"\|\\|\N\\|H$l\\$l I i , , — __—_— — —__—— fl— — i 7 _ ———' — ,_’_’ __’__——— ___——_—— — __—_4 MNIWIHN 1 1 é \M FURTHER STUDlES ON MEDIA FOR THE COLIFORM BACTERIA Them; for the Degree of M. 5‘ WCHIGAN STATE COLLEGE Eleanor Laverne Gilmore 1944 IHESIS 5).“), N. '1: ‘z’a, A~ -‘i' *7 14“»‘Imn 6‘: 5‘ p‘. .v I; V; . ~ g sid‘isfif n X ‘ ' . _ng~}&‘9$§; ,r' I < s.“ . . 3" 19m §~J~1 ‘. ._ _ ‘_. ._ . {hw‘fi' 34-- A; . " ht...‘ A .‘ (“pit-CA 4 .H ‘t:.’ Ipfi‘L-v' 3‘ p150... .fiT‘Jn“~'\”; u I1"..- .3 '0‘ _' 'Q‘fik.‘ .V’v‘ \ 9., 9.37:1 . 5"k'\" k 5' 3L)? , 53 .,‘_ ‘ ‘3'“. 2+: “3"“; its. .. V ' :+,3"’i*‘-.‘s-~1.-,- ' ..,WJ ' w. :. 'l . 1.:.j-::‘«s;n.§-',u‘..g -; . '_ V 0} j 5 .13: . . '1. cs" ‘ ~-‘-‘+.’9’vk- . ‘ ‘ A-‘q or- ' x \l" 1:921; "i ..,* .H.’ :0"? L‘URTIER STUDIES 0}: mom FOR 173*? 0014130117; momma by anHOR mvnmzrn elm-10m P A TIESIS Submitted to the Graduate School of Michigan State College in partial fulfilment of requirements for the degree of EASTER OF SCIENCE Department of Bacteriology and Public Health l9hh 'I'H ES‘S -‘ C’I‘C’T‘ 213* A‘L l‘ e I J yr“; “it I wish to xpress my sincere apprec- iation to Dr. N. L. Hellmenn for his many helpful surwcstions on the \J C) 1 material presented in trio thesis. II III IV VI VII VIII IntrOQUCtiorloooo0000000000000...o HiStorical I‘lGVieWOOOOOOOOOOCOOOOO Procedure...OOOOOOOOOOOOOOOI0.00. —D ResultSOOOOOOOOOOOOOOOOOOOOOOOOOO DiSCuSSiOI-100000000OOOOOOOOOOOOOOO Summary.......................... D RefeltenceSOOOOOOOOOOOOOOOOOOOO0..-D APPeIldiXCOOOOOOOOOOOOOI...0...... The fellovring studies were initiated by the questions arising from the use of lauryl sulfat te tr"ptos e broth as a preSInptive L'LGQIUH for the isolation of eoliforn ergon— iSIIS free water sujplies. Inasmuch as lauryl sulfcte tryptose broth has been accepted by the Co; tee on Standard Iethods for the Exem- on of aster end Sewage of the American Public meeitn H. .' .. .1. 1.118 U $~~uOClLLlLfl, it is iiperutive that its .ropeJ“t {.40 C.) U) H. :3 H (D I '0 gsrd to tne '“o't; of coliform organisms be fully evaluated. If a bet W31 fornuletion can be developed it should be done h.) before the present formula becomes S‘sntcid procedure. One of the first questions to arise hes been cost. Lauryl sulfate tryetose broth costs approximately four times more than standard lactose broth. Laboratories where marked economy in operation is necesse )4“? hesitate to use this medium unless they are fully convinced that a cheaper eredium could not be substituted SLCCUSUlulIVo A ~ number of ,I. eOple he ve esxe whether it would be possible C1 to reduce the conCentretion of tryptose from two percent to one percent thus reducing the cost by half. To date the answer has been that the labor costs in the use of this medium.would be less in laboratories where positive pre- - 1 _ sumptive tests are encountered. in addition media costs would not be materially different in that confirmatory med- ia would be unnecessary. This is true, but if a reduction can be hede without reducing the efficiency of the medium, it should be done. Eurther research is necessary to deter- mine the eileet 01 various concentrations of trvotose in the medium. Another question presented has been that of niniral numbers used in comparative studies of lauryl sulfate tryp- tose broth and standard lactose brot . In tile ori inal work oy Jarby and hallmann (2) the minimal numbers used were from 40 to 50 per ml. rhis is not a minimal nuzlber when ceiupared to the coliform concent ation in water supplieS‘where the coliform index is less than one per 100 ml. further re- search is necessary when the coliform concentration is app- roximately one or less per ml. rhis still would certainly be a much nearer approach. A lower concentration would be difficult to evaluate because a higher incidence of n g'tive tests would be encountered. Still another question arises concerning the relative growth rates of soonericlia coli and Aerobaeter aerogenes. rhe original studies of oarby and hallmann (2) were made with B.coli and no tests were carried out with A. aer 0 er es . "o 9 4w -_ “'— Inasmuch as both organisms are present intiater and are both CwiSifiOIed representatives of the celiforn group, data should be Obtained on growth rates in leuryl sulfate tryp- tose broth and standard lactose broth. Along with studies planned to ensuer the above 03’s“ ions, it was also thoudht advisable to deternine the value a 3 ‘ \ 01 euiin; so ium cnlOLi e to both lauryl sulfate tryptese broth and standard lactose broth. Only tryptose oroth new tested in the St;dies n media for the coliforu >~~ by Jerby and Tellnunn {2). 1131 C111 C111 113118711113 '1! In thl Darby and Hallmann 03) presented a new medium, lauryl su‘fate *ryptose lactose broth, for the isolation of coliform organisms fron water supplies. rhey reported that t11e use of a formula containing tryptose, sodium chloride, and phosphate buffer grew out more members of the colifor m group present in the water as indicated by the higher colon indices obtained with this medium. Further, they reparted that thee'HC1tion 01 sodium lauryl sulfate in concentrations of 1:5000 prevented the growth of gram-positive lactose fer- menting bacteria without any appr e01aole inhibition of the growth of the coliforn group. 