Effect of Temperature on Transport and Plating Media for Enteric Pathogens

Transcription

1 JOURNAL OF CLINICAL MICROBIOLOGY, Oct. 1975, p Copyright ) 1975 Americn Society for Microbiology Vol. 2, No. 4 Printed in U.S.A. Effect of Temperture on Trnsport nd Plting Medi for Enteric Pthogens WELTON I. TAYLOR' AND DOROTHY SCHELHART* Est Jefferson Generl Hospitl, Metirie, Louisin Received for publiction 24 April 1975 The effect of wide vritions in incubtion tempertures nd long periods of incubtion on trnsport nd enrichment broths nd plting medi ws determined by exhustive nlysis of 132 dirrhel stools for slmonelle nd shigelle. Homogenized stools were streked onto eosin methylene blue (EMB), Slmonell-Shigell (SS), nd xylose lysine deoxycholte (XLD) gr pltes, nd into sline, Cry-Blir (CB) trnsport medium, nd Selenite F nd grm-negtive (GN) enrichment broths. Incubtion tempertures were compred t 20 C, 35 C, 40 C nd mbient, nd over rnge of 4 to 52 C for medi incubted in n insulted picnic cooler in n uto trunk. At 1, 2, 4, nd 7 dys the pltes were observed, nd the broths were subcultured. Ech stool ws streked to 12 pltes for 48 observtions nd pickings, nd to 48 tubes, subcultured to 192 pltes, for totl of 240 observtions for pthogens. Anlysis of dt from 6,246 Slmonellpositive pltes showed direct streking to be most effective fter 2 dys of incubtion, but broths were eqully effective t 1 or 2 dys. By dy 4 mny pltes were overgrown, nd both pltes nd broths showed diminution of positives by bout 10% nd t dy 7, 19%. The 2,434 Shigell-positive pltes were more demnding in ll times nd tempertures of incubtion thn slmonelle. Although t dy 2 best results were obtined on direct streking, shigelle dieoffs in broths were excessive, with positives declining 23.7% by dy 2, 49% by dy 4, nd 60% by dy 7. Direct plting of both pthogens ws poor t 20 C with bout 48% success, but slmonelle preferred higher tempertures (35 nd 40 C), wheres shigelle chose 35 C nd mbient, which verged 28 C for the 10- month study. Temperture ws immteril to slmonelle in broths with mbient slightly better thn 35 C, but shigelle preferred 20 C nd showed 50% filure rte t 40 C, mbient being equl to 35 C. The preferentil rnk of broths in efficcy ws GN > selenite > sline > CB > direct for slmonelle; for shigelle, GN > sline > direct > CB > selenite, with selenite proving to be unsuitble for shigelle. Plting medi preferences were XLD > EMB > SS. Ten of 39 shigelle strins could not be recovered from the selenite nd SS medi combintion, the mny replictions notwithstnding. The effectiveness of slmonelle nd shigelle detection t mbient tempertures in Louisin during the 10-month study period, s compred to controlled incubtion tempertures, indictes tht stisfctory enteric bcteriology cn be done in wrm climtes without constnt temperture incubtors. A tcit ssumption by microbiologists tht the optimum temperture for growth of enteric pthogens equtes with the obligtory temperture for growth probbly exists simply becuse lbortories hve lwys hd incubtors nd they hve lwys been used t the optimum temperture for the growth of humn nd niml pthogens. In retrospect there must be cogniznce tht the norml life cycles of mny humn pthogens include survivl outside of 1 Present ddress: 7621 S. Pririe Ave., Chicgo, Ill the humn body. Slmonelle, in prticulr, my survive ll of the rigors of food processing, including heting, drying, tretment with chemicl gents, chilling, freezing, nd long periods of storge t mbient tempertures, nd yet remin infective when ingested by mn or nimls (10). Their bility to survive under the most dverse of conditions is undoubtedly mjor fctor in their ubiquity nd their involvement in n estimted two million cliniclly pprent slmonellosis cses per yer in the United Sttes, where, presumbly, the wter is 281

