. .. .. + p.. 148 + -- 193 + - + .. + .. .. .. + .. + I- - 200 + - + - +- .. + g. I: + + I- - + - 4- - -. - + .. .. + + I... .. BC]. + - + - + - - - + + + I- - ,202 I + -. + - -. .. .. - + _ + I- .. ‘ + + + .. + .. - - - .. + '- - I I I_ — 4 _ i _' I I Table VII- broth in comparison with tubes in disagreement with 5 media used.. Reactions of fuchsin lactose broth positive Confirmatorj 33.3 ”)1? E II: 3 I __ T .-_ I III; II lo”: . :tef. F. “5.2.. 30v. :joro GIOV. .‘iv. iLI‘I if I I v . l v t ‘I j ‘4 ‘t 1 y La 7% I3 .545: ‘1 .3 : A 1 _I/I' 5-41 .4 v w: TJ I I 97 I 98 + + 4 - I- .. .. _ -, .. 4- .. - 499 4 + .. 4- -. 4- 4- + 4 + .. - I100 + - .. _ 4- - .. .. 4- - + I .. - I 2 I - - - I .I' I I No grow h - + - - - - . _ + - + I , I I — - - I 10f) 4- L _. + - 4- .. 4- I - + - .. 103 .- .. - I... 4- - - + + 4- + - 4- 110 4- - + .. 4- .. - .. + + + .. .. I 4- 4- 4- .. 4- - .. .. - .. 4- .. .. I + - 4- .. -. .. 4- - 4- I - 4- .. -. I114 4- + + - + - -. - + I + + - 4- I _ 4- 4- 4- - 4- - + - + 4- 4- .. .. Illa - - c- - + - - - '0 - -0 - - I ‘ 4- - .. - 4- - 4- - .. I + 4- .. - I107 + 4+ + .. 4 + + I - - I“ I - + ' I IlN 4- 4- 4- - 4- .. - .. + - 4- - + I + - + - F. - + - + - 4' - -. ‘- -— I I In I L I I 3 I I_ ‘ I 4_ .__I_ fi_ L Table VII emedie used. Reactions of fuchsin lactose broth positive Confirmator broth in comparison with tubes in disagreement with 5 I .- I ”miéie I digéi. Ref: ?. 7.5. ”.v. 3.r. G.v. Si 48’ ZI'T 19 4o JZ'EB L4'48 ZIIIB fifig4 48 ' - I- - 4 4- - .- .. 4- - 4- .. - .. - .. .. 4- .. - .. 4- - 4- - - o u u- U + - -0 - + _- + - I00 I O - I- -' 0'0 - a - -0 - -o - '- 13:) - - - - + - - - 4- - 4- - - 133 No growth - 4 - .. .. 4 - 4 - .. 133 " " 4 4 - 4- - - .- 4- .- 4 1754 " " - + - +' - + - 4- I - - u n _ + _ _I __ - _ _ _ _ I 135 "’ - - " + - - " " ' " " '- I137 + - - " i ‘ i ' ' ' * " * I138 + - - - + - - - - - - - - ‘139 4- : 4- .. 1 - —. - - - 4- - - 140 No growth - 4- - .. 4- 4- — - - 4- n n _ + _ g . _. + _ _ _ _ 141 No growth .. 4- - .. I - .. .. - .. - 14g 4 - - - 4 - .. ' .. .. .. 4 - .. 14,3 4 - - .. 4 .. 4- . - .. .. .. I .. - 4- - - .. 4- - .1 I 4 - - .. .. 144 No growth 1’. 4- I - - .- .. .. - - - I145 NO growth - + IT - + I - + - + - .. I 4 - 4- .. 4- ‘ .. 4 I - 4 - 4 - E; I 4- - E .. 4- ' .. 4 - 4- - 4- .. - 14:5 4- - 4- - + - — .- + - _4; - - 147 4 4 4 - i - 4 .. .. 4 4- - 4 ‘136 4- _4: i - 4 .. 4- - 4- .. 4- - - 14-9 ' ' " " + I ’ " " " " " ' ’ lél + " + " i I i :1 I ‘+ + '* f i 4 - 4- .. 4 I - .. _4_-_ 4 - 4 .. .- 4 - 4 - 4 - - - — - + - - 153 "’ ‘ * ' E I " “ " + ‘ + " " L + - + - 4- ‘ - .- - + 4' + - + I 4 - 4- - 4 I .. .. - 4- ‘- 4 - .. I 4 - 4- - 4- I .. 4 - 4 - 4- .. .. I I155 + - + -. 4- -. + -. + . - + - - ‘ I - - - 1 + I - + : + 1 + - - I I .. .. .. I 4 - -. 1'. 4- 4 4 - .. I I 4T 4 I Table VII Reactions of fuchsin lactose broth §6§I3333“663fffifia£orw broth in comparison with tubes in disagreement with 5 , .media used. .Zfi'wnl'aT 4.42.?“ I I :5. I L"9r I “ef°j 3° 10:0 34V. F.r. , 30v. 4 n I; w L 3 4—4;; i .2 I :0 ie N r ‘* :5 .6 i ‘t 5 L25: ‘7 3 e t ‘3: " 12x3 .. —_I - .. .. 4 .. -. - - i 4 - .. 4 .. 4 -. 4 .. - - 4 - 4 .. .. 4- - 4 .. 4 .. .. .. 4 - 4 - .- 159 - .. Z .. -. .. - .. - - .. - .. + - - - + - - - + .. 4- .. 4- 160 4 .. .. .. 4 m - .. 