A STUDY OF CHEMICAL AGENTS FOR SELECTIVE GROWTH OF THE COLlFORM ORGANISMS Thesis {far that Dawes 91‘ M. 5%. .V‘JCHJGAN 5139353 COLQEGE 3 “d. " v- Y} male :mn 8mm m. A 1* At ‘J ' . O}, ‘3‘ p - I . This 1. to certify that the I“ thesis entitled A Study 01' Chancel Agents For Selective Growth Qt The Conform Organism. A * presented by Dale Bordt has been accepted towards fulfillment of the requirements for ‘ _H.3.._degree m_§s_cter1_olosy '2 '1 j .j . .i _‘ Majot professor ' 3 i , t ! j Date-W Dale Emil Tordt A STUDY OF CHBKICAL ACEITS FOR UJL3“”WV” GR UTE OF TH: COLII‘ 01:: 0:1CIJ-"IQ 1:3 (An.Abstract) (10 since the first recogt'tion that drinking water which had bec ne cont amit.at ed wit;1 1°ecal material ray be the source of dissemination of certain diseases, tiere has bee en a search for a suitable zxeans of testing waters for bacterial purity. Many selective media have been devised for the detection of the coliform organism . Of this host of r1edia,most have been abandoned because of ire ccurate results ob ained by their use. These inac cur— acies wer mainly due to too great a toxicity to the coliform organisms. Even the confirr atory :nedia most widely nsed today, brilliant green bile broth, and eosin methylene blue agar, have been shown by Hallnann and Darbyl to be inhibitory to the coliforms. Thus false information is given by their use, and a false impression riay be ob- tained regarding the condition of a water source. As a result of these findings, it seemed advisable to continue the search for a selective agent which would effectively inhibit all non-coliform org anisns wlich may give false positive tests. At the same time this agent must not in any way interfere with the growth or gas production by he ner=.1:ers of the coliform group. Seven substances were tested which were known to exhibit this selectivity to a greater or lesser extent. Of these seven compounds, preliminary tests showed five to be inhibitory to growth and gas production by the coliforms at a con— centration which would effec uively inh'bit th e two gran positive organisms, Staphylococcus aureus and Bacillus su} tilis. {I‘HEEJe b These five compounds are: 1. Potassium ferrocranide 2. Potassium ferricyanice 3. Potassium cyanate 4. Potassium arsenate 5. Potassium arsenite The other two compounds tested were ethyl purple and 2,2' Kcthyl- ene bis-A-chloro-b-isopropyl phenol (K-7643). Both of tVese compounds exhibited a remarkable selectivity in preliminary tests. Ethyl purple effectively inhibited the gram positive organisms tested at a dilu Mi shigha sl-l.l million, while allowing the coliforms to grow at concentrations from l-A0,0TO to l-l0,000. Fur- ther studies wit11 ethyl purple showed t} M1 a concentration of 1-333,000 would effectively inhibit the gran positive organisms with— out noticeable inhibiting gas predicti' on by+ he coliforms. Compound K-7643 was shown to inhibit growth of the gran positive bacteria tested in a dilution of more than 1-1 million, and allowed abundant growth of the coliforms at l-lC,OOO, the highest concentration tested. Further studies using l-lO0,000 and 1-400,000 K—7643 showed it to exhibit no apsarenti nhibi ion of the coliforns, while effect— ively inhibiting growth of the gran positive bacteria tested. Actual field tests run with media containing these compettds in proper concentration on chlorinated sewage effluent showed them to be comparable in their selective specificity. When compared with bril- \. liant green bile broth it was evidelit that 1-333,000 ethyl purple and 1-100, 000 K-7043 eliminated certain non coliforn organisms which produced gas in, and discolored brilliant green bile medium. These two media also were capable of allowing the growth of certain coli- forms which, due to their attenuated condition or natural sensitivity 208303 Dale Emil Bordt were not able to initiate growth or p oduce gas in brilliant green bile medium. The promising results in these limited studies of these two media seems to warrant their further and more intensive investigatiOn as a possibly more accurate and dependable confirmatory medium for the detection of coliform organisms. (l) Mallmann, N.L. and C.W. Darby Uses of a Lauryl Sulfate Tryptose Broth for the Detection of Coliform Orgeiisms., A.J.P.H. 31:127-134 (1941) A STUDY OF CTTTIGAL.AGBKT FOR SELECTIVE GROWTH OF TLL COLIFQRMO ORGAIISLS By Dale Emil Bordt A THESIS Submitted to the School of Gra Lduate Studies of IZichir n State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of 1"“0 ‘l h 1" .luhhd O SCI3NCE Department of Bacteriology and Public Health Year 1051 AC K2 3 ONLFJDGI 2.1I T I wish to express uJ appreciation for the generous guidance s a , .. given me in this work by Dr. ‘J. L. .Lallmann, Professor of Bacter- iology and Public Health. TABLE OF COBTEH 3 Historical Background........................................l Purpose of the Problem.......................................8 Experimental Proceedure......................................9 Descriptive Listing of Laboratory Cultures Of COlifOI‘InS used-COOCOIOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO.QOOOOOOll List of Compounds Tested, and Their Sources.................12 Experimental Results Obtained With Potassium Ferrocyanide, and Conclusions Drawn From These Results....................13 Experinental Results Obtained With Potassium Ferricyanide, and Conclusions Drawn From These Results....................15 Experimental Results Obtained With Potassium.Cyanate, and Conclusions Drawn From These Results....................l7 Experimental Results Obtained With Potassium Arsenate, and Conclusions Drawn From These Results....................l9 Experimental Results Obtained With Potassium.Arsenite, and Conclusions Drawn From These Results....................21 Experimental Results Obtained With Ethyl Purple.............23 Discussion of Results.................................24 Experimental Results Obtained With 2,2'Nethylenebis- 4-chloro-6-isopropylphenol (K-7643)...................,.....26 Discussion of Results.................................28 Comparison of Results Obtained With 2 Percent Brilliant Green Bile, l-333,000 Ethyl Purple, and l-lO0,000 K—7643 on Chlorinated Sewage Effluent..............................29 References Cited-OOCOOOOOOOOOI...000.00.00.6000000.0.0.0...0.32 In the last half century there has been a his t eWifi devised for the selection an‘ differentiation of organisms of the coliform group. The stimulus for the development of these many media was the recogm tion 01 a need for the detection of fecal pollution in water sources intended for domestic use, and later the reed to test Che ef- ficiency of purification of known cont amine ted water sources being used for domestic consumption. The assumption and reasoning here being that in the case of fecal pollution, members of the coliforr group will be present. Theobald Smith (1) was the fi.rst to at tempt the use of glu- J- 1.