2 282 TAYLOR AND SCHELHART sfe to drink. If these pthogens re ble to survive not only under wide rnge of nonoptiml tempertures, but even under deleterious conditions, then it is likely tht the restriction of temperture to 35 C for detection of these orgnisms my not be mndtory, nd tht there is wider rnge of tempertures which my not necessrily be best but my still be stisfctory for stool nlyses for these orgnisms. In the previous report in this series of studies (9), trnsport nd enrichment broths nd primry plting medi t rigidly mintined incubtor tempertures, optiml nd otherwise, were observed for efficcy in detection of stool pthogens. It ws concluded tht the temperture rnges exmined, 20 C, 25 C nd 35 C, t 1- dy nd 2-dy incubtion times, were less importnt thn the choice of medi for the nlyses. In the study here presented, the lest efficient broth medi hve been bndoned but wider prmeters of incubtion tempertures, including mbient tempertures for southern Louisin, nd longer periods of incubtion hve been exmined. (This pper ws presented in prt t the 73rd Annul Meeting of the Americn Society for Microbiology, Mimi Bech, 6 to 11 My 1973.) MATERIALS AND METHODS Dirrhel stools received by Est Jefferson Generl Hospitl, Metirie, L., during the period 23 August 1972 to 15 June 1973 were nlyzed using four trnsport or enrichment broths, three plting medi, four incubtion tempertures, nd four incubtion time periods. Becuse of the gret number of replictions involved in this study, nd since negtive results re unrewrding, freshly collected dirrhel stools from inptients nd outptients, nd occsionlly some from other hospitls, were nlyzed by routine methods for slmonelle nd shigelle, nd the stools were sved nd refrigerted. If the stool proved to be positive, then it ws subjected to the experimentl protocol described herein. In mny cses, fresh stools were collected from ptients lredy known to hve n enteric pthogen, so tht stools utilized rnged from freshly pssed to those refrigerted up to 3 dys. During the periods of infrequent stool positives, some dirrhel stools were employed in these studies without prior screening nd these ccount for the negtives in this report. An emulsion of stool solids, liquid, mucus, nd blood ws mde from the ptient's specimen. Approximtely 3 ml of this ws suspended in 15 ml of sterile sline nd thoroughly mixed on Vortex mixer. Four tubes ech of sline, Cry-Blir (CB) (2) trnsport medium, nd Selenite F nd grm-negtive (GN) enrichment broths received 2 ml ech of the stool suspension. A sterile swb dipped into the remining suspension ws used to inoculte four J. CLIN. MICROBIOL. pltes ech of eosin methylene blue (EMB), Slmonell-Shigell (SS), nd xylose lysine deoxycholte (XLD) grs. One set of ech of the broths nd pltes ws incubted in 20 C, 35 C nd 40 C incubtors. The fourth set, t mbient temperture, ws plced in styrofom picnic cooler in the trunk of the cr of one of us. The insulting properties of the styrofom cooler were used to diminish the temperture extremes which would result from the metl skin of the cr prked in the sun. Tempertures in the continer were recorded by clock-driven circulr chrt, 7-dy recording thermometer, -30 F to 120 F [c. -34 to 49 C] rnge (Bchrch Instrument Co., Pittsburgh, P.). The pltes initilly inoculted (direct) were red, nd suspect colonies were picked fter 1, 2, 4, nd 7 dys of incubtion t ech temperture. These pltes were returned to their vrious tempertures fter ech observtion. The direct method used only 12 pltes, but with four observtions