4 - 4 - : Ilol 4 4 4 - 4 .. .. .. .. .. .. - .. IlU3 + - + - -. .. - - + - .. - .. I163 + - - - + .. - .. - .. 4 - .. 4 - 4 - 4 - .. .. 4 .. .. - - 1514 + + + - I -. 4- .. 4 4 4- .. - + Z. 4 -. 4- - 4 - 4 - 4- - - I + -. 4- : 4 - 4 - 4 4 4- .. .. 165 4- - + - + — .. - .. .. 4 - _, + - + - + I - .. - .. .. 4- - .. 100 + -o -. - + -. + .. 4- - + _ _ 4 - — - + I .. 4- - + - + - - 172 No growth .. 4 I - - .. .. .. .. .. - 1715 + - - - -. - -. - -. 4 4 - - 174 + - + - -. 4 4 4 4 4 4 - .. 175 + 4- + 4 4 I .. - 4 4- .. - .. .. + + + - +1 - - - - — .. I .. + 4 - -. - 4 4 4 4 4 .. - - .. .. 4. L 176 4- - — - -.I 4 4- - _4_ 4 4 .. .. 4 - .- 4 4 - - 4 4 4 4 - .. 4 .. .. i 4 - — 1'. 4 1- 4 - .. ,177 + - + - -... - - - - - - 178 4- 4 4 4- +. 4 4 4 4 4 _ _ 1'79 4' + + + +I 4 4 4 + 4 4 - .. I 4 .. 4 4 4I .. -. -4 4 - 4 - .. 13]. + - + - + .. -. - 4 - 4 - - 8‘7 + -. + — +I - .. .. - .. + - - 4 - 4 .. 4l - -. - - - - - - 4 -. 4 - 4 .. .. - .. .. .. _ - | 183 4 - i I - -.II - - - .. - 4 - - I I ; 1'84 4 .. _4_- I - 4 - .. .. 4 .. 4 .. - 4- .. i _ 4- - .. -. 4 - 4 - - positive ConfirmatoryI Table VII Reactions of fuchsin lactose broth broth in comparison with tubes in no u m a .1 ' ,IIIII. t n“ m m . e e r g a . .1. n a * .V.V.. 1va 4.4.44 44 l 2 1) A. \J 6...]. .7 DJ 1 l l l 5 1217 d le l~- Table VII Reactions of brilliant green bile lactose broth fiEEiiiv Confirmatory broth in comparison with tubes in disagree- Wthfimedia use . _ . SQJI‘EL)1€ ~40~~io%or ’1‘ o 4 , a ‘1 1 4 ’ '.-,n'1 “31-. 30". Dev. I 4‘0 ~02. ’ovo ~~OQ .- val - V‘ 24 :8 ”L34 4:? 234 4'3 $4 49 24 13 Earl/T 24 48 33 - 4 4 4 .. 4 - 4 4 4 l4 4 - - A IQ I7“ - 4 4 4 -. 4 _4 4 - I 4 4 - - I I :P + - - - + - - c - o + u - Ilu‘é I '- 4 I I I I: ' I lC‘J .4. + + _ +I .. 4 - - + - - I — I — - l I 1.00 4. _ _ I - 4 - - .. - - 4 - 4 I - .A, f I 1C” .. - .. I 4 4 4 4 - .. 4 4 - 4 I — I 115 4- - + I - + - fi - - - + I - I - I + - + I - + - *0 - -. - + - -. i I I ' I 114 4 4 4 I- 4 - 4 - 4 4 4 .. -. I l — — _- I 113 4 - - I- 4 - - - 4 4 4 - - . I — _— p l 1130 4 .. 4 I- + - 4 - -. — + - -. . I _ I __ I I ' I 131 4 4 4 I 4 - 4 .. - .. 4 - 4 4 4 4 4 4 4 4 .. - 4 4 - - wh‘h 4.33 4 q 4 - 4 - 4 - - 4 4 4 1130 4 4 4 .. 4 - 4 - - 4 4 4 4 I r $1433 .. .. - - -. - 4 .. 4 - 4 - -. n - - - + a— - I- - o -. - - 133 INO growth .. _4_- - .. I- - - - .. 4 I" " j - 4 .. - 4 4 - 4 - 4 Table VII Reactions of brilliant green bile lactose broth positiv Confirmatory broth in comparison with tubes in disagreed ment with 5 meQia used.__ Sggple Ef‘AO". )Tgf‘. 3’ ‘0. :OVO‘ LP. ifloI'. I GOV. lo. near ‘ C : ‘ J__r 4‘ 24 4—3 24 43 24 48 ‘34 46" 24 48124 48 244—8 ,1 1.34. No growth - 4 _ 4 .. 4 - 4 - I I f139 ‘ .143 4 - - .. 4 - .. - 4 - - - 145 No growth - 4 .. 4 .. 4 - 4 - 4 - 4 .. 4 .. 4 .. 4 - 4 .. 147 4 4 4 .. 4 .. - .. i ‘ + 4 .4. 149 .. - - 4 4 4 4 .. - - 4 .. I I . I 1433 * - + Oi + - + -. 4 - + I .. - - - - + + + + 4 4 4 I - - - - - -, 4 4 4 4 4 4 I .. I 134 4 - 4 4 4 i 4 .. - 4 4 . - 1‘46 " ' " - -o - - «- + i + I - I + I - + 4 4 .. - .. - .. + + I I I I i . I I 5:3 4 4 4 I 4 4 4 4 - - 4 4 4 I I I149 4 - 4 - 4 - .. .. - .. - 4 I164: 4 i 4 I -. 4 .. 4 - 4 4 4 .. ‘ 4 -. 4 I .. 4 .. 4 -. 4 - 4 - 4 -. 4 I - 4 .. 4 4 4 4 4 .. 166 4' -. -. — 4 - 4 .. 