1hey recongended that the med- ium be use d in place of standard lactose broth, A. “. H..A. (31) in the presumptive test. They also recommended that the medium be used in place of brilliant green bile lactose broth as a con11ruat0’" medium. '\ ficCrady (17), in a summation of comparative studies made by a number of laboratories distribL1ted throughout the United States, reported that la1ryl sulfate tr, gave a higher incidence of true presunpt standsrd lactose broth. is reports that a sunnation of re- sults obtained from both 1n1nisi1ed and finished waters showed t.1at lauryl sulfate tryétose broth wave 56 to 58 percent less primary pas positives t1an standard lactose k} .1 broth. this would indicate that the statenent by sarby and allmunn was correct in that lauryl sulfate tryptose broth - h - allowed the growth of greater numbers of colifora organ- isms initially present in the waters examined. KcCrady also showed fro; the collective studies th u~t lEUlfl sulf- ate tryptose'broth; field ed far less false presumptives as measured by the final identification on eosin methylene blue agar in the completed test. Levine (16) in lth demonstrated that lauryl sulfate tryptose broth gave a higher precentage of true presumpt- ives than did standard lactose broth. ‘ Perry and Iajna (20) in a comparative stuuv of E. C. ‘1 3 {)4 1e J : inn and lauryl sulfate tryptose broth also found the latter median as well as 5. C. mediun to be superior to stand? I'd lactose broth in the number of true presumptive tests obtained. Mupp (9) found on 1nu1unaool1s plant effluent that laury l sulfate tryptosc broth gave no false positives whereas false positives were frequently encountered in standard lactose broth. STANDARD LZCTOSE? ROT 2h hrs. #8 Hrs. Total Confirmed COMpleted 5 £16 51 5 o 1RYPTOSE LRC TOSE ERCTI UIiK SODIUT LAUN'L SU'EKTE 2A Hrs. LS hrs. Total Confirmed Completed 0 O O O O w q ”The above confirmed results from lactose brot11ui1u not pass the completed test because of their failure to _. >- grow in eosin methylene blue agar.” The Counittee on Standard Ketimo~ s for the Sxemination of water on nd S wage reported to the Labeletory Section the following reco 1endntion for the use of lauryl sulfate tryp- tose broth. "In these stande1d tests laur"l sulf te tryptose broth may be substituted for lactose broth in the e: CdiULUlOfl ofa ll waters e:cept £11131 filtered, treated and filtered-treated waters. It may be substituted for lactose broth also in the e""‘1n tion of final, filtered, treated and filtered-treated waters provided the laboratory worker has emvly demonstrated by correlation of positive completed tests (isolations of coliform organism ) secured through the use of lauryl sulf- ate tryptose broth vit1those secured through the use of lactose broth, in t; 1e examinatiOn 01 such waters, 'het the sub titution results in no reduction Iron the ”38 it:,‘ of col- iform organisms indicated by the standard procedure using lactose broth." The report was approved by the Section and reconxended for inclusion in the next edition of Mt ndord Hetl1ods. At present this medium is in routine use in theznuni- cipel water p ants of Lichige- for the determination of colifo 1 iLditjes in raw water without confirnetion by brillent green bile broth or eosin methylene blue ag€1r. > see 713m“ VA-J VA“ The cultures usc1 in this study were Sscherichia coli, Aerotacter PCIU east and Pseudononas aerv»1noe1. A 15 to 17 hour cultzre 3r wn on a pla in agar slant was used in seed- ing the var‘ous media. in cider to obtain minimal nunbers, or :anisns were transferred fr 3 a 24 hour asar slant culture to a tube of sterile saline solution. Sufiicient or3€nisms were added until the first opalescent turbidity which was visible to the naked eye was obtained. ln t11e ca se of S. coli the app- roz_nate number at thi 8 density is 50,00u,000 per ml. rhe dilution was then further diluted by decimal dilutions of l to 100 and l to 10,000 until the count would be approxim- ately one or less per ml. rhe last dilution was made by transferring into a flask containing 99 ml. of the medium used instead of sterile saline. One ml. samples of this broth in each flasK was plated immediately to obtain the initial counts. rhe flasks were placed in the 3700 incub- ator and one ml. samples were plated at 2, h, 6 and 2A hour intervals in apprOpriate dilutions. All plate counts were made after 48 hours incubation at 3700. .1. Care Wes tM en in pret ation a1d ste rilizat “n of the 1—!- media. hn adjustment by the col01w1r1etric method vus made of all the media to a pm of 6.3- ; 7 _ The selection of the pH 6.8 was made because Derby and Hall- mann (2) found that the greatest increase in growth occurred at this pH. In the first series of experiments a comparison of the growth rate in various percentages of tryptose was made. Bacte- tryptose in concentrations of 0.5, l, 1.5 and 2 percent was used in a medium containing phosphate buffers, sodium chlore ids and lactose in the same concentrations as used in lauryl sulfate tryptose broth. One ml. portions containing E. coli were plated and incubated. In the second series one a nd two percent tryptose con- centrations were selected for comparison with lauryl sulfate tryptose broth. The same technique as previously mentioned were employed. Twenty nine sets of determinations were made with E. coli, 25 sets with.A. aerogenes and 18 sets with Fe. aeruginoSa. A large number of separate determinations were made to eliminate variabilities obtained in individual sets of determinations. In the next series, instead of measuring the effective- ness of the media through the determihntibn of total numbers of organisms, the first appearance of gas fermentation and rates of gas production were used as measurements. Minimal numbers of organisms were obtained in the same manner as for the studies on the growth rates by plating. How- ever, the dilutions were carried out entirely in sterile -8- physiologyical salt solution instead of usin media for the U) final dilution. ren r . of the desired dilution was seeded into fermentation tubes containing double strength media and the tubes were read at the end of 13 hour and 24 hour periods. H.) In the irst series, one percent tryptose broth, two percent tryptose broth and two percent lauryl sulfate tryp- tose broth was compared. Due to the variability in the rate of gas production 37 sets of fermentation tubes of E. coli 1 . ’ .