- UOOJ'l. cose broth in the estima tien of Bacillus coli in water, He advantage of the ability of g. 32;; to ferment glucose with the production of gas, and by inoculating tubes with various dilutions of the water sample being tested, lze estimated the approximate n1a1- ‘ bore 01 organisms in tne sample. It became evident however that many other organis 118 could also produce gas in glucose broth. tackson (2) in 1907 recompended the use of lactose broth n -le for the e-Iivni1a ti on 01 non coliform gas prodwc Is. 1.]. contair ing b In 1912 lactose bile broth was tie stanéhrd medium for the presum— ptive test in water analysis. various workers, using different con- yJo «q-u a +1- .~? '.0 uh- .‘ii‘) 011 male C~Ct.‘.'j-L.- A H. centrations of bile broth reported conflict .“ o< V A. ability ofI usin: bile as a presumptive medium. Some workers reported bile a U) being inhibitory to merbers of tIIe co oliform group, and as a result lactose broth wi thout bile was rec omrxen ed by tie Sta.d.rd I-Iethods Committee in 1917. Nany species of has oer ia occurring in water have the abilitv to ferment lactose .hile not belonging to the coliferm group (T). a" Among these suecies may be found nowhere of the anaerobic s;ore form- ing genus Clostridiun,n ans y Cl. rerfrir" 4:3 an Also certain aerrbic seer tose. Lanye Htr st ococci 1-1 - ”4. °.- hydrolyze lactose into it + viola 1;"1 «“ “ms nn+ JO ut-\ 4 vL )-L..L.a.‘-a' _‘\:J. ‘2; \av‘ Lav US r1 0 gasses. rhis srnerristi c U tive presurlptive coliforn Klete mi ll.a and Jruinea may also fermene 01 gas, but their rare oc being c'1e'niiicaat as inte It should be note d great majoritv of false e u L 7.. in water ancJ3 sis should tive organisms at a conce the growth or gas product [.10 1H8 interest n and analysis rroceeded in the Nurtz (4) in 1892 an indicator for the uete isms. ihe litmus would t OFf‘ilsJS, and theren" in Drigalski and Genre Wurtz had devised. DJ 1. sporosenes. e formin3 hacilli,ma y fro ucc 3as from lae- ard so“e staphylococci have the ahili+ v to '5 component nonosacche ides lucor e and sugars ma1 then be aUUathd air] brok on such as me :e non,ers f the fenus Eroteus . Among these resultant products day be h combination may then result in Ia :p—J we posi- tests. Varioas members of the genera ose with the production currence in water eliminates then from o .L rferin" organisms in water bacteriology. _J 4.1 .1. 4.1- from the in_oruaulon just given tnat e_.e ositive tests involve at least one grai -ore, a desirable selective medium for use he one ukich world inli it all «ran posi- N \J ntration which would not be inhioli torv for ion by the celiforns. '31 eve10fnert of various media used in watzr :oll e"ir n3 general manner. renarv‘ lac os eager (or*w1nln" limblu as ction of intestina lactose fe :‘. entin3 organ— urn red with acid 3rodue ctmi by the coliforn dicate the:Ir gresence. di (5) added gentian Violet to the medium 1he gential violet bein” added to inhibit K‘. the growth of gramp no sitive or anisns. Endo (6) did his classical piece of work by using basic fuchsin decolorized with sodium sulfite for the isolation of Sn - monella typhosa. Later various workers modified this medium for their particular purposes, and it became widely used for the de— tection and differentiation of the coliform organisms. The pro— duction of an aldehyde by’E, ggli on the medium restores the color to the Easic fuchsin and produces a metallic sheen on the colony. The chie disadvantage of Endo's medium was it's insta- bility. The color returns to the medium in a shcr rt time even at ice box temperatures in the dark, and much more rap idly under warm, light conditions. q The disadvantage of instaeility was on ercome by Holt-Harris and Teague (7) who used eosin and net1nlene blue in t11eir me dium. This mediu11 also underwent various modifications and simplfi1cations. The molification by Levine (8) is stilllaeing used extensively to- day in th e confir:.atory test for 0011? orms in water. Eisenbcrg (9) studied the action of 115 dyes on various micro- organisrs and he cor ncluded that the gr-m positive organisns are gen— erally much more sens1mt ve to those dyes than are the gram negative organisms. Many other workers also reco nized this fact and several 9...... [3. [0 dye media were devised which showed t specific selectivity to a greater or lesser extent. Brief men+ ion of some of thee e media devised for the isolation and/or detection of bacteria of the colon—typhoid group follows. Conradi (10) in 1908 conzbin ned picric acid with brilliant green in a selective medium for the ccliforms. Krumweide, Tr tt and he- +5 Williams (11) also recommended brilliant green, while Teague and Clurrlln (12) favored eos in and hr in 1.nt green for use in the iso- lation of s. tvnlm sa from stools. Loeffler (13) used malachite green, and Ditt r(1/) us ed china blue malachite greenzigar. Glassner (15) employed china blue meta- chrome yellow agar. Many other workers used various combinations fl these ands similar selective dves. host 0: these dves however - ° - .. : W. .I. 1 roved to be 1nh1hitor3 to mentors of the colifozi 3r.up as tLe con- centrations necessarv for use. file or it's as ts were extersively employed also because of their marked selectivity. Ftic C onkey (16) used bile salt a3ar with neutral red while Hector (1?) used dried whole