4 - 4 -, 4 _l Table VII Confirmatory broth in comparison with tubes in disagree Ivheactions of brilliant green bile lactose broth positiv .3 9,121 e f ‘r‘ "1 "W H ‘ p91 . flefo 3. :10 40V. Jo 4‘. 1. IoVo I .0; --.~’_"3.I‘ 1 I 234. 49 f2”: 48 24 48 214 4-5 :31 48 24 48 24 43* I I 17-3 O I - - - -. I - -. - - - o - c + - _..- .- -. I - -. - -. + + - - 174 4 - 4 4 4 4 4 .. -. 4 4 - .. . I I 170 4 I .. - 4 4 - 4 - -.I 4 4 - - I _ I 17:3 4 4 4 4 4 4 4 4 4 I 4 4 - .. , 173 4 . 4 4 4 4 4 4 4 + I 4 4 - - I I : 18]. 4 . - 4 - -. .. 4 I- + .. 4 . - .. 182 4 - 4 - -. .. - I .. 4 .. .. .. .. 180 4 ' 4 4 .. -. .. .. ' .. -. .. 4 I .. -. + + + - -. - - o - - + I - -. + + + - -. - - I - -0 -0 + I - -0 I I 88 + - + - -. - - - -. - - I- - I 1:15 + + + + + 4- + l + + + + I .- 17K.) + ‘+ + I a + - -. - -. + + - - + + + .- + a. + .— - - + .- + 200 4 - 4 I .. 4 - 4 -. 4 4 4 - .. I i I Table VII K' 3 -o of gentian violet oile lactose brotl positive C“ T Itory broth in axnjariconxvith tacos in disagree- ment with 5 media used. 7 ' i ' 4.1.3. I 3.3. ‘ C.v. I I i€?r L.J. ¢.v.b.l.b. b.l.b.I l.b. F.l.b F.r.p. T 234 111‘s.1 Be 48 24 48 24 48 1.4. 48 ;BI 48 24 4? I , , a. 30 l - - - .- 1 - - - + - + — + 1C1 I 4 4 4 - I - - - 4 - 4 - 4 1027 I + + + .- + + + - — - i c + . No growth - 4 - - - - - 4 - 4 ' 4 4 4 - 4 4 4 - .. - 4 4 4 us i + 1 + - + .- 1 - 1 - - - 1 I 4 - i - : - at - 4 - - - 4 106 I + - - - 1 - " - - - - - + 1C7 I - - - - + + 4' 1 + — .- + 4' ICE, ‘ - - - - 1 - - 1 1 - 1 1 + I - - 1 - - 1 1 - 1 1 + 110 I + + + i .. .- -. + - + - + 11]. ' ' " " 1'. 1'. " " I i ' ‘ i I. 115 ¢ - + — -. - i .- + - -. - + 114 "’ I I - 1 - -. - _4 - _I + + 4 4 4 - -. - I - I. - i 4 4 ,1 - - - - - 4 - - - 4 - 4 - 4 II; I + + + - + _ - 1 I - + 1 I 130 4 4 4 - 4 - - - 4 - 4 - 4 121 + + + - + + + - + - — — 4- 12,5 + i + - + - + .. + c - O 4' 1:35 4 - 4 - 4 - - - 4 - 4 - 4 136 + + + I + '0 - + - — 4 4 125 - - - - -0 - o —- + - + - + _ .. - - -o - -. - + - + - + - .- - - 4 - - - 4 - - 4 4 3123:; No growth - 4 - I - - - - - - "' " " , - 4 - 4 - - 4 4 - 4 m + - - - + - .t - - - 1 - + 140 I No growthI - 4 - - 4 4 - 4 - - 145 I No growth - 4 - 4 - 4 - - - 4 . 4 4 .4 - 4 - 4 - - 4 4 4 1?? I + - - .. + - - - + - : - - 1‘15 I I — _ I 4 I- 4‘ - 4 - 4 - 4 - - - 4 1?“ I + I+ 1 - I - + + + - - - - 101 I + . 4 I .. _ I 4 4 4 152 ~ ' ' ' " ‘ ' - + I- 1I — 1 - - 1 + - - - + ‘1'”? I 4 - 4 - 4 4 4 - - - - - 4 lg? I 4 I4 :7 - I I I 4 4 4 - - 4 4 ’lUd I I ' I I _ I L I MI v..— Table VII Reactions of gentian violet bile lactose broth brctu irlccxnoarisorlvdJM1 tines iziriisagree- {>381 ti ve ment with :3 1:18 die. used. bmple 8.61.13. Gov. 3.5:. . JOVO I DID. fizg’ar I L01). 0]..le bolobo 1.00. :fiOLODI FOrOUO 3 2:4 hrs; :34 4; 234 48 24 48 24 48 24 48 4 48 ' l 166 4 -. -I - -. - 4 .. 4 - 4 - 4 3.173 .. .. -I .. -. .. -. 4. 4. .. .. 4 4 190 4 4 4? .. -. - - - - - - - 4 184 + 1 + - 1 - - — + - + - - 1:10 4 I 4 4 - 4 .. .. - .. - 4 .. ,4. 17:) + I + + u- + i o I - — — + - I__ ___4 noim oe ricinoleate oroti :ositive Confirhatorv Tests Coaparel with the OC‘GP 5 media. Total Hot n . . . 