— and 29 sets of A.aerogenes were seeded. In the next series these same media were compared with standard lactose broth to determine the rate of growth and the amount of gas produced. we determine the influence of lauryl sulfate in the stimulation of growth, two ml. of a 5 percent solution of sodium lauryl sulfate was added to standard lactose broth. This is the same amount of sodium lauryl sulfate that is added to lauryl sulfate tryptose broth. Standard lactose broth was used as a control. RESULTS In the first series of experiments involving the various percentages of tryptose, a 0.5 percent concentration of tryp- tose broth showed consistently lower growth rate as compared to the other concentrations of tryptose. fhe 2 percent conc- entration has proved to be the most favorable for the growth of the organisms used in this study. However, the 1.5 per- cent tryptose broth gave plate counts closely approximating the counts for the 2 percent tryptcse broth as may be seen in graphs I, II, III and IV. The six hour plate count has been found to be a more reliable indication of the growth rate of an organism in tryptose media than the 24 hour plate count. She trend of the 6 and 24 hour counts in a series, frequently does not coincide as may be noted from the graphs and tables dealing with plate counts. For example, lauryl sulfate tryptose broth shows a higher count in 6 hours than standard lactose broth, whereas- the latter medium may show the higher count in 24 hours. These differences in plate counts would indicate that the organisms has changed growth phases from the IOgarithmic growth phase to the negative growth acceleration. Thus having reached the negative growth acceleration phase 24 hour count is less re- liable. Tryptose broth stimulates the growth of the viable cells to a greater extent than d9? standard lactose broth which fact is more evident by the 6 hour plate counts. Phe bacterial pepulaticn of the flasks varied at differ- - 10 - m.HH asses w.nH pczoo .>m Om!ma "w .mHiHHu m .oalm "N .mto ”H QOHmmm moom m .memsem homeowmsm menace Hewpepown an cmopazsp mo meowpmhp smocoo macapmb mo somfipmasoo .>m s m m . mm H.@ pesos - m N v H - am.s a v m m as s.a peace .>m , m H - mm.o \l.’ .Haoo canvasenomm muse: H caste é ILL - QOHmmm msom v mesne .mma-Homum .oom-HmNuN .omNuouH .maeewm socesesne mam pesos .>e , s N H an Nam peace .pw e v m N H am.H mmN pesos .>m m N - H . sH oNN pesos .>m - m N , H m.o .Haoo canowhmcomm msflm: mussoo Hmfipmpomn an mmopampp mo mcoapmnpccosoo mSOpr> mo confined Boo HH compo J -12- Qonmm mmsom m xHOmsus .OOmsuHoomum nooomnHomNuN .oomNuouH .mmmewm seemseona ommq mpssoo .bm v m N H an mmmv mpcsoo .>w w m m a sm.H mama mpszoo .pe ., m m H ‘ ma mmHN mpfidoo .pw N H em.o .HHoo eH:0Hnenomm meHms mussoo Hmflhmpoen an mmopdmap mo msOHpespsmosoo escapw> mo somfismmioo HHH manna - Jig - QOHmmm moom ww HomHum .oomHuHooHuv .OOOH-Homum .oomuHOHuN .OOHuouH .mmmssm seemsemna Seam pence .>m m m N H aN Emma pesos .>w m m N H am.H swam pesos .>m m a m N H aH same pesos .>m m a m N H mm.o .fiaoo manuaheaomm msfimo mpcsoo Hwflaepown an mmopaapp mo meowpmhpcooaoo mSOHHwb mo somflpreoo >H gamma - 13L - ent time intervals in the same series and for difierent series. A c; it ice 1 survey of the data in the tables listed in the appendix show definite trends but are better under - steed graphically. 'nany methods of graphing the data were tried but in eve cry instance only a three dinensional graph would rive the desired picture. because three dimensional graphs are impossible to depict clearly on a flat surface, other means had to be devised. I- J !.J ows: Bacterial H» S O- :1) rhe wr. he used were obtained L- counts for each time interval were arraa nged serially from the lowest to the highest counts. The figures were class ified into four or five representive groups as for example in cragh I, the first group included counts r8 nging from O to 5, the second group 6 to 10, the third group ll to 15, and the fourth group 16 to 20. The number of plate counts ‘1 falling into each group were recorded. block graphs were made based upon the number of sagplesa a ppearing in each .L g group. Secause it was desired to show which - edi u1 rave the highest bacterial counts, increased importance was given the frequencies in the higher brackets. :hus in Graph I, O to 5 was given a value of one unit; 6 to 10, two units ll to 15, three units; 16 to 20, four units. Sherefore, a medium with a high count frequency would give a wider block than one with a low count frequency. If the counts varied consi rably, the block graph would still shit the extent of the high counts by the width of the block. -15.. nverages of the counts were also used, although the writer ’1 is aware or the danger oi averaging widely diver ent bact- erial counts. Lowever, the avera ges arree fail lyv ell with C‘ the block graphs. In justification of averaging the counts, A it may be stated that to a very large extent t;‘1e counts varied less than 25 percent from the mean. such data can be averaged without danger of oste1n_nh a suers not representative of'the growth rates. In a second series of experiments where one percent tryp- tose and two percent tryptose were eel red wit: 1 L