ech, for 48 exmintions for pthogens per specimen. In the indirect method, the four broth tubes, incubted t four different tempertures, were ech streked onto EMB, SS, nd XLD pltes, respectively, for 48 such pltings. The tubes were returned to their respective incubtion tempertures, but the pltes were incubted uniformly t 35 C nd observed for pthogens only t 24 h, so tht only the broths were subjected to temperture vrince, not the subcultured pltes. By contrst, in the direct method, the pltes themselves were incubted t the vrious tempertures. Ech tube ws subcultured fter 1, 2, 4, nd 7 dys of incubtion for totl of 192 pltes nd observtions for the presence of enteric pthogens. The combintion of direct nd indirect methods produced 204 pltes nd 240 observtions per stool specimen. The nlysis of 132 stools resulted in the inocultion of 26,928 pltes nd 31,680 recorded observtions. Of these, 6,246 pltes were positive for slmonelle nd 2,434 for shigelle Ṡuspect-pthogen colonies were picked in duplicte, or more, to dulcitol lctose sucrose iron (BBL) (7) gr slnts, lysine iron gr slnts, or r/b tubes (Dignostic Reserch, Inc., Long Islnd, N.Y.), usully in combintion of two of the three medi. Isoltes resembling Slmonell or Shigell biochemiclly were typed serologiclly to group designtions, nd mny of the slmonelle were sent to the Public Helth Lbortories of the Stte of Louisin in New Orlens for further identifiction of serotypes. RESULTS The distribution of slmonelle isolted fter vrious periods of incubtion on pltes streked directly, nd in trnsport or enrichment medi, my be seen in Tble 1. Pltes streked directly yielded significntly better results fter 2 dys of incubtion thn fter 1, 4, or 7 dys (P < 0.01). By dy 4, pltes were frequently overgrown to the point tht colonies of pthogens were no longer recognizble. The dy-2 isoltions represented 32% increse over 1 dy, but

3 VOL. 2, 1975 TEMPERATURE EFFECT ON PATHOGEN TRANSPORT 283 the dy-4 nd -7 isoltions were decresed by 10.0 nd 21.6%, respectively. By contrst, the efficcies of the liquid medi were diminished less t 1 or 2 dys, but by dy 4 declining numbers of recoverble slmonelle resulted from ll four medi. Sline positives decresed by 11% from dy 2 to dy 4 nd CB decresed by 10.4%, but selenite decresed only by 3.6% nd GN by 9.3%. By dy 7 the filure to recover slmonelle ws even more mrked. CB, which ws lest effective in isoltions on dy 1, showed 24.3% decrese, lmost onefourth of its positives by dy 7. GN, the best broth, dropped 18.8% nd sline decresed 19.4% but selenite, which ws comprble to sline on the first 2 dys, ws superior to it t dy 4 nd 7, decresing only 14%. The preferentil order for the broths then is GN first, with greter recoveries thn sline or selenite nd still best fter 7 dys, followed by selenite, lest ffected by the 7-dy holding period, then sline nd the lest efficcious, CB. Thus the inhibitory broths proved to be better for slmonelle thn the noninhibitory broths when long periods nd wide prmeters of incubtion tempertures occurred. The distribution of shigelle isolted is seen in Tble 2. The recovery rte of shigelle is poor by comprison with slmonelle under the sme conditions. For exmple, the die-off of shigelle by the second dy in the broths (23.7%) exceeded the filure rte in slmonelle on dy 7 (18.8%). By dy 4 shigelle recoveries hd decresed by 49.4%, nd by dy 7 positives by ll methods hd decresed by 59.7%. In preferentil rnk of efficcy, GN nd sline were superior to direct stool plting for shigelle, with CB less effective thn direct, nd selenite so poor s to be uncceptble. Direct streking of shigelle, s in the cse of the slmonelle, produced the mximum