4 1. _ onfirmed Boeitive goniirned” I 4.1 . + .. Sosin methylene blue agar 104 1C4 Formate ricinoleate broth 104 51 73 Crystal violet lactose broth 73 6 67 Fuehsin lactose broth 72 25 49 Brilliant green bile (2,5) ’19 8 41 lactose broth Gentian violet bile lactose 30 7 2 broth Eosin methylene blue agar 53 32 Formats ricinoleate broth 33 5 29 Crystal violet lactose broth 21 5 lo Fuchsin lactose broth 18 5 l5 Gentian violet bile lactose 10 10 broth . Brilliant green bile (Sp) 7 7 ~lactose broth Eosin methylene blue agar 20 26 O Formats ricinoleate broth -26 5 21 Fuchsin lactose broth 14 2 12 Crystal violet lactose broth 12 1 ll 3rilliant green bile (2p) 9 9 lactose broth Gentian violet bile lactose 5 5 2 broth Dosin methylene olue agar no growth 8 Formats ricinoleate broth 8 5 3 Fuchsin lactose broth 5 5 Crystal violet lactose‘broth 5 l 4 Brilliant green bile (23) 1 4 lactose broth . Gentian violet bile lactose 5 l d #_fbrot11 - 34 - 1'1 1' VT 1‘ L9.Jle - O. IAEL Crystal violet lactase oncth Positive Confirmatory Tests Compare-i w ith th e ot her 5 been a Tot . Total 1 . "unfirmed + _ uOl’lfl I'Zi’lei U Josin methylene blue agar 79 79 Crystal violet lactose bnath 7a 9 7O Formats ricinolca e croth 72 ll 61 FuchSin lactose broth 53 ll 47 Grilliant greec bile (25) 45 6 37 lactose broth lentian violet bile lactose 21 2 19 broth losin methylene olue agar 24 24 0 Crystal violet lactose broth 24 7 17 Formats ricinoleate broth 21 2 l9 Fuchsin lactose broth 11 6 5 Gentian violet bile lactose 9 2 7 broth . Brilliant green bile \2,) 5 5 lactose broth Eosin methylene blue agar 2O 20 0 Crystal violet lactose broth 20 3 l7 Fuchsin lactose broth 15 3 2 Formats ricinoleate broth 13 2 ll Rrilliant green bile (2,) 4 l 3 lactose broth Gentian viole bile lactose 3 l 2 broth Eosin methylene blue agar no growth 7 Crystal violet lactose broth 7 3 5 Fuchsin lactose broth 5 l 4 brilliant green bile (2p) 3 3 lactose broth Formats ricinoleate broth 0‘3 (3 t O rh F‘ 44 F" ()4 Centian violet bile lacto' broth 1‘9 7’1’ 8 -/ o 3’11: Fuchsin lactose "= it ve Confirmatory Pests Com- oarei with th other 5 meiia Tgtal Hot Confirmed Confirmed Eosin methylene blue agar so 85 Fuchsin lactose broth 86 27 59 Formats ricinoleate broth 7O 19 51 Crystal violet lactose broth 57 7 50 Brilliant green bile (2“) 56 8 2 lactose broth Centian violet bile 24 3 21 lactose broth Sosin methylene blue agar 22 22 O Fuchsin lactose broth 22 8 14 7ormats ricinoleate broth 18 5 11 Crystal violet lactose broth 2 2 10 Brilliant green bile (3w) 9 1 8 lactose broth Centian violet bile lactose 2 O 2 broth eosin methylene blue agar 21 21 Fuchsin lactose broth 21 8 13 Formats ricinoleate broth 19 2 17 Crystal violet lactose broth 15 4 11 Brilliant green oils (2”) 4 4 lactose broth Gsntian violet oils lactose 5 2 5 broth Losin methylene blue agar no growth 11 Fuchsin lactose broth 11 4 7 Crystal violet lactose bnoth 5 2 3 Formats ricinoleate broth 5 2 5 Brilliant green bile (3g) 3 3 lactose broth 0 Gentian violet oils lactose 3 l broth ' I'. r " _‘i, .‘-. ‘ J“ ' ‘ i.