S.T. (laur- yl sulf;te trvptose) broth graphs V, VI, VII, and VIII in- dicate that two percent tryptose broth is slightly more eff- icient than L. .T. broth. dowever, the difference is nes- ligable. Because these two breths contain the same iusreaients with the exception of sodium la ryl sulfate being add d to L.S.T. broth, the plate counts should approximate each other. In another series of experiments, sodium lauryl sulfate was tested for its stimulating or inhibitory pronerty. It eund that sodium.laur fl sulfate, when added to standard lactose broth, occasionally showed a slight inhibitory act- ion but when used with tryptose, a very slight stimulation. action was noticed. This is shown in the Appendix: in Tables VIII and 1X. Tilerefore, the difference between L.S.". broth and two percent tryptose broth may be attributed to eXperinental error. Lauryl sulfate tryptose broth is un- questi01ably a better medium than one percent tryptose broth -16- 0.0m< N.mom pesos .>w $.0mm peace .>w w OOHmmm maom m xHOOHum .oooH1Hmsue .omsuaomnm .oomuammum .omwuo«H .wmmcem assessmea v.ama peace .be m e a .r.: m WGSOO .bw ‘l T‘Z‘i CQ LO CR2 H . F1 1; ‘—' E" H n.0wm pqsoo .>.mw msfims menace Hwfihepomn an eaves mmopoea one ewues .HHoo efiflowaenmmw mmopmhnp mo confistEOU > gasps -17.. mmsom vm xaooaum .oooauamsne .Oms1Homum .oomuamwnm .omu-ona .mmmqmm seamsemna av 9:300 .>w m fl .L.d .ecapm m.mb panoo .bw m H HQra mad m.oma pesos .>w e m H _ ea meH PCNHOU o 52w. m «V m .H “o“ ®.WVH P2500 o>u m m —N — H . .U.J I .Haoo mflnOHpenowm .cflm : mpssoo Hmflaepomn mp eases mmopowa paw memos emopmkpp mo semflpwdsco H> sawpo - 113 - QOHmmm mDom m xaooeum .oooe1HOOmue .ooom1aoomum .ooow1Hooanm .oooH"H .mmmcem seemsemae e.mama pesos .>a m e m m H swmz x 1 1 . a.u v.HmvH 9:500 .bs m e m m H eH m.mme assoc .>a m 1 w m N H Va W.H was pesos use m _ e m m a .e...n .mmeemdnew smpomnoped mcflms maesoo HwHampown he wamE emopowH pew eases emopdhap mo somfipwesoo HH> sumac - 159 - QOHMmm maom ow xHOOmHum .ooomanaoooaue .ooooH1Hocmum .000m1Hooanm .oooH-O“H .memsmm mecmscehm H o mam PSDOO o>w N H 0mm NH cm 0 Jr m.ammm 9:300 .>w m w m N H WH m.ummm 9:500 .bm m w m m H an v.mm¢u upcsoo .>e m. o m m H . .2.w .mmneMOpew pepompope¢ mcflms mpcsoo HmfipmpoqL an waves emopomH one waves enopmhap mo nomwamaeoo HHH> geese - 20 for all organisns studied as may be shown bv plate counts fermentation reactions. {)1 8. I1 ? fermentation studies reveal L.S.T. broth to be the sup— erior nediun.far both mscherichia coli and aerobacter aero- \J .- 5enes. more was was produced in L.S.T. 'roth than in one percent or two percent tryptose broth. n bubble of gas would often apoee r in all three of these media in thirteen hours)whereas cloudiness was the only indication of growth in standard lactose croth. .At the end ofthe 24 hour period, as much as 100 percer t gas was present in L.S.T. fernen tstion broth, whereas, the ahount of Fas in standard lactose broth seldom exceeded 50 percent. sewer negative tubes were en- 1 countered with L.S.T. broth than with standard lactose broth. It i ossible that these negative tubes were not seeded due on ’U to the minimal nunaers present ra'her than failure of 5:31oh. fables IV and VI in the Appendix give the initial count along oh the 53s production. These tables ware graphed in the aforementioned manner us in seven ranges offrequencies. The first range include s sall tub es vhich either failed to grow or pr01uced growth without 5as formation. The second range included all tubes con miiing a bubble of gas; the third range, 10 percent gas; the fourth ranre, 25 percent 5Cs; the fifth range, 50 percent gas; sixth range, 75 percent has; and the seventh range 100 percent gas. Graphs IX, X, k- XI and III clearly show the fermentation rates of E.coli and.a. aerogenes. .mmm womus J A C0 (U [3.0 30‘ LC N 00 V‘ a U} (11 b0 QOHmwm mbom ma mwm mooauu .mwm meum somnm .mmw mo manpzp mum .mwm ocua .meMCmm zoneswesm pom sowposmopm mam we assess msflsenm eflwme coapmpsessem mo cemwswmsoo .flaoo «shows ‘nomm xH gusto season 950: ww mam soosms .mwm ensue .mmm voc.m .mmm mmwuv “woaum .mmm mo mannsn mum .mmm aqua .momcmm hosmzcmhm b m m ¢ ma , i 1‘. Hana m e m H m.r.o b _®**.b.uod .flaoo mwmofisezowm new soapozeosa mam mo pesoem esp mswsonm waves ceflpwpsessem mo somflswdsoo x noose -23.. memos ms .mwm wooauu .mmw mmvuw .mmm momum .mwm acwuv .mwm moaum .mem mo mapnsnum .mmm ocua . memsmm monsoompm . . N H .7014 U .— .Tsepm mo .NrWOWP owe \ v m w w H _.E.m.q .mecmmosew sepowfiesm¢ . pom seapozcosa mmm mo pcsosm flawsogm warms :oflpmpceEhmM esp mo Cemflsmoeoo HN nomho 24-- acemmm msom em .msm vocaus .msm mmsum .mmw memuw .mmm wmuuq .mmm moaum “mam mo manpspuw .mmm osua .mewcsm hocezomhm .weCeuesmm sopomfiesm¢ sow coflposeoso mam mo pcsosw esp mzflsonw waves soapspmessem mo somflmeEOQ HHx :awsu -25.. A. aerogenes did not produce as much gas as did E. coli in any of the fermentation reactions. The plate counts for A. aerogenes were considerably higher than those of E. coli thus it would be logical to assume that the fermentation rates, would correspond. Sodium chloride was found to be definitely a stimulating agent for the growth of organisms. When added to standard lactose broth, sodium chloride almost doubled the plate counts of the standard lactose broth..At times these counts equaled those of L.S.T. broth. The rate of growth and amount of gas produced increased noticeably as shown by the fermentation reactions. However, at no time did the gas production equal that of L.S.T. broth. This stimulating preperty of sodium chloride is considered to aid materially to the value of trypt- ose broth. In the studies involving Pseudomonas aeruginosa, it was found that this organism gave approximately the same reactions described above. However, the growth rate was considerably slower than that of either E. 0011 or.A. aerogenes. The growth rate seldom exceeded that of E. coli under 2h hour period. Graphs XIII and XIV show the rates of growth for the 6 and 2h hour periods and more detailed growth rates may be found in the Appendix, Table X. -26- wcflms w wmbom m \oovunomne .oomuaomum .oowuflosuw .ooauonfi .mmemm mommSthm “.mw pesos .