number of positives fter 2 dys of incubtion, there being 22.3% nd 24.3% fewer positives, respectively, of the two pthogens recovered fter only 1 dy of incubtion. The mjor fctors here were (i) the effect of the low temperture, 20 C, which resulted in pin-point colonies with no identifying fetures on EMB nd XLD t 24 h of incubtion, nd (ii) the distinct preference of SS gr for higher tempertures, s it usully showed no growth of either pthogen t 24 h t 20 C or mbient tempertures. The recorded mbient tempertures which occurred in the 10-month durtion of this study re shown in Tble 3. The temperture extremes from the Februry low of 4 C to the September high of 52 C certinly fforded the specimens sufficient temperture rnge to exert ny untowrd effects on the vibility of the pthogens tht one could resonbly expect. The verges of the highs nd lows produced 20 C differentil, greter thn the difference between the 20 C nd 35 C tempertures of the incubtor. For 6 months the mens TABLE 1. Distribution of 6,246 Slmonell-positive pltes Dy Direct Sline CB Selenite GN Indirect totl (24.3) 354 (0.6) (0.3) 1, (4.1) 352 (1.7) 389 1,421 (1.2) (10.1) 317 (11.0) 289 (14.5) 339 (5.3) 353 (9.3) 1,298 (9.7) (21.6) 287 (19.4) 256 (24.3) 308 (14.0) 316 (18.8) 1,167 (18.8) Totl 922 1,314 (9.1)b 1,207 (16.5) 1,357 (5.5) 1,446 5,324 Number of positive pltes from ech nlyticl method; numbers in prentheses indicte the percentge of decrese in positives. b Numbers in prentheses indicte the percentge of decrese in positives compred to GN totl. TABLE 2. Distribution of2,434 Shigell-positive pltes Dy Direct Sline CB Selenite GN Indirect totl (22.3) (21.5) 131 (26.0) 49 (36.4) 182 (19.8) 537 (23.7) (36.9) 118 (47.1) 89 (49.7) 35 (54.5) 114 (49.8) 356 (49.4) 7 57 (72.3) 81 (63.7) 72 (59.3) 32 (58.4) 99 (56.4) 284 (59.7) Totl (4.0)b 469 (24.6) 193 (69.0) 622 1,881 Number of positive pltes from ech nlyticl method; numbers in prentheses indicte the percentge of decrese in positives. b Numbers in prentheses indicte the percentge of decrese in positives compred to GN totl.

4 284 TAYLOR AND SCHELHART were 20 C to 26 C, nd for 4 months the mens were 32 C to 36 C. It ws not redily discernible tht there ws ny difference in the dt in the colder months thn in the wrmer, however, becuse in the colder months there were fewer dirrhel disese ptients nd the negtive stools exmined cme lmost entirely during those winter months. It ppers tht, when the seson for dirrhel diseses exists, the mbient tempertures re lso the best for the lbortory detection of those pthogens. The gretest number of slmonelle isoltions occurred in April (eight cses) nd My nd August (nine ech); the fewest occurred in December through Februry (only two). Shigelle were most numerous in April (ten) nd My (nine), but rther mild Februry produced six cses lso. The negtive stools occurred most frequently in September nd October but the only Arizon ws detected in October. The effect of temperture on the distribution of positive pltes is shown in Tble 4. Neither TABLE 3. Ambient tempertures of incubtion Month Recorded temp (C) Low High Men September October November December Jnury Februry Mrch April My June Avg The low nd high recorded were the temperture extremes for the month, wheres the men is the verge of the weekly temperture records for tht month nd therefore my not be the midpoint between stted highs nd lows. J. CLIN. MICROBIOL. pthogen grew well on direct pltes t 20 C; slmonelle isoltions decresed by 46% nd shigelle by 50% by comprison with 35 C. However, in the indirect method, shigelle gin (9) showed preference for tempertures lower thn 35 C, with 20 C producing the mximum isoltes nd