bile. It was found however that the cones mt - ions necessary for the desired inhibition of gram posi ive or3anisns :as sozexmh t inhititory to the coliferns as well. Dominik and Lauter (18) described a methylene blue—tron crescl purple combination to be used as a con firnatorv me dium for colifor.s in water. Hall and Ellefson 19) used rentian violet in lactose bro h and obtained better results t1 an with lactose oro alone. In subsequent studies (20) these same investigators found tfat increas- ing amounts of dye gave fewer false res itive presumptiv es, hut at the same time tended to innioit members of the coliform group. A little prior to this worlccronfien.renne Welles13cr and Soletsw (21) reco3nized that rosolic ac 1.1. Q: } J. '3 p. vv-5 1 p. (1‘ C') Q; ('7 3 f0 ‘5‘ r) «‘5 O C) p d" 1‘" (g (D organisms, but not the 3ram negative ones. he susscsted it's oossi- ble use as a differential ned11u1 constituent. Meur and Harris (22) showed a brilliant 3reen bile medium to have marked selective properties. This medium was extensively in- vesti3ated and lied i fied by various workers. (23,24) 1he final conclusion regarding this medium was that 2% brilliant vreen aile inhibited somewhat certain attenuated forms of the coliform bacter— ia, but at the same time it is probab y slightly more efficient in detecting and differentiating the coliforms from other bacteria than is plain lactose broth. Salter, (25) in his obs erv vations on the rate of growth of 'f-I n. 0011 found tt at even at dilutions of one to one million or 1 0 more, brilliant green and crystal violet inhielt the growth of E. 0011. He also found that bile salts stimulate the growth of E. coli in a concentration of 0.5 percent. Advantage was taken of this finding by Hajna and Po, rry (26) in the formulation of their "E. C ." medium. Recently also other selective media have been investigated. Cowls (27) describes the use of Drene shampoo in a modified lac- tos e broth as a possi ale improvement in the test for the coliform ETOUPo Faulkner (28) showed that the female hormones diethylstil- boestrol, hexoestrol, and stilboestrol are much more selectively inhibitory for the 3r am positive than for the gram negative organ- isms. He showed t}. e colife rms to grow in concentrations of 1 to 5,000, while the gram positive ore 3ani sms and some spore formers were killed in the range of l to 100,000 to l to 250, 000. Naghski, Copley, and Couch (29) showed quercetin to be in- hibitory to certa ingram positive or3anisms at concentrations of .075 to .l millig wane nor milliliter . , the action beinv much 1833 K.) on those of the gram negative group. The actual tolerance of these gram negative organisms was not determined however, since the prac- tical limit of solubility of quercetin is about .15 milligrams per milliliter. Recognition of the fact that many antibiotics show a remarkable selectivity quite naturally led to their investigation as a possible constituent of media to be used in the analysis of water. Rittenberg and Silliker (30) tested three antibiotics against several organisms. They found that streptomycin had about the same effect on the coliform and non—coliform organisms, and eliminated it as of no possible value for thecietection of coliforms. In testing penicillin howeVer they found that all coliforms tested could withstand 50 units per milliliter, while 9;, welchii and the enterococci were inhibited at 30 units. Five of the seven aerobacillus strains however were unaffected by concentrations up to 400 units per milliliter. It was found in the case of tyrothricin that all 45 strains of coliforms tested withstood 100 micrograms per milliliter. All 9;, welchii and enterococci were inhibited by 30 microgram per milli- liter or less, while 4 out of the seven aerobacilli produced gas with as much as 50 micrograms per milliliter. Actual field tests subsequently run proved antibiotics to pos- sess no particular advantage over the presently used brilliant green bile broth. It was concluded by these investigators that at least for the present, the use of antibiotics in water analysis is not practical. Chapman (31) discussed the remarkable selectivity of sodium alkyl sulfate (called Tergitol 7). He claims that th only gram neg— ative organisms inhibited by this substance are members of the genus Proteus. Stark (32) in 1936 evaluated the use of formats ricinolea broth for the detection of coliform organisms in milk. Later Fred- eriq and Levine (33) showed all cultures of g, gel; tested as well as Aerobacter and Citrobacter species would produce acid and gas in formats ricinoleate medium. quglln, Levine, and Smith (34) describe a buffered boric acid 'W lactose medium for enrichment an ad identification of L. coli. The 9th edition of Standard Methods (35) includes crystal vio- let lactose brot‘, for: ate ricinoleate broth, fuchsin lactose broth, and brilliant green bile brothels well as eosin me+hylene blue agar and Endo's agar as confirmatory media for {O itive lactose pres ulptive tubes in the test for coliform organisms in water. Ruchhoft and lorton (36) found brillian gr en bile, crystal violet, and formate ricinoleate broths to 1e less fr odu cti e for coliforns than star dard lactose broth Hallmbnn and Darb3, (37) in their studies on the use of lauryl tryptose broth for the detection of the coliform organisms in water, found that eosin methylene blue Ovar and brilliant green bile broth “(1- cannot be used if it is desired to confirm all positive presu1rt1ve tubes which contain coliforms. Lvidence to support tleir statement came from the fact that th,y were able, il’l nany cases, to isolate coliform organisms from lauryl tryptosep osi mix presumptive tubes which had failed to<3onfirm by the use of eosin nethylene blue afar or brilliantm ween bile bro 0th. C0 The failure of these coliforns to coniirm on eosin methylene ndica tes the er oeability that these .8. :1. blue or brilliant green bile media are too inhibitory to certain members of the coliform group ClCh as mi ght as found in marginal a11d insu;ficiw . Ml treated waters; these waters whi.h may contain coliforns in rather s all numbers, or