>w m m w a sa wm Panes .bw m w a mu m.mma pcsoo .>w m w w H Haw: “RON; 0H m.0wa QSSOU .>w w v —m — H .B.m.q .mwocflMSsmw mmCOEO©Smmm pcsoo Hwflhopomp an waves mmopoma vow «fives mmOthhp mo COmHsmasoo HHHx :thm -27_ QOHmmm whom vw \oomuv .OOmuomwum .Ommnooamw .ooauoua .mmacmm soctsemam sh.au pesos .>w_ m — m H ts Ea.cmu panes .>a a ti.) CU H F‘- (. - EEm.moH pszoo .>m v w a em.sMH oases .>c—e# m a a .e.a.m .«moawmssww masosomsmmm meww: O {-4 C‘ C) \L magnoo Hmwpmpown an waves owovowa was waves maOpompp mo somfipwmsoo >Hx sumac - 257 - "C o nah. -0 C ' . C} (U DI A good diagnostic medium sH101ld stimulate the growth of viable, dor 3.nt cells as r-pidl;v as possible. After growth and develoPment has begun, the organism should re- produce in such numbers as to be recognized by their physi- ological action upon the medium. to date, lauryl sulfate ryptose broth and 3.0. medium are the only media that stimulate ra :M growth in C shor period of time. ‘1*he oomp- osition of these two media is practically the same with both cez1ta111ing a tryptose base. ln order for an organism to grow and reproduce it must go through a number of phases in the life cycle. Buchanan (25) defined th fir st four phases as fol lows: 1. Initial stationarv phase - the number of bacteria remain constant or nearly so. (‘0 . Lag phase or positive growth acceleration pha se - the average rate of growth per organism in reases with the time. 3. Logz'titnnic growth phase - the rzte of Prowth per C1"- orvunism is constant in a geometric ratio. C A. Kega tive rrowtl acceleration - the average rate of growth per organism decreases. Reproduction begins after an organism has overcome its stationary phase by adjustment to environment and satisfying —28- 0 1t S fOod recuirements. fhe cell undergoes fission and the two young cells begin growth. rhese young cells are quite susceptible to adverse conditions during this period as demonstrated by huntington and Winslow (8). many viable cells fail to reproduce when transferred from one environ- ment to another, particularly a radicallyd fferent environ- ment be cau se of the inability of adjustment. rhe coliform organis 21s fro; 1 fecal material a1 e not normal inhabitants of water supplies. fhese orgtnisnsbecohe proportionately att- enuated with the kength of time they remain in the wa C1" er, due to such factors as ina de uua te food supply and temperature conditions. when too, the water greatly dilutes the number of organisms per ml. rhese factors must be taken into con- sideration when choosing a good diagnostic medium. hahn (25) found that an increase in the number of org an- isms caused a shortening of the lag phase, when comparatively small numbers were used for seeding. this is true as ob- served from the tables in the appendiL A relatively small seeding remained in the stationary phase for about four hours after which a noticeable increase in growth was observed. In some experimental studies by Salter (25) on the rate of 1 growth of 3.00;; in peptone water, the first three hours of a nine hour period showed con Mi wably slower growth than the last three hours. In the atter period there was always a lengthening of the generation tim which would indicate -29 that this time included a part of the negative growth phase. At the on at of the period of maxinum reproduction there U) |——Jo S33 p 4 7:1 H V l" I (D :4 H. n reasc in 1eta bolic activity with greater heat 0 production, oxygen consumption, and a roduction of carbon *J dioxide and annonia. The metabolic activity apparently de- clines before multiplication ceases to tzke place at a maximal rate. Since this is true, lauryl sulfate tryptose broth ev- wth of viable cells to maximum Ho :1. ’ N! 'L‘ —P ‘1. . '-~~ ‘1. ~ "“ " ‘-‘ . 1 10 den ply stinuiates the ra. 1d - ' 0 Fl epi oduction and increased metabolic a.ctivit y much more quick- ly than standard lactose broth. although, all concentrations of tryptose broth studied increase the growth rate of an organisn to a.greater e12tent than sta11dard la ctose broth, a two percent tryptose concen- tration is re001ne rnded for Sta1dar d methods. These studies show that 1.5 percent tryptose grow out the organisms almost J. as well as 2 percent tryptose but maxinun growth is obtained with this former concentration. Laboratories where economy is of much concern are Urged not to reduce cost by using a 1.5 percent concentration. 3\ecause la uryl sulfate tryptose broth elihinates false presunptives, labor and media costs are automatically cut. rhis would be especially true in lab- oratories testing reatedc nd f.iltered waters Under no cond- '— it ion as is a concentration below 1.9 percent to be used. Pseudononas aeru :inosa 18 an organism antagonistic C!” O fischerichia coli and is usually ass ciated With it when H 0 U) I - 50 _ ol ted from water supplies. ss.aerug:inosa outgrows E. coli 0‘ ' 1 _’ . (‘1 -‘ ° .