“ - \ _‘I - - .ctoee broth lQolulVC ~coniirrnau jrilliait reen oile tor; 1 i tory Fest: Coigerei v-2h the ”the“ 5 beiia. +’i‘otal tot Confirmed ' CooCirhei Eosin methylene blue agar 54 54 Brilliant green bile (3w) 5% 9 45 lactose broth Farmate ricinoleate broth 48 7 41 Crystal violet lactose broth 42 4 58 oucmein lactose broth 55 7 2o Gentian violet bile lactose 20 2 l8 broth Sosin methyelene blue agar 11 ll 0 Brilliant green bile (2p) 11 l 10 lactose broth Ebrbate ricinoleate broth 9 2 7 Fuchsin lactose broth 8 1 7 Crvstal violet lactose UnDth 5 l 4 Ceatian violet bile lactose 2 2 ‘rotP. Josin nethlene blue agar 8 8 Brilliant rreen bile (2r) 8 8 lactose broihi Formats r13inoleate biota 8 l 7 szstal Violet lactose oaoto o o Fuchsin lactose broth 4 4 Centiah violet bile lactose 3 5 hrcth Losin methylene blle aiar Yo prowth in. 5 jrilliant green oils \2,) 5 5 lactcmse brotii 4 Formats ricinoleate broth 4 Fuchsin lactose broth 3 5 Eryst violet lactose broth 5 3 Centian violet bile lactose 3 l 2 1‘1 ‘_ . -rw- llJlJ .o. 111I I" + - ‘ ' e. ,- 0 1 1 -, _. ' .. - '1 i - - L ..- . oenoian Viol-u oiie _actose crota -~: 3 c-4i1r finJfiJ « _ A a - ‘ u L -- -‘ (p.21 .2 w awhi 1* f'. - 0 n7 .u‘n 3‘ 5 ~ I. .. J\.v g. _ . - kn / .1 . ' L A —j- ..n + +_ 'fifi ~.‘f.o_ ' _ 1 ~CL3 Cu u"-.'... _L' Li'u. .‘N . 4' - 1; Iain; 1 .~ .4“. 1 ’ r 1 - ‘ ~' 0“ \ (j I- 40: n iatoyijn? oiie a‘ur 23 “J ft . 4. - ,- - a. - 1 ‘ + up A r) ) '. r~ . V ‘ J.nitial 'vzolet, _ile lnxctoeea ca 4 so \L‘ Ol'ot gl ‘1 ‘ . P . 3 ’ +‘ ‘-.I 3,. . . r. I - I donnate r cinolea e or on N7 4 co 1 fl ' -‘ ‘0 A , v" , L' Crystal VLOll’Jt lactose growl f1 ‘JBhB r" n l ctJLe broth SI ‘) 3rilliaot {reel bile \2p) lactose broth H F" [\f' Cl 0 "J H EC: 1‘ O ’4 l“; ll 1‘0 1 , 1" _ ,1»“ “F JOSLH me fly ene bile i=1? . Fuchsin lactoSs broth Crystal violet lactose broth Brilliait green bile (by) lactose broth 4 Jentian violet bile lactose broth N r? {‘0 F0 N} l4 H bJ o m r“ ...‘-‘. ‘r -.~1 m ,.. ,n mfibfifltde olue efler broth 3“illiaft green bile \cfl) lacto se oro tn Formats PlCLflol"&te broth no growth 4: [\L - 38 - n J ._ O ’ 1‘ :l v. .9 . '. 3. onaerstanlinr of the leviations can be cotaiiJc. In gave positive completed tests from eosin nethvlehe blue ar. One hunlrei snl eleven tubes were positive in rystal vio ole t lactose broth' 103 were 9 osi ti ve in Eachsin lactose broth; 09 were positive in brilliant green bile oositive in g: htian violet a _ (2;) lactose broth; and 43 were bile lactose broth. rortv-four of the 170 cultures in formater icizm leate broth, 13 of the 111 cultlres in crystal violet lactose broth, 28 of the 10: cultures in fuchsin lactose broth, 8 of the 63 cultures in brilliazt green bile (at) lactose broth, anl ll Ol the 43 cultures in qentian violet bile lactose broth did not confirm. ihe following reactionsxvere produced by these cultures: Formats Brilliant Fachsin Crystal Jen' ‘ a ricinoleate green lactose v.1. v.b.l. broth_ bile llbl broth, broth b‘glh_ 5 l 7 Lactose 5 9 5 3; Dextrose 7 3 4 l L-o+-vr+uza+0+14- so, .3 14 s L r—D e’v'P-ZIR- C-r-I‘ 13 l 4 23 L+D +3.7 9-,.I'i- Cf l" l 2 * These or3anisns are identified only by the characteristics obtained on lactose (L), dextrose (D), vo»cs-:cosxalei\19) ethyl red (£3), citrate Jelium (C), ani insole (I). In crystal violet lactose broth, 130 of the 215 elltures produced Comparing the other nedia with (n, in (D 0 crystal violet lactose broth, it was found trmt 7. gave ’0 ositive completed tests from eosin nethylene blue agar; 110 produced {as in formats ricinoleate broth; 89 formed gas in fuchsin lactose broth° ’ 55 fermented brilliant green bile (2;) lactose broth; and 35 prodcced