possibly coliforms somewhat attenuated bv age, exposure to c11lor1 nation and other adverse conditions It is recognised that occasional ou tbrea1:s of water borne diseases occur even toda" from prm ablv tested and vs ified safe sources. This fact also ray suggest that perhaps the waters in— volved in the etiology of such outbreaks are of this borderline type, and are missed by the routine media and methods of confirm- ation in use today. A recom i ion of the above facts and indications then seems to deem it advisable to seek a confirmatory radium which will effec ively inhibit the greatest proportion of non-coliform lactose fermenters without in any way inhibiting members of the coliform group. The purpose of this work then is to survey a nu ber of com- pounds with the hope of findiig one which at the proper concen— tration will exhibit the desired degree of selectivity. EYFJRIVTITlL 3100333313: The e 5pc: rinents conducted in this wor: were des igned tozsatisij a threefold objective. 1. (a) To deterAine the maximum cono ntration of cheuiCal which would allow growth of the coliforus. (b) To deterr ine the minimum concentration of chemical which :ould effectively inhibit the growth of the gr J1 positive or- ganisns. Due to low limits of solubility of some of the compounds tested part (a) above could not be determined. 2. To determine lie optimum concentration which wouJ.d efiect— ively inhibit the gran positive organisms without perceptibiy inter- fering with gas prodAHcgion if the coliforzls, if suc}1 an opti: um con- entration exists. 3. To deterrlin ne tie actual inhibitory action of concentrations of die chemicals within the range of dilutions be tween nzr nun tol- erance by the colif orns, «nd minimum dose inlib itor" for the gran positive organisms, as dete mined in (2) above. This was done by using mirAiral known inocula and plating known dilutions oftbemfl- ture and determinin3 growth o‘rves over the initial stages of growth. A typical E, coli culture was used in t?ese growth curve determinations. The base medium used in all determinations throughout this work was one formulated by Barb; and Kallmann (38). Thor shou ed t at it's composition and pH provides optimum conditions for growth and gas production by the coliforns. The composition of this bise medium is: Bacto tryptoso —— —2.0fi Bacto lactose 0.5% Elia-13C4 - 0.2739 A KaflPO, 0.275; F Cl ==C.5p n“ t?" .6 ‘: 1"”h t f‘ 7'; rmvnht . "- G m‘oio 757;- «"1“ Ail-e F’I: Of Alb.) 1"“ d-L‘ltm ‘Ij U 01*. “Ck.J uh; -.CLA. 13 U. U. 111 L.) 1‘1 - (, ‘9 shown by Darby and Kallnann to be optimum for the early growtn phases of the coli "I The proceedure for deterrzfi nine the moximua concentration or chene ical tolerated by the various organisms tested involved the preparation of 10 milliliter portions of neoium containine V.e;ious ci mti ns of the . - .1. .Lfio ion in the autocla at 121 C for 15 minutes, were seeded with a 4 millimeter loopzul of a vigorously growing 24 hour culture of ti e croonisU being tested. rowth was deter- mined after 24 hours by observing for visual t*w1rbiclit3 of tte nedium. . a a o Incubation tegpo atnre was 35 C. in en ch case. Once the approximate tolerance of the orfanisns to he c exical has 1 oh ained in the wearer ce. escribed asove, fernen c+ ation tubes were prepared containing concentratior s of the chemice l or .nracing the ranje from maxi- mum tolerance of tie coliforms to minimum concentration 1m militorv to the gran po si+ ives. These tubes then were also seeded Lith 4 millimeter loop- fuls of 24 hour 017 ures 5 several strains of 2, coli,.g. aerorenes ,anl Ho H .4 ci‘ 0 "5 (g Q: } " {3 ts col.if orms. Otservation 0: amount of gas produced in ‘hese various concentrations, aid comiarison with gas pro lucticn by tee sen organisms in the control tubes containing only base medium, estarlishes the highest concentration of the chemical which apparently would not in- hibit has production when observed at 12 and 24 hours. The laboratory cultures of coliforns in this study will larfely be referred to by nunoer. 11 The following is a brief description of these cultures. mflhmenmwm' l. Organism E. coli . coli E. coli E. coli s. coli E. coli a. coli E. coli E. coli A. aerogenes E. freundii E. coli E. coli E. coli intermediate A. aerogenes intermediate intermediate A. aerogenes A. aerogenes A. aerogenes Source water water man cow rumen cow feces cow rumen man orange juice water water orange juice man(atypical) cow feces water water water water water water water water water water INViC formula //-- //-- xx- x/-- //-- xx-- /¢—- xx—- //-- --¢/ x-/- xx- xx— xx- xx- //- xx- //—- xxx- --x¢ #xx- x--/ --// --x/ --// 12 The chemical compounds tested in this work are: Potassium ferrocyanide Baker's C.P. Potassium ferricyanide Eimer and.Amend C.P. Potassium cyanate Baker's C.F. Potassium arsenate baker's primary crystals C.P. Potassium arsenite Baker's C.P. Ethyl Purple (Ethyl Violet) Harleeo-Iartman Leddon Co. Hexaethyl triamino triphenyl methane Philadelphia, Pa. K-7643 (2,2 Methylene bis—A-chloro-isopropyl phenol) from Dow Chemical Co. Hidland, Michigan. The experimental results obtained with each of the above com- pounds and a discussion of these results will be taken up in order in the folloring section. l3 :r‘f-:-rv— *7 a'11‘f'1 v ~‘3 MC I‘fim ' "f" «" '. T" (“1" ‘n (‘F-T“ ' F‘T - v" ‘,. MT Fa P 1’- ' C P ) LUJALJ. ...L .11.: Kile/”Li; iii 1... Li.‘.u.LJ will; 1:0*1-Uu-Lbls ill)...“ “.1. .L'J...‘ 18.3.01' 5 o o . “J- Table I Influence of Potassium Ferrocyanide on the Growth of o, Organisms. (Observed after 2; hours incubation at 350 C.) Concertr ation Culture Control 1-25 1-50 l-lOO 10. A. aeroeenes ll. 2, freundii 12. I; 0 1'3: Ho \\\\\ \ I \\\\\ es visible growth. catcs no visihle growth. t growth. The above table of results shows that Staph. aureus is inhibited by dilutions as high as 1-80, 2, suotilis beinv more resist tan';, showing ’ J slightg newth even at a concentration of 1—25. The three colifi orns tested here s:zowed growth even at the 1-25 dilution. To be of any use for the selection of coliforns, potassiur ferrocyanide must be used in a concentration higher than 1-25. 