- — . ‘._. V 13.43ver a lon:w e1 period of tine. erowtn is slowed up through the 2; hour period but after L8 hours, t1ere is a dc cided increase in the grout 1 rite over that of E. coli. Here again the aVvantage in using a nediun.wnich stimulates the rapid growth of s. coli and A. aerogenes can be observed. Q The addition of :ndiu1 1auryl sulfate to a medium prod- uces a very slight stinuiating eflect upon the coliforn or- "anisns. Cowles (1) found that sodium lauryl sulfate exerted (A U uhibited H. a selective action for the coliforn rganisns and other gram-positive lactose ferhenting organisms. Perry and W ' .1. U Hajna (20) presented some data 0 show the superiority of E. C.mediu1 over lauryl sulfate tryptose broth. In E. C. medium bile salts have been substituted for lea aui yl s1lfate. 1h- *" . 1'-‘ . “L ‘ " 011281115118 mlo have 1—1- (D Aile salts also inhibit the grah«wusit v been prove. to be inhibitorr to the coliforn organisms as well. To ate, no literature is available concerning the inhibitory or stimulating property of laury sulfate. From» this study it is evident that lauryl sulfate tryptose broth sodium chloride has never been added to standard lact- ose broth because it makes the medium hypertonic and it is, therefore, reasoned that a large number of the organisms would fail to make an adjustment in this new type of environ- ment. rhe marked increase in growth as denonstrated by these studies shoz tit the organisms not only made tnzugu.t1ent 31- but were also stimulated to increased growth rates. Because of this marked increase in the growth rate a 0.5 percent concentration of sodium chloride in standard lactose broth is recommended for Standard hethods. The experimentation carried out for these studies is not considered complete. More work should be done with mixed cultures of E. coli and A. aerogenes as well as with several antibiotic organisms common to polluted waters. - 32 - l. ififlflflfiflf The growth rates in 1.5 percent tryptose and 2 percent tryptose broth approximate each other. A concentration of 1.5 percent tryptose should not be substituted for a 2 percent concentration. Two percent tryptose broth is recommended for Standard Methods. Lauryl sulfate tryptose broth is superior to standard lactose broth. Lauryl sulfate tryptose broth stimulates fermentation reactions more rapidly than standard lactose broth. Escherichia coli grows more rapidly than Pseudomonas aeruginosa during the first 24 hours. Sodium lauryl sulfate has little, if any, stimulating or inhibitory action upon the coliform organisms. Sodium chloride shows marked stimulating properties in aO.5 percent concentration. The addition of sodium chloride to standard lactose - broth is recommended for Standard Methods. - 33 - -; raw-1" wrn R43F.hit'_'4l“ (.1 330 (l) gcwles, Philip B. A.Kodified Lactose Broth for Use in “9 Presumptive Best. J. Am. Water works Assoc. 30:5, 979 (1940) (2) Darby, C. W. and Kallmann, H.L. Studies on Nadia for Coliform Organisms. J. Am. Water Works Assoc. 31:4, 659-706 (1939) (3) Green, Glenn Studies in Bacterial Counts. 20th Annual Conference on Water Purification. Oct. (1940) (4) Greer, Frank E. The Sanitary Significance of Lactose Fermenting bacteria Not nelonging to the B. coli Group. J. Infect. Dis. 42: 6, 525-536. (1928) (5) Greer, Frank E. fhe Sanitary Significance of Lactose Fermenting Bacteria Not Belonging to the B. coli Group. I I l'" (f) J. Infect. Dis. 42: o, 568-y74. (1920) (6) Griffin, A. K. and Stuart, C. A. An Ecological study of the Coliform Jacceria. J. hact. 40; l, c3-lOO (1940) (7) Hajna, A. A. and ?erry, Alfred C. Comparative Study of Presumptive and Confirmative hedia for Bacteria of the Coliform Group and for Fecal Streptococci. Am. J. Public Health 33: 5. 550-556 (1943) (8) huntington, Evelyn and Jinslow, C. - E.h. 'A Cell Size and Yetabolic Activity of Various ?hasas of the Bacteria Culture Cycle. J. Bact. 33: 123. (1927) (9) Hupp, E. Iemorandum From Indianapolis Water Co. Jan- uary 8. M40) (10) Janzig, h. C. and hontank, I. A. fhe Elimination of false Presumptive Bests. J. Am. Tater Jorks Assoc. 20: 5. 684-695- (1925) (ll) Johnson, 3. R. and Levine, Ta“ Characteristics of coli - like Ticroor‘anisms from the Soil. J. Sect. II: 4. 379-401. (191/) (12) Kahn, Reuben L. Bhe Differentiation of Lact - inf hnaerOJes from E. coli. J. Bact. III: 6, 561 (1916) - 34 - (16) (17) (18) (19) (2o) (21) (22) (23) (24) (25) Ibii. Examination of E. coli in Water, in. J. Tuolic 7r _ ~ 1" , , / neOltF- IA: 5. 313-330 (1919) d $im1lified (1916) “ ' v: '1 '1 ° J- s ., ,. .L .r:- . I Old. 0 -i '19 01.11 11.1719. U011”: :‘C .3 J .1 31,3). -3act. III: 6, Levins, Tax. Bacteria Termcnting Lactose and Their Sign— ificance in Hater Analysis. Iowa State Coll of Agri and Hech Arts. IL: 31, 5-119 (1921) Determination and Characterization of Coli- Jaters. Am. J. Public Health Levine, flax 3. form Jacteria from Chlorinated 31: 351-8 (1941) McCrady, I. H. A Practical Study of Lauryl Sulfate fryptose firoth for Detection of the Presents of Coliform Organisms in Water. 33: 1199-1207 (1943) Mallmann, W. L. and Darby, C. U. Uses of a Lauryl Sulfate Tryptose Broth for the Detection of Coliform Organisms. Am. J. Dublic Health. 31: 2, l2/-l34 (1941) Parr, Leland W. Coliform Sacteria. 3act. Reviews. «3: 1, 1-48 (1939) Perry, C. A. and Hajna A. A. Further Evaluation of E. C. the Isolation of Coliform Bacteria and Escher- Am. J. Dub. Health. 34: 735-8 (1944) Perry, C. A. and Jahna A. A. A Modified Eykman Kedium. J. Bact. XXVI: 4, 419-429 (1933) Fedium for ichia coli. Ritter, Cassandra fhe Presumptive Best in Water Analysis J. Am. Hater Works Assoc. 24; 3, 413-424 (1932) Ruchhoft, Kallas, Chinn, Coulter Coli-aeroSenes different- iation in Water Analysis. J. Bact. XXI: 6, 407-440 (1931) The Differentiation of the 0011 and AerOgenes Salle, A. J. 3 J. Am. Water Works Assoc. 