gas in crystal violet lactose broth. Twenty-one of 151 cultures which produced gas in crystal violet lactose broth were not identified as members of the coli-aerogenes group. In the other broth media the following number of cultures was not confirmed: 16 in formats ricinoleate broth; 19 in fuchsin lactose broth; 8 in brilliant green bile (2}) lactose broth; and 6 in gentian violet bile lactose broth. The reactions of the organisms identified are listed below. Formats Brilliant fuchsin Crystal Gentiin . . rreer 'ac ose v . v o. RieligiiiteOiie ljb. Lbroth broth bioth' Lactose 5 4 2 Dextrose 2 4 5 5 L-D+VP+TR+C+I— 8 4 9 8 l L4—D 41.72-33’ITZ-C+ I— 5 l 5 l L+D+ve-:a-C-I- 1 2 4 Studying the 140 cultures which produced gas in fuchsin lactose broth it was found that 88 gave positive completed tests from eosinznethylens blue agar. one hundred ten produced gas in.formate ricinoleate broth; 89 produced gas in crystal violet lactose broth; 52 le ( _ L n k ‘ fl) lactose orotn- [0 Ho formed gas in brilliant green b and 34 produced {as in gent ian violet bile lactose broth, Fron 47 cultures of the 140 cultures, menoers of the coli- aerogenes group were not isolated and likewise from the a the follmvin; numoer of cultures was sot identi- pa 7—“ of. her .116 cultures in formats CI) fied as belonging to this group: 2 ricinoleate broth; 14 in crystal violet lactose broth; 9 (I) cultures in brillim1t green bile (2H) 1 cto e broth; and p) '71 8 alltures in gentian violet bile lactose oroth. ihe cul- tures gave the following reactions. ‘Tuchsin W.illiuit Crystalv. 8T0rmate Gentian actose,. green lactose ricinoleate v.o.l. bile b. l.b. broth broth broth Lactose 7 3 3 g 2 Dextrose' lO 4 5 3 L-D+VP*IR+C+I— 19 L+D+V3- u-o r? \J ) + H 1 m HHCJJH a) L+D4VP-$R-Cé}- 3 CD [—5 In brilliant green bile (3g) lactose oroth 7 o a T',‘ the 215 cultures fermentel with gas proiu ction; be 0 H) this rroup gav eitive co;nplet ted tests on eosin methy- ‘v \D L} O lene blue agar. Sixty nine oroduced gas in fornate ricino- leate broth; so formed as inc ystal violet lactose broth; 48 formed gas in fuchsin lasts :se broth; anl :38 produced gas in :entian violet bile lactose broth. In each of the melia, cultures were found which did not confirn. Ihe nfirh were as O cultures in each medium whichtiil not c - 41 - follows: lC in brilliant green tile is.) lactose broth; I 1C in fornate ricinoleate broth; 8 in fuchsin lactose t" broth; 5 in crystal v olet lactose broth; e.ni 5 in O'\ broth. She cultures «ere cla sified (i. (D violet oile la cto as follows: lrilliant Ljrgstsl Tucasin Formats Ljiitiaa green Vin lactose riciholeate J.J.l. b.l.b. broth broth brotn bi th lactose 3 3 l f stress 2 l .i 2 L-“¢Vtu.12+3+I- 3 3 4 2 13 L +D+Tr?-':.:Z-C+I—— 2 3 L+O+UB- ’-3-It_ l l a Forty eight cultures proiuccd gas in :entian violet O D. P; bile lactose oroth fron this grouo of 215 cultures vii sent. In formats ricinoleaJe {.L r—Jo p. ’— ,4 C) Cr U) f O O K G L_J ( D c+ CD \_ 1 r3 LI) (1 broth, 4d of the cultures, in crvstal violet lactose broth . +m -n:'_.