14 Table II Influence of 1-25 Dilution of Peta siun.Ferrocyanide on Gas Production by Several Coliform Organisms in 24 Hours at 350 C. Culture Control 1-25 1. E. coli 70* I 0 / 2. 3. coin. '75 o ,1 4. 3;. 0011 60 o ,l 5. E. 0011 70 o / 17. E. 9931 45 O / 3. g, aerocenes 65 O SI. 12. E. coli 55 5 1:3. subtilis ”1 ,1 51. Staph. aureus )1 - # indicates growth. — indicates no growth. 81. indicates slight growth. * denotes percent of gas nroduced in Durham fermentation tubes. The above data indicate that potassium ferrocyanide at concen- trations sufficient to effectively inhibit the growth of g; subtilis and Stan} . aureus also is inhibitory to the eroduction of as bi members of the coliform group. Consequently it would probably not be of use as a selective agent for the detection of he coliform group. 15 BA in IEHTAL LESULTS RITE :GTASSIUK FARRICYXKIDE. Eimer and amend C.P.) Table III The Influence of Two Concentrations of Potassium Ferricyanide on the Growth of Several Organisms. Concentrations Culture Control 1-50 l—lOO 10. A, aerosenes ll. E. freundii 12. E. coli \‘k‘k‘k E. subtilis \Xk‘k‘k \\\\\ Staph . aureus 2 indicates growth. - indicates no growth. From the above table it is evident that concentrations in excess of 1—50 must be used to effectively inhibitlg. subtilis. Therefore, to be of use as a selective agent for detection of coliforms, a concentration of more than 1—50 is necessary. 16 Table IV Effect of 1-50 Potassium Ferricyanide on Gas Production by Various Coliform Organisms. Cultur-e Control 1-50 1. E. coli 70* 0 51, 2. E. coli 75 0 51. 4. E, coli 60 0 SI. 5. E. coli 70 - 17. g, coli 45 _ 33. A, aerogenes 65 ' O 51. E. subtilis { . _ Staph . aureus ,1 _ * indicates percent gas produced. / indicates growth. - indicates no growth. s1. indicates slight growth. It may be concluded from the results obtained that potassium ferricyanide is inhibitory to growth and gas production by the members of the coliform group at concentrations necessary to effect— ively inhibit growth of the two gram positive organisms tested. l7 EiPL:RIIEIITAL PESLTTS OBTAINED WITH POTASSIIDE CY’ZETE. (Baker's C.P.) Table V Influence of Potassium Cyanate on Growth of Several Organisms. Culture Control 1-25 1-50 l—lOO lO._£_X_. aeronenes 1‘ ll. E. freundii 12. E. coli \‘k‘kR 13. Serratia marcescens \‘k‘k‘ix‘k 14. E. subtilis ‘k‘kk‘ix‘k \‘Kk‘k‘k‘k 15. Staph._auzens The data in the table above show that concentrations of more than 1-50 are required to effectively inhibit Q. subtilis, while Stan} . aureus is inhibited by that concentration. 18 Table VI Influence of 1-50 Potassium Cyanate on Gas Production by Several Coliform Strains. Culture Control 1-50 4. E. coli 60* 5 5. E. coli 70 O / 6. g. 0011 80 0 / 10. g. aerogenes 60 - 13. E. coli 70 O / 15. E. coli 60 5 17. E. 22;; 7O 7 * indicates percent of gas produced. / indicates growthl - indicates no growth. Since marked inhibition of gas production and growth by the coli- forms is evident from the above data, it is unlikely that potassium cyanate would be of use in a selective medium for the detection of coli- forms. l9 EXPERIIENTAL RESULTS OBTAINED WITH PCTASSIUM.ARSENATE. (Baker's primary crystals C.P.) Table VII Influence of Two Dilutions of Potassium.Arsenate on the Growth of Several Organisms. Concentration Culture Control 1-50 l—lOO 10. g, aeroeenes 11. E. frelndii XXX 12. _E_. coli \‘1\‘1\‘1\\ \‘kk‘kk S taph. aureus / indicates growth. - indicates no growth. The above table shows that potassium arsenate at 1-50 dilution inhibits growth of the two gram positive organisms tested, while at the same dilution the three coliforns were able to grow. 20 Table VIII The Influence of 1—50 Potassiun.Arsenate on Gas Production by Several Members of the Coliforn Group. Culture Control 1-50 1. E. coli 70* 25 2. E. coli 70 o ,1 44 E. coli 60 - 5. E. coli 65 Q / 12. T. pol; 55 5 17. 3;. coli so 0 / 33. A, aereeenes 65 5 ‘2. ggytilis_ ‘ / _ Staph. aureus / - * indicates percent of gas production. —- / indicates growth. - indicates no growth. It becomes obvious by examination of the above data that potassium arsenate in concentration necessary to inhibit the 4 am positive organ- 9% isms tested also inhibits the gas production and growth by the coliforms. For that reason then it would not be satisfactory as a selective medium for the detection of the coliforms. 21 EELJRI‘. 3231-11.. IZLSULTS 031111th RITE: EC’L‘ASSIUI: 311531113 (Baker's C.P.) Table IX Effect of Various Concentrations of Potassium.Arsenite on the Growth of SeVeral Organisms. Concentration Culture Control l-ZOOO l-AOOO l-ZOOOO lO. 5, aprosenes 11. E. freundi' XXX ‘1\ 12. E. coli E. subtilis \‘k‘k‘kk \ \\\\\ Staph. aureus / indicates growth. — indicates no growth. 51. indicates slight growth. The above results indicate that concentrations in excess of l-4000 are necessary to inhibit growth of Staph. aureus and E. subt°li§. 22 Table X Influence of Various Concentrations of Potassium Arsenite on the Production of Gas by Several Coliforn Strains. Concentration Culture Control l—lOOO l—ZOOO l-BOOO l-AOOO l. E. coli 33* 5 55 40 55 2. 30:11 75 o % o ,1 o ,1 o ,1 4. _13. coli 55 o ,1 o ,1 o sl. 0 sl. 5. e. coli 40 O / O / 1., / 7 17. _.;. coli 90 o sl. 0 ,1 e ,1 5 33. e. flame 65 O 7‘ 5 20 35 Ser. marcesce s / sl. sl. / / Q. subtilis / - sl. / / Staph. aureus / - sl. sl. sl. * indicates percent or gas produced. - / indicates growth. - indicates no growth. sl. indicates slight grou h. The above results show a varying degree of inhibition of growth and gas production of several coliforns to effectively inhibit growth of Staph. egreus and E. subtilis. reason it seems unlikely that potassium arsenite would be of use as a constituenttin a medium selective for the coliforns. at the concentration necessary For that 23 LXPLn LLLTl RLSTLTL CELL. .LD UL_‘H LTKTL PULFLL. (Lth 1 Iiolet) 1he res1 ts shown in table AI agree with the findinfis of Darby (39). He also found that ethyl a1ibitory to the gran positive organisms at very high dilutions, and that members of the coliform group are able to grow in relatively high cone ntrations of he dye. The data given here