21: 1, 71-76 Groups of Bacteria. (1929) Salter, Raymond C. Observations on the E coli XVIII: 42, 260-84 (1920) Rate of Growth of The Relative Thermal Death Rates Stark, C. N. and Stark, P. _ J. Bact lo: 333 (1929) of Young and.Mature Bacterial Cell. '-35- (27) (28) (29) (30) (31) (32) Stuart, C. A. Sanitary Significance of the Coliform Bact- eria in Water. New England Hater works. J. LV: 355-362 (1941) . Stuart, C. A. Mickle, Friend Lee, Borman, Earle K. Sugg- ested Grouping of Slow Lactose Fermenting Coliform Organ- isms. Am. J. Public Health. 30: 5, 499-508 (1940) Tonney, Bred 0 and Noble, Ralph E. fhe Relative Persistence of B. 0011 and B. aerogenes in Eature. J. Bact. XXII: 6, 433-446 (1931) Ibid. Colon-Aeroaenes Dypes of Bacteria as Criteria of Fecal Pollution. 24; 9, 1267-1260 (1932) Year Book. Report of Standard Methods Committee for Water and Sewage to Laboratory Section. A.P.H.A. (1943) Young, C. C. and Greenfield, M. Observation on the viab- ility of the Bacterium 0011 Group Under Natural and Art- ificial Conditions. Michigan Dept. Health. No. 12; 1-5 (1923) -36.. TABLE TABLE '71 K ‘3 7,1 -113) _‘J ti E) t4 .4 .fl \ j ’v’w LiijJ—LLI EABLE TABLE PABLE TABLE PABLE I II III IV VI VII VIII IX APPENDIX Plate Count for E. coli comparing various, concentra- tiOhS Of tryptoseoooooooooooucooooooooooooo Page 37 Plate Counts for E. coli comparing 1 percent and 2 percent tryptose, L.S.T and L.B............ Page 39 Plate Counts for A. aerogenes comparing 1 percent and 2 percent tryptose, L.S.T. and L.B......... Page 44 Fermentation of E. coli in 13 hours comparing L.S.T., 1 percent and 2 percent tryptose and L.B... Page 46 Fermentation of E. coli in 24 hours comparing L.S.T., 1 percent and 2 percent tryptose and L.B... Page 50 Fermentation of A. aerogenes in 13 hours comparing L.S.T., 1 percent and 2 percent tryptose and L.B......OOO...OI.OOOOOOOOOOOOOOOOOOOOOO0.. Paége 52 Fermentation of A. aerogenes in 24 hours comparing L.S.T., 1 percent and 2 percent tryptose and L.Boooooooooococooooooooocooooooooooooooooo Page 53 Fermentation of A. aerogenes in 13 hours comparing tryptose media with standard L. B. and L. B. with sodium lauryl sulfate...................... Page 54 Fermentation of A. aerogenes in 24 hours comparing tryptose media with standard L. B. and L. B. with sodium lauryl sulfate ..................... Page 55 Plate Counts for Ps. aeruginosa comparing 1 per- cent and 2 percent tryptose, L.S.T., and L.B. Page..........................o.........o.. Page 56 xxxvii Ti". 3 1.3:; I Plate Counts for Escherichigg coli nouns 0.53 r 13 T 1.53 T 2; r L.B.-N801 Initial 10 5 7 8 2 2 5 ll 6 6 5 h 79 12 130 122 169 6 98 1660 2235' 2362 1797 24 1650M1776§ 850K 18005 331% Initial 8 l2 8 6 ll 2 13 10 9 6 8 4 94 105 138 234 245 6 1050 1511 1060 3213 2600 21;. 952:; 10503.: 152211 15881.: 1101;: Initial 12 11 2 17 13a 2 ll 13 10 l7 15 A 82 197 306 500 568, 6 1n60 2288 6000 5750 5080 24 1270L 889M 696K 692M b73fi Initial 11 ll 12 18 10 2 10 18 8 10 16 n 298 295 293 236 268 6 1570 2150 2890 1130 1210 24 120E 130M 230K 190M 80M Initial ll 7 10 13 ll 2 9 7 5 6 6 h 547 540 392 179 337 6 LILO 5910 2330 1500 1200 2A 503 100M 9011903 20H Initial 3 l O h A 2 3 8 13 l5 12 A 182 283 559 902 802 6 2286 2730 65A0 1165A 9779 24 3 0:: 671;: 6511 81;: 292: Initial 5 7 10 A 6 2 6 6 n 9 7 % 178 170 203 413 A92 1226 2540 4000 8000 5060 2A 2837 SALE 3911 811 2211 - 37 - Plate Counts far Escherichia coli (cont'd) HOURS 0.5% T 1:5 1‘ 1.573 T 275 T L.B.-14301 Initial 10 11 7 5 12 2 10 12 10 23 15 4- 296 4-45 1115 480 518 6 4254 4572 11239 5461 6096 24 37 0M 3 3 01:: 4001-1 6801:: 903.: -38.. .Plate Counts for Escherichia coli 3:00:23 Initial 2 l» 6 24 Initial 2 h 6 24 Initial 2 . a 6 24 Initia1~ 2 h 6 24 Initial 2 1+ 6 2h Initial 2 h 6 21+ Initial 2 A 6 21+ 1»; T l O O 133 an HON 30 1184T 2th m}! T‘ "7“ w-w- JJ'oiJLL‘J JUL 2273 'I' 4’- l O 0 5 5 2 9M ' 23M - 39 - 0 8 2 189 12M \J'c \OOWOO 108 T F'FND #95 9M 6L1 Plate Counts for Escherichia 901i (cont'd) HOURS Initial 2 L 6 26 Initial 2 2 24 Initial 2 h 6‘ 24 Initial 2 h 6 24 Initial 2 h 6 24 Initial 2 A 6 24 Initial 2 A 6 2A 700 10500T L.B.T. 1 1 3 189 22M 100 5970T 200 5780T N00 500 10500T (DOM) 1030 SM N 139 9M 10 10M L.B.-13301 O 2 35 #59 6M 6 1:1 15 141 6M Plate HOURS Initial 2 4 6 24 Initial 2 2 24 Initial 2 h 6 24 initial roam IO Initial 2 A 6 24 Initial 2 2 24 Initial 2 A 6 24 LET In Counts for dschericnia coli (cont'd) 2%T 5 h 20 117 2M L.S.T. l4 0 10 115 ZIf‘I 409 6M 1.1351201 5 2 l6 1” 8M 3 11 49 %9 99 100 14 4W 14M 18 224 29 513 CM 12 571, 11 Plate HOURS Initial 2 4 6 24 Counts for 1‘: J T 2 o 13 438 111; Escherichia 2% T 2 l 12 298 511 - 42 - coli (cont'd) LOB o "l‘lLQCl l 0 O O 12 18 379 358 nouns Initial 2 A 6 24 Initial 2 A 6 24 Initial 2 h 6 24 Initial 2 A 6 24 Initial 2 4 6 24 (:7 r1 21:) J. l 0 21 205 623M 13 451 938m 16 389 66711 86 717 890M 569 773K 2‘56 T O l 28 860 62511 17 532 1174M Plate Counts for Escherichia coli 0 ' O 10 495 5 M 0 2 33 588 65 2M 0 3 25 332 7141-1 0 l 30 1290 468M 0 o 12 85 101311 -43.. 112-. l O 4 LOB 0 “11-8501 350M Plate Counts for.Ae:gpacter aercgenes RCURQ Initial 2 2* 24 Initial 2 A 6 24 Initial 2 A 6 24 iInitial 2 A 6 24 Initial 2 t 24 Initial 2 t 24 Initial 2 A 6 24 (3’ I"! 19 1 1 1 21 8255, 12065T 0 0 32 9652 10160? 22 15875 19050T O 2 59 18615 11430T 23 7239 64202 10 195 4500 on 66 1600 150M TABLE III 23 T 0 1 2 26 1651 13335T 111 3000 1* )T A“. 129 2800 2 5 OH L.B.T. 0 2 22 696 82551 O l 18 9775 146OOT ll 48 22225 184151 635 127001 232 6200 0h 0 0 144 1000 200M L013 0 -i\i'8-Cl 26 12392 3112T 22 9843 2620f 20 1016 3160T 52 1932 278OT 38 3048 3280T 470 94 2600 v 111 Plate Counts HOUR S Initial 2 h 6 24 Initial 2 A 6 24 Initial 2 4 6 24 Initial 2 A 6 24 Initial 2 h 6 24 Initial 2 2* 24 Initial -2 4 6 24 16 T 1 1 45 1300. 