- , «0 OfObi (qt) 0:. t-l3se ()3 (J1 O H.) C’- 6.) O 5.. H C+ p t ‘5 (D U) U 0") L) [>4 p J y. H F) ’._J L13 ('3 c f O ( . u. cultures, and in brilliant 3*een bile (2”) lactose broth 28 prciucei 305. Den of tee 48 cultures in gentiafl Violet bile lactose orotn fail:i_to 1H3 ilentifiel cs moloers of -aero- d :JJ (D r4. I \0 CD *3 O 3 (J {L U) '1 P Q 0 I 4 [3 cf- (D C (1' ’ 4 ’- \L *3 L H r4. 5)) O O H 0 col. genes cultires were not con iriel in the IQ ollo-xi n; cultures; 8 in formats ricinoleate broth; 5 in crgstal violet lactose broth; 10 in fichsin lactose broth; “hi 3 in brilliant green bile (2}) lactose are L. ?hese cultures gave the following reactions: / brat. rl .‘ 5-3.1. Ll ,'.,‘ \J. -- J. "a 0-41.}. x v u . . J‘J-§U .- 1.: 7d 5 n3 1.. +2. 3 3 5'7 is} ) 4. [L --u+; A.— ,— “ h+uj+' T T 1 tot V L v.-. 1‘ U a ‘ v 3. ‘ O . 2 7‘ 1 1* ‘— .llv 3.4 My; LU .«u nv 7. .11. I — rTJ _' 1 A 0 5 N to; numbers q '1 .& aha ‘ ) .E‘t, C CLlOfls “3 1‘} a I-;. «aols .a. *1 3. .l .11.. Il'.‘ rmatoi, o I L .N’ I UU. d iarl J A+ J L» ‘d hate n .L. so ‘1 A ac 0‘;— Q L Du HI! 1: 1‘ .., i. l; 4. .0 ”.3 l 4. inf. \Hd 10 19 e by 01 r .l nav so I—13 f‘. 1-24 13-":- - J+‘. L-D+V?+£Q+C+I- .1; r. a; as CU "I U C}: .1 .\ 31 .‘ A. I 3 WWW .ru C 9 43 l & i i; or elk A... k r: 3.. 3.. Meiiun I. C V 01L .0 . .3. 3 “ a! is v a... V .1 a." .0 Jr. .1!“ IHA no 0 ; _nl . “a nf—A Ow I). .11 fl 3 .‘J' .4 Nov 1"“) -JJ~ n3 o1* mu _U Q.» ..U .9. L U 3 .1 a]. a U t. 3 cl n aroiuc ‘ On ‘ .1". 1‘, I? V S from any 9. ‘1. ll) 'ollow' .L in.) .LV "1 L districts. V8 . l 1. soect v .- etueen loc. L) ’l ."\p U. i ’10 rel at ‘1 L AI shows 0 L Table LI lelction of tecilts of Tests Conpared to the Locality Sections Location Results of Tests Pesos Number fiercentabe i tests not in in Aireenent Arreeacnt Arreement .. " + — U " O l Vorth—west part of 8 9 18 94.44 Ltate and Upper Peninsula 2. Worth-east part of 14 2 28 57.14 State 3. South-west part of 10 2 29 4l.37 State 4. South-east wart of 32 5 45 b.28 State "xvi'fi If: Tr gr H‘s/J; 4d-v.l 7‘ In this s3ries of coiifirnatory tests made on £03 water sanjles, it was found that no medium was more specific in testing sangles from one locality than from another part of the state. ihe results obtained were prooortionately in s fir enent and in disagreement in the variOis media. No one section of the state was in total disagreement in any meiinm. In two parts of the state the total samples in agreement were aoproainately one- half of the number 'n disagreement while in the other two sections the total niznoer of samples in agreenent and in disagreement were about equal. These facts new be of interest; only, due to the fact that a limited number of samoles were received from any one section their value may cc of little significance. The first group of cultures, those in total agree- ment, indicated that in 365 cultures the liquid media and eosin methylene blue agar were equally good as con- firmatory media. On 24 eosin methylene olue agar plates colonies, which were produced, appeared to es menoers of the coli-aerogenes group. Dhese cultures did not ferment 3 lactose broth. 'Prami fe rs fro; .