also show that L, 221; is apparently more susceptible to the inhibitory properties of ethyl purple than is either e. aerosenee or E. f ”r ' . The next ste3) in the in"estigation of the effects of ethyl purple 2 aeternination of he influence of 1 on the coliform organisms is tha various concentrations of the dye on gas production by this grou The method employed,i1nvolved the preparation of Durham fermen— tation tubes contaix ling progres mi'ely r'reater concentrations of et yl purple in the base medium. Inoculations were then made 'nto these tubes using a 4 millimeter loopful of ?4 hour 0 1tures of coliforns obtained from various sources. Gas production was recorded as percent ofr gas in the fermentation inserts, readihss bob made at 12 and 24 L,) £3 0'! Zours and compared with control tubes similarly inoculated, but con- aining only the base medium. It is evident from the results shown on table XII that ethyl purple in l-L0,000 concentration in some way inhibits the production of gas by coliform bacteria. T1118 is eswoec ally antarent after 12 hours incubation, where indeed, 4 of the cultures even failed to show growth. The fact that many of those cultures which showed less gas the an the controls at the 12 hour interval actually showed con arable percent of gas in 2/ heir 3, indicates that the ethyl pur;le may act .APSoam o: mopsofiwzw I .3930.0 eePQOflwsfi \ I I I I I I \ gamma .smwpm Mela mmIH ”QIH E Ia SNIH a”. HIH HPSQOQ 0.39.30 Cowpdapzoomoo \ \ mUu.¢OceOonw ‘ 65 0 <1: 0 V0 (V ‘I’ I‘I megoucsom .4 .0H Haeasone .: .HH \k \k \k \k \k \k \k \k \k \k \k Laos .a .NH \k \t \k \k \k \k \k \k \L I \k \t \k \L \L H00 .m .H oa 9017,.“ Hm MIH BOMIH mt NI BONIH Em HIH HOHIH Hopvmo o 0.59.3 o SOMJGHPQOCCOU .mSmfimduso Hdho>om mo xpzono one no oamnfim ngpm me meoflpshpmoomoo meowne>.mo sesame mam P\ r HM 0.3qu Table XII The Effect of l—S0,000 Ethyl Purple on Gas Production by Several Coliform Strains. Control 1—BO,DOO Culture 12 hrs. 24 hrs.' 12 hfs. 24 hrs. 1. E. 321; 60* CO 60 90 2. 33.291; 20 60 5 5o 3. E. coli 60 75 2O ”9 4. g. 993.; 60 so 10 1,0 5. E. coli 60 75 - 7 6. _L. c011 50 75 a ,1 90 7. L. 9g;; 3 9O 10 60 _. a. coli 40 75 5 63 9. E. coli 60 75 7 83 11. g. freundii 2O 85 20 SO 13. g. coli 65 do _ _ 14.. 3;". coli 45 60 45 '75 15. E. coli 60 80 - 2O 16. g. 991:: 60 80 40 so 17. E. coli 60 CO - 4O 19. 33. coli 30 5) 7 '75 * indicates percent of gas produced. / indicates growth. — indicates no growth. 24 by inhibiting or slowing down growth or o.ivision of the cells rather than actually supressinf Ede gas Lori; n3 me hanisnl proper. 0f the sixteen strains of coliforns STO'nl in 1— 0,000 ethyl purple, seven straixs showed rath r marked innibition. rhese s ven strains were seeded into two higher dilu ion: of ct yl :Lrple, 1—100,000 and 1-333,032. Gas rodJctio on we.s rec rded after 12 and .34 hours in- cubation at 350C, as before. Table XIII The Effect of Two Concentrations of Ethyl PL mp'e on Gas Production by Seven here ocns tive Colif ea‘n Straius. Control 1-100,00T l—3f3,“70 Culture 12 hrs. 24 hrs. 12 hrs. 24 hrs. 12 hrs. 24 hrs. 4. g. coli 29* 45 o / 10 L5 be 5. Q. “2;; 2 53 5 55 20 57 6. E, coli 45 60 25 75 55 75 7. ,1“; coli 55 8-7) 25 55 60 5 13. g. c .11 40 c5 0 10 35 :37 15. _E_. coli 50 so 5 55 40 to U’I V”: \O 0 UI C‘ 1 O \f‘ U \1 O 17. g. coli g / indicates growth. * ildicates percent of gas produced. By the reszu ts of the above table, it is s own that ethyl purple in a dilution of 1-100,000 still inhibits Pas production by the coli- forms to approximatelyt 1e sa“e 5t nt as 1-80,000. however, at 1-3? 3,000, gas production is comparable at both 12 and’ 4 hours to that produce d in the corresponding control tubes. Ethyl purple demonstrated a remarkable selective action against the gran .ositive bacteria tested. Also at he proper concentration, ethyl purple has practically no demonstrable toxicity to the coliforms, and for that reason it may be valuable as a selective agent in a medi— um for the detection of coliforus in water. Revis (40) in 1011 in his work with malachite green, found that most coliform strains are inhi‘ited Ly concent“ations of the dye as low as .03 percent. He also found that on serial transplant in this dye, coliform strains lost all power to produce gas even when re— iLoculated into ordinary nedia containiig no dye. These strains how- ever would show vigorous growth without producing gas. This production of permanently atypical coliforms may be due to an actual alteration of the cells metabolism by the dye, or by a selective process in which the non gas producers are favored. Since ethyl purple ("exaethyl triamino triphenyl methane) like malachite green, is a triphenyl dye, it was thought that possibly this same gas supressing phenomenon could be demonstrated by it's use. The proceedure employed in this experiment was much the same as that used by Revis. Two coliform strains (2. £211 1 and 2) were serially trans— ferred each day in tubes of base medium containing 1-80,000 ethyl pur- ple. This dilution had previously shown some inhibition to certain coliform strains, among which was . coli 2. At the time of this writ- I til ing these cultures have undergone 32 transfers, and still continue to produce abundant gas when re-transferred to base medium containing no dye. 26 QIPLR”I;11AL ancJTTS 07”ainn NITH 2 9'““"T’LLIBBIo—4~C LORO—o-ISOrn PYL‘ELIOL (h—7 43) This substance was obtained by courtesy of the Dow Chemical Cone pany of Ki‘land, Lic7.1igan. The manufacturers state that K—7643 has a phenol coefficient against StaLh. aureus of aoout 390, and against g. t”1hosa of from 1-10. They did not indicate how t11ese coefficienw were obtained, but it should be noted that K—7643 is no ot soluble in water to any appreciable extent under ordinary conditions. In my attempts to render K—7643 soluble, several facts were ob- served. he substance is soluble in alcohol, glycerin, strong inor- ganic bases and dis Lilled water wlie. heated to boiling. However, none of the solutions obtained by these meth>ds were satisfactory here, be- cause as soon as the alcoh lic solution was placed in an a ueous medium it precipitated out