110M 94 1900 35 8000 140M 59 1111 23m 66 1118 1 5M 12 387 30m \IOO 1911' for Aerobacter aerogen§§_(cont'd) 29 T :1... 0 3 122 2000 21011 54 1600 240M 102 131.1 ‘ - 45 LOSC-T; O 2 49 1500 170M #2 1400 90M 1005 2+ 21 305 28H £0 B 0 “138109; l h 61 1800 10M . 98 ' 1822 6M 1 2M Plate HOURS Initial 2 4 6 24 Initial rat» 1‘) Initial 2 4 6 24 Initial 2 t 24 Initial 2 4 6 24 Initial 2 % 24 Initial 2 4 6 24 Counts for.Aeropapte§_§§;oge e .47 , , m 0 J. 1 9M 2% T L.S.T. 1 1 3 3 17 '18 1651 184 29M 3 8M 0 l l O 5 5 279 173 17M 16“ l 1 1 O 12 13 362 231 2 O 1 1 10 37 241 231 101.1 7M 0 l 1 O 16 14 292 191 2 9M 7M 1 2 O 2 33 17 391 444 10M 14M 2 O l 1 l 26 44 1890 2560 640M 631M - 46 - (cont'd) IJOBO “13.801 1 O 23 1308 4M P100 Plate Counts for Aerobacter aerogenes (oont'd) gpuns Initial 2 4 6 24 Initial 2 4 6 24 Initial 2 4 6 24 Initial 2 4 6 24 O 2 19 2470 1106M 20 1430 729M 2610 789M 38 3310 103 511 c" 20 T 0 4 38 2140 377M - 47 LOSOT. l O 54 2050 16 1500 _567m 40 3250 539M 72 5050 602M LOB o-IqaCl 0 2 39 2140 332M \100 660 388M 43 3990 656M 22 2080 532M T513133 IV Fermentation 01“ Escherichia coli --- 13 hours ' J Initial Sample No; L.S.T. L.B.-NaCl 2% T 1w T Count 1 x ,1 - ,1 o 2 / 7‘ - - 0 3 10,3 - - - 0 1+ - 2‘ - - 0 5 21 7! 10513 21 2 6 ,1 / 10;:5 - 2 7 10,13 / 10,23 26;; 1 8 1073 ,1 5;; 1053 0 9 / ,1 - 5:75 0 10 2533 - - 30;?) 0 11 20;; . 533 5:3 ,1 1 12 10;; 5:3 2;; ,1 1 13 / Growth Growth ,1 3 14 10% / / f l 15 Growth - - 5% 0 16 10% - ‘Growth / 3 17 5 056 59-3 2 3 05:3 2 075 1 l8 / - Growth - 8 19 ,1 5‘5 71 / 3 20 7‘ - :1 71 2 21 5:35 / 3523 ,1 8 22 - - ,1 333 1 23 - 7! :1 / 0 ~48- Fermentation of Escherichia coli --- 13 hours (cont'd) Sample No . 24 25 26 27 28 29 3O 31 32 33 34 35 36 37 L . S . T . 2053 151,15 105:6 15:3 Growth ,1 ,1 Growth ,1 ,1 Growth Growth ,1 LOB o "ITaCl \‘K‘k‘k ,1 G r out 11 Growth ,1 ,1 Grovrt h ,1 -49... ,1 ,1 ,1 10;; Gr oath ,1 ,1 ,1 Growth ,1 Growth Gr ovrth ,1 ,1 ,1 40,5 1 53:3 ,1 ,1 ,1 Gr ovrt h ,1 G r 011' t h ,1 ,1 Growth Growth ,1 Initial Count 2 3 0 2 O O l 2 3 O O O O O rermcntation of Sample No. l \OOQQOWPWN :4 P‘ +4 id F’ F’ +4 +4 \1 O\ \n 4-“ W N H O ‘0 ., .L 111 4.114 'P‘fl V Escherichia coli L . s . T . 95; 100,3 90,5 90:5 9553 9756 100;; 100,3 100,3 805 90;; 855 1006 75% 100;; 100523 8 573 100526 1005 100% L 0B 0 “ICECJ. - 50 - 115,3 305 303 3055 305-5 4013 30:15 506 z, 2023 --- 04 2% T 855-5 953:; 8053 6073 10 0;; 50$ 1006 75% 9 525 100% 8 55:3 . 5 0;"; 5 065 9033 10 05"; 9 0;; hours 60;: 50,323 455.3 5 52% 100% 9553 2 533 100,123 Fermentation of Escherichia coli -—- 24 Hours Sample No . 24 25 26 27 28 29 30 31 32 33 34 35 36 37 L.B.-EaCl 30:3 15;; 10:3 2 53.5 60,3 40;} 30:3 50:3 2075 15%; 30:3 2 555 23.5 T 100:5 10 0-7-3 90;; 10073 10 0:3 3 0,13 75373 95:"; 8 5:53 10 0:3 100,53 115:3 7023 I 95%? 12.313113 VI Fermentation 01‘ herobacter aerogenes -—- 13 Hours Sazrxple 1‘30 . l fiOWi—"wm O) 10 ll 12 13 14 15 16 17 18 19 20 L.B.T. ,1 ,1 Growth ,1 Cr out h ,1 ' 1 G r owt 11 Growth ,1 ,1 ,l ,1 C-roxv'rth Growth Growth Gr owt h Growth LoB . 511101 ,1 ,1 \‘k‘k‘kX‘k‘kX‘kl \ Growth G r owt 11 G‘ r 010.340 h G r owt 11 Gr owt h -52.. ‘K‘k‘k‘k‘kQ ,1 Growth \‘k\\ ,1 Growth Growth Growth Grouth ,1 Growth ,1 ,1 ,l ,1 ,1 ,1 ,1 ,1 ,1 ,1 Growth Growth Gr owt h Growth Growth Initial Count '1 a - ~~~~1 ~71“- L-:._3...J_J .‘.L.L Fermentation of i‘zerobeeter aeroiienes --—- Sample No . l \ooaxaome-‘wm NHHI—‘l—‘HHI—‘Hl—‘H OQDIQOW-{T‘WNHO LOSOT. 9,3 593 15;; 5&5 1,035 A >0 2033 I 155:: L”, LOB 0-133101 10,1; 10,5 5,3 105% 1053 30;; 75-5 2‘,’ 3 211 Hours 35/3 20;; 20,3 2 5; J 1533 30;; 10,5 10".?) 2 5‘; a 103:; 10,15 2 54,3 5,3 5;:3 10,3 TABLE VIII Fermentation of Aerobacter aerogehes --- 13 hours Sample Stand L.B. & Initial No. L.S.T. L.B.-RaCl L.B. 2% r 1% T L.S. Count 1 - / Growth Growth - Growth 0 2 - / - - - Growth 0 3 / - Growth - { Growth 0 4 / / Growth { Growth Growth 0 5 - - Growth / Growth V Growth 0 6 - - Growth / - Growth 0 7 / Growth Growth - / Growth 1 8 Growth - Growth - - Growth 3 9 f % Growth / { Growth 0 - 54 - *J 2131* IX t Fermentation of Aerobaoter aerogenes --- 24 hours Sample Stand L.B. & Ho. L.s.T. L.B.-H301 L.B. 2% T 1% T 1.3. 1. 65% 50% 35% 75% 60% 1 % 2 65% 50% 30% 20% 80% 15% 3 60% 60% 45% 100% 80% 10% 4 70% 45% 25% 60% 50% 25% 5 100% 40% 10% 100% 65% 10% 6 65% 40% 40% 100% 70% 10% 7 100% 50% 40% 60% 80% 25% 8 65% 50% 40% 50% 60% 10% 9 80% 65% 45% 75% 60% 10% - 55 - Plate HOURS Initial 2 1+ 6 24 Initial 2 2 24 Initial 2 1, 0 2h Initial 2 A 6 24 Initial 2 h 6 24 Initial 2 A 6 2h \OUYHOW {—34 n-n ”3T ‘2‘ Ugh—J“) fi-F‘NOO ’5” I -—‘! \nWOON ~q¢wbh40 FHA F’WI‘QW M m [j-F‘NHO 5010100 0; QOHNO h -56- V 4.8. Counts for Pseudomonas Aeruginosa h-rl Fifi w EQOQOFND is _ tgunohao FZ—‘t L 0B 0 “'I‘l—aCl l O O 7 10M tucna ll 1 2M Plate HOURS Initial 2 2 2h Initial 2 2 24 Initial 2 h 6 % Initial 2 ‘1‘ % Initial 2 A 6 24 Initial 2 h 6 24 Initial N #0PN Counts for Pseudomonashheruginosa (cont'd) 200 2OL1 32 100 207M (’0 :— sQCMOCM» \J'ION 170M OM L.S.T. 2 1 h 67 on 15 fi 111 29 19 OM 200 184M 40 lZOOT 2 2 3151 200 21811 300 193M 1 l 2 50 1M 0 85 5 16 2M 615M Plate Counts for Pseudomonas Aerucinosa (cont'd) HOURS 1% T 2% T L.S.T. L.B.-NaCl Initial O 1 O 2 2 O 1 O O 4 3 2 l 3 6 7 9 10 11 2h 6m 502E? 167M 81M Initial 2 O O 0 2 O O O 1 A O O 1 O 6 9 10 10 2 21 3 31m 1815: 1291: BM Initial O 3 O 1 2 O O O O 4 1 1 1 4 6 4 1 8 3 24 283 930M 155M 211M -58- ' " '-’7°1L‘NIK‘K,L1. , Filt' V'PIL‘ ..\.- If. , In I .1 III!!|.1\|.I III:I.|-'0§I1IQ--I|IO .. [EtillIIl-IIIIII.I.II"$ lilollurr . l, l l l l l l I ll