— - the lactose presumptive tests made from these cultures into the five liquid media produced no gas. The reaction of thi sgroup of cultures indicate the fact that the iieitifi cation of colonies on the eosin me hylene clue agar demands extreme exactness. The possioility of not identif ;rinf menoers of the coli- aerorenes groug and of‘not transferring such meM*o rs of the Jsc‘ericdia—herobacter group to lactose broth tubes is inevitable with.zrny cultures. orty cultures failed to grow on eosin ne thzlene blue agar and also failed to produce fas in the five broth media. Dhese cultures showed that fro n all of the confirmatrry meiia used resxlts sinila r to those from eosin.nethylene blue agar were ootained. The failure of the coli-aerogenes grouo A. 1 to grow on eosin methylene blue agar is one of the out- standing criticisms of the medium; the above showed that _J ere e.nent in that no members of (O y. the liquid media were in the coli-aerogenes group were identified. From 2 cultires in methylene blue agar members U) which did not grow on so of the coli-aerojene 8 group vere found in the liyiid media. These cultures did show that the liguid media were superior to eosin rethylene blue agar. lie 39 cultures which showed.dumrepa ncies but were not identified were classified as being questionable; hese rec ults were not considers further. Fr om the cultures which shov.ed disa reeme nt in the li1uid confirmatory media and on the eosin methylene blue afar, it was found that fornate ricinoleate broth gro- duced 12 6 coli- -aerogenes cultures from the Slb positive (I) ctose broth tubes. Forty-four cultur (D . presumptive l s pro- Dr. duced gas in formats ricinoleate broth but thes'e were found to be false positive tests. - 46 - eosir nethylene blue agar as the coniirmatorj Using medium 121 of the 215 cultures in disa to be genbers of the coli-aerogenes group. more cultures of this group were identified as coli-aero- genes organisms on the eosin methylene blue agar; these cultures did not produce gas in lactose broth. Forty- one of the 215 cultures did not produce t pical or atypical growth on the eosin methylene blue agar. :ifteen cultures produced no growth on the eosin methylene blue a ar. Josin methylene blue agar groducei 7 fewe oositive co;- firmei tests than fonnate ricinoleate orotn. methylene blue agar gave 38 false negatives. The liquid confir story nedia in order to cohuare in Specificity vith eosin methylene blue agar must us in- cubated at 3703. for 4s hours. This period of incubation L '1 n oi the 0 thus makes the total tine required for isola i coli-aerogenes group in liguid broth media of the same duration, 72 hours, as needed to identify the organisms from eosin methylene olue agar. A shortening oi the period reiuired for the detcrnination of tne presence of the coli-aerogenes group from water is desirable. In order to isolate the coli-aero enes group from eosin methylene blue agar it is necessary that the plates be streaked very carefully from the lactose broth presumptive tests tubes. Overgromth of the coli-aerogenes iroup by other organisms results if care is not taken. She failure to transfer all tyoes of colonies on the eosin me hglene the sli , v .Q 0th v9 1 . however, or o ’ V ‘ - C -‘u ’. a M , . ffiil Q47 8 D ‘J .4 ngared V 1 \r r 331‘042' 8X1 (1 1.. ol ’3 a rgstal violet lactc ESQPTKDIQGI “'1‘: Ll l um a J ’J J he a V a Q s. b o ”I I u -ield -J 01 "‘31 ‘1 O J. (Y O 0 .1L — e P t O O a 3 t O n 3 W s .1 .i U .L .l .8 P. 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