in the form of an “HOTUJO as, guxmv substance. A similar reaction is obtained when the basic solution is bTOUfht near the neutral point, or when the hot solution cools to incubator temperature. Ho true solution has thus far been obtained, but a rather stable, milky suspension was accon lisl1ed by the following means .0.l gram of solid K-7643 was dissolved in the smallest amount of ethyl alcohol nec- essary to render it soluble. Then 0.5 milliliters of Tween 80 (Atlas Products Company) was added and thoroughly shaken to produce an emulsion. his emulsion then was diluted to 100 milliliters, well shaken, stonpered and kept as a l—1,000 stock emulsion. Preliminary tests with this substance showed that K-7643 does ex— hibit considerable selective action against the gram positive organisms tested, while allowing th e gram negative orb anisms to grow in the highest concentration used. 27 The Effect of Various Concentrations of K—7643 on the Growth of Several Organisms. Table XIV Concentration Culture Control l-lOT l-lOOT l-5OOT l-lM l. E. coli / / / / / B. subtilis / - - _ _ Staph.Aureus / - — _ - / indicates growth. - indicates no growth. Also at dilutions of l-ZH, l-4M and l—5H, the tubes did not become turbid with E. subtilis and Staph. aureus, but minute, granular growth showing bizarre forms under the microscope precipitated to the bottoms. To determine the effect of two dilutions of K-7643 on gas pro- duction by various "typical”and intermediate coliform strnins, fermen- tation tubes containing 1-1oo,ooo and 1—4oo,ooo K—7643 in the base med- ium were prepared and inoculated with a 4 millimeter loopful of the organisms being tested. Observations were made and recorded after 12, 24 and 36 hours incubation at 35° C. From the results shown on Table XV, it appears that K-7643 in either dilution tested has no inhihitory effect on gas production by the various coliforms as compared with the amount of gas produced in the control tubes. Both these dilutions however did effectively in- hibit the growth of both E, subtilis and Staph. aureus. Further investigations on the influence of various concentrations of K-7643 on a strain ofIE. coli (No. 1) involved the more precise technique of seeding with minimal inocula, lOO milliliter portions of ‘base medium containing the various concentratidns of K-7643. After 28 O, 2, 4, and 6 hours incubation at 350 C., nutrient agar pour plates were seeded with measured volumes from the previously inoculated flasks of media. The plates were incubated for 24 hours at 35° C, and the colonies found to have developed were counted. From the known dilutions used for seeding the plates, the numbers of viable cells per milliliter were calculated for each concentration of K—7643, and for each time interval. From the results obtained and recorded in Table XVI, it is evi- dent that multiplication occurred at a comparable rate in all concen- trations of K-7643 tested. This rate was also comparable to that of the control. It may be concluded from this set of data that K-7643 shows no appreciable inhibition of growth of E. coli at the concentrations tested and the nrevious data show it to be markedl inhibitorv to : . y .1 the gram positive organisms tested with these and greater dilutions. Therefore K-7643 shows considerable promise and should be further in- vestigated as a possible selective medium for coliform organisms. .mpsog 2H asap soapwgsocfl wemefibsfi % .mwm mo oHQQSQ HHsSm mepmowwsfi 9:3 .fipBOpm on wepwowdsw I .£P:opm meprfiunH \ .beoswonm new mo accused merOflpsH # Na Na Nu MK mumpsw .mmdpm mfiaaenee .m me 3 3 mm em 3 mm ,3 e gums .a .mm me me OH ma oe 5 mm om pee eeeeeeeneeew .em mm pen 0 cm .esn 0 ON men use mmmmmmmmm .a .em £5 as 0 £5 as 0 see, 23 o g a. .3 me me ma no oe CH ow as me Haeo em .e on me ea on on e co Om om wHeo .m..m me as mm on me me mm me em mmmm .w .H em em NH em em NH on em aNH massage ocoqoeesfl occqoofiufi Hoapgoo .mcwwhpw Enomwaoo Hdhebem an nOfipQS@0hm who so mfioblm mo mQOfipmhpceonoo 038 me poemwm one >N mamas Table XVI Comparison of Growth of E. coli (No. l) in Various Concentrations of {—7643, with Growth in the Base Medium.Alone. Incubation Period Concentration 0 hr. 2 hrs. 4 hrs. 6 hrs. l-100T 33* 67 1950 138,400 1.4001 28 60 1855 140,400 1-6001‘ 34 56 1816 138,400 1-11: 30 52 1949 107,900 Control 27 47 2317 113,700 * Number of organisms per milliliter. 29 Comnaris on of Results btained WithQ Perce11t Brilliant Gree1Eile, 333, 000 Ethyl Purple, and l—lO0,030 K—7C43 on Chlorinated Sewage d. E Efl 1ent. The following discussion involves 1n oraation gained during the course of work concerned with deterri 31ing the most efficien n .et‘ods of effectively chlorinat ting sewage effluent at the Huskegon, Michiga an Sewage Treatrent Plant. In this work lactose broth was used as the presumptive medium, and 2 percent brilliant green bile broth as the confirmatory medium for the detection of coliform organisms at various points in the treatment process. It was noticed, especially in samplw from chlorinated final effluent, tlat many lactose po.iti1ve tubes failed toc:onf irm in brilliant green bile broth. Microscopic examination of the contents of these tubes usually revealed the presence of gram variable to gram negative large spore-forming rods, with oecas nal gram posi- tive cocci and also usually many gram negative, non sporing short rods. Limited studies were made of 236 positive lactose tubes which failed to confirm on brilliant green bile broth. Tests usin ng l-100,0 K—7643, and l—333,000 ethyl purple fermentation tubes respectively were run in parallel with brilliant green bile and observed in 24 and 48 hours. 0f the 23 0 tubes which did not confirm with brilliantg Teen bile, 18 confirmed with l-100,000 K—7643, and 17 confirmed with 1-333, 000 ethyl purple. It was possible to isolate, by streaking on solid nutrient agar, gram negative, non s} oring, lactose fermenting, short rods from each of the tubes which had 51 own gas in either ethyl purple or K-7643 media. These data then indicate that of the 236 positive 30 lactose tubes which had failed to confirm on brilliant green bile, at least seven percent actually did contain coliforms. The bril- liant green bile apparently was inhibitory to their development or their gas production. It was further noticed that among the brilliant green bile positive tubes, there would frequently be produced a marked dis- coloration or yellowing of the medium. Two 4 millimeter loopfulls from 84 such discolored tubes were planted into tubes of ethvl purple and K—7643 broths, and the results obtained compared and presented in table XVII below. Table XVII Brilliant Green 1—333,ooo 1-1oo,ooo Bile Discolored Ethyl Purple K-7643 t. 84/ 65/ 19- 63/ 21- * number of tubes. / indicates presence of gas. — indicates no gas. Microscopic examination of the contents of the brilliant green bile tubes which failed to produce gas when transferred tecethyl pur- ple and K—7643 broths always revealed the presence of gram positive spore forming rods, often associated with shorter gram negative rods. It was thought that possibly the gram negative rods were coliforns responsible for gas production in the brilliant green bile, the gram positive spore formers merely being respon51ble for the discoloration. 10 check this possibility, tryptose agar plates were streaked from hose brilliant green bile tubes which did not produce gas in K-7643 or ethyl purple media. The colonies which developed were picked 31 and placed in lactose broth fermentation tu growth took place in these tubes, in no case was gas produced in tee inserts. These orsanisms were gram negative short rods usually, wi a occasional aberrant fi anentous forms “redo““notinr Failure to is- olate the gran po Mi ive rods was prol)aolv due to the fac: t‘ t st"ea« plates were used, and since thes gram positive rods did not develop, they were probably anaerobic forms. Lack of suitable anaerobic equip- ent made it impossible to ml anr further study these organisms. It appears however that there are orfiaafsns, or a svne r istic co m ination of or~enisrs which Irey reduce gas in brilliant green bile while not belonging to the coliform group. Twenty of the discolored tubes which also produced gas in efiLyl purple and K-76A3 media were streaked on tryptose are . Sevem ml- onies which developed were picked and seeded into lTCtOSO fermentation tubes. In every case it was poo ible to isol te cram nefative, non '-..J {J Sporing, lactose ferxentinfi rods, although in three cases from K-7hll, I'P 7-1 and in two cases from et”v purple nedia, peculiar gran nefiative dip- werOi id forms were pres at, and apparently responsi fléle for th .e "as production. It is realiser that no broad, general conelus ions can be so sfelv drawn from tne limited observ 713 .tions made to date, but the ir 1fo rmation which is available fro n this single source indicates that l-; “9“ ,OOO ethyl purple broth, and l-lO0,000 K-7643 broth ezd;ibit comparatle es el- ectivitv for the col ifor :1 organ isms, and appear to eliriinete to as yet an unknown extent, false positive tests which are obtained :ith bril- . 0 fl 0 l‘ C o liant green bile broth. also these txo selective atom n h“ Or“ to &110U Jl-'-..« \A. ' \rL‘J ‘- A one growth of certain coliforms wLiCh, due to their attenuated 32 or naturally sensitive condition cannot initiate growth or gas pro- duction in brilliant green Tile. Further studies with these selective agents should be under- taken in various localities, and under all practical conditions to determine if they actually do continue to show more accurate res lts than the presently used confirmatory media. \o (‘0 \_/ 33 T”r~‘*:>“"c;‘.s crest .. J- -1 .lJ_.~ «1" 5 ~w S:ith, Theobald cited by Greer, J.u., L.n. Toble, F.V. Ernsn and A.E. C'neil The Sanitar; Si nificance of Lactose Fermenting rganisns Lot Pelenging to the P. Coli Group. 7. Mediurs and Nations, Jr. Inf. Dis. 42:556 (1 23). Jackson, D.D. cited by Greer, F.3., 3.3. iohle, :.V. Yyha~ and A.3. O'Yeil TAO Sari‘ ry Significance of Lactose Fermenting Crban1sre Not Pel enging to the F. Coli Grirp 7. r- iuns and hethods, Jr. Inf. uis. 42:55; (1:23). a r w p r “ ‘Jrepr’ .:...J., --.&J. _ ‘1 '1 VT ' -J‘. . '1 r .‘J rl‘vh 3.1.1., 1' . . 2.”.‘1-.3.I‘1 allot 1L .._J. C': Oil .18 s nitar" Sisnif-canc- L4. --- b—S-—- —. I _- U o f Lactose Fernenting Orpanisns not Belonging to the E. Coli Group. 7. Leiiuxs and Net eds, *0 A ‘ rr} Jr. Inf. A13. 44:556 (15;; . f 0 _V i :1 . T11 ' . '1 Kr '_,., .‘.. '. ‘3 Jurts, Cited b, Greer, ;.E., 3.4. otle, -... .‘Ian aLd n.1. " . ‘5 r‘ W '- ’. ‘0: n ‘7 -1M \ 4"." 'o :11 The Sanitary Si niiica nee of Lactose -eIhcncitl . u“ ...- ,. - 1 f‘ ' r . ., H H Organisis I.ot Ielenpinp to tMC P. uOll Group. /. neoiums ‘1 U. D “I r‘ :l/‘ f‘f“\ and IHet rods, J1. Ini. 313. 44:)c0 (194oj. Dri*alski, V. and T. C nrldi Ueter ein VerFahren sun Tack eis n o _ ‘ a. _ '° Tv_‘_’_~o r if.. ,wf-t . der Tuneu"“1CWIlen., ~e1tscni1ft fur itwiune, ,,.4o3—jd0 (1902) F" '2 ‘ w I. v . 1 fl"" ‘1 A . ends, 8. Ueter ein Verfa‘ren sun IachJezs e: .JphusoaCJIlon., '. — n meLZT ‘Lt or We ’ Coo Ceit .1 l. iur ta.- iol. Act. 1, ,;:1c9—llO kl, ,). " 1 71.. ' “'7' .1 r“ -. .- v 4- T 9' D -3lt-Marris, J.n. ani O. IeaDue A .ew Culture Lcuium ior « 13 . o _. -- o - (u. 1' . . ' d. "of. Isolation 1 F. truaosus Iron stools., vI. Inf. 1s. lc.2)c— £10 (1915). Le'ine, N. Differentiation of P. coli and T. aerocenes on a CI 0 7‘1‘ fl : _fi'" fl . ' 7? l‘ "r VI 1 "fl . H (7,: o '7‘ 7" "I (I. (1‘ Ul‘»-iz‘.~-i -lEd .J.:L.," . ‘LULLL . , Jl . I11... . D D. QJOQJ-A,‘ -L/]-I--') . 71¢ ‘ ‘ J .L '. "-,\ H a o -. . n. .C" ' o nisenge15,T. Uncersuchunuen urer alb; pe siiiscle Desinie’J1ons . _ _I- ‘ _ ’0’ 1— f~vv° F.“ p . v‘ (- Vo organge., Centraltlat. fir Frit;.1ol. I ici. 42. ( yTt). Conradi, H. Lin Verf.1ren sun IIac h~eis spari ‘I! l c e Centralblat. fur Pakteriol. I def. 42: (l§09) if “'1 ’? 'T3 .. "J 9 1.4 {J [I H. H 4-..: '0 f3 . v Krunweide, C.K., J. S. Iratt and H .I. Vclilliams The U o liait " reer. for tI1e Isolation of ngho 1 a1. acilli'fro" Feces., Jr. Inf. Dis. lCzl—lB (l~l4i J Tcague, O. and A.U. Clurn an A Kethod of Preserving Typioid Stools for Delayed Lia.1u tio n and a Coivar tive Study of the 3_fie- ieney o; 2.K.P. A ar, eosin an' Iril Green *"ar, and 31