mbient being equivlent to 35 C in efficcy. By contrst, t 40 C 50% of shigelle were missed. Slmonelle in the direct showed little preference between 35 C nd 40 C (- 1.1%) but were less tolernt of mbient temperture (18.2%), but indirect preferred mbient by slight mrgin over both 20 C (2.3%) nd 35 C (0.8%). Even 40 C showed only n 8.5% decrese in positives, indicting the flexibility of the broths in comprison with direct plting methods for slmonelle isoltions. By contrst, t 40 C one would isolte only 50% of the shigelle found t 20 C. By both direct nd indirect methods, nd for both pthogens, mbient tempertures proved cceptble s the lower temperture ws preferred to 40 C in three of four ctegories. In Tble 5 the performnce of plting medi is shown. The superiority ofxld for both pthogens upon either direct or indirect inocultion is obvious, with recoveries from 90.0 to 97.9%. Shigelle preferred ELMB to SS by more thn two to one, but EMB still found but 63 to 65% of the totl. Slmonelle showed slight preference for SS over EMB (68.9 versus 59%) on direct plting but exhibited reversl on indirect with EMB (71.9%) over SS (63.7%). Selenite broth nd SS gr proved to be excessively inhibitory to the shigelle. Nineteen of 39 strins filed to grow on ny of the 78 replicte SS pltes per specimen, nd 20 were not recovered from ny of the 48 selenite broth subcultures. Five of the six S. flexneri filed to grow on SS, four of six filed to grow in selenite, none of the five S. dysenterie were detected from the selenite-ss combintion, nd only one grew on ny SS plte. For tht mtter, only 10/39 shigelle strins could be recovered Incubtion temp (C) TABLE 4. Distribution of8,680 positive pltes by temperture Slmonelle Shigelle Direct Indirect Direct Indirect (46.0) 1,339 (2.3) 95 (50.0) ,360 (0.8) (18.7) (1.1) 1,254 (8.5) 127 (33.2) 302 (50.0) Ambient 224 (18.2) 1, (25.8) 484 (19.9) Totl 922 5, ,881 Number of positive pltes from ech nlyticl method; numbers in prentheses indicte percentge of decrese in positives.

5 VOL. 2, 1975 TEMPERATURE EFFECT ON PATHOGEN TRANSPORT 285 TABLE 5. Performnce ofplting medi Method EMB SS XLD Totl Slmonell Direct 245 (59.0Yb 286 (68.9) 391 (94.2) 415 Indirect 1,640 (71.9) 1,452 (63.7) 2,232 (97.9) 2,279 Shigell Direct 195 (63.3) 78 (25.3) 280 (90.9) 308 Indirect 674 (65.1) 265 (25.6) 942 (90.9) 1,036 Totl 2,754 (68.2) 2,081 (51.5) 3,845 (95.2) 4,038 Totl number of positives from ny combintion of the three pltes. b Number of positive pltes resulting from ech nlyticl procedure; numbers in prentheses indicte the percentge of totl. from selenite-ss nlysis even with 16 replictions per stool. One must conclude tht this is highly unstisfctory combintion of medi, lthough these medi re still widely used for this purpose in gret mny clinicl microbiology lbortories. The 132 stools resulted in isoltion of one Arizon hinshwwii, 39 Shigelle sp., 47 Slmonell sp., nd 45 tht were negtive for these pthogens. The slmonelle were identified s: S. gon (two), S. ntum (two), one ech of S. give, S. infntis, S. jvin, nd S. mississippi, S. st. pul (five), S. thompson (four), nd S. typhimurium (nine). The remining slmonelle were typed by group only: B (eight), Cl (nine), nd one ech of El, E2, G, nd I. The shigelle were: S. dysenterie (five), S. flexneri (six), S. boydii (two), nd S. sonnei (twentysix). DISCUSSION The purpose of this study is to delinete the prmeters of efficcy of times nd tempertures for detection of slmonelle nd shigelle in dirrhel stools. If those prmeters re wide enough to encompss the mbient temperture rnge for the tropics, neotropicl, nd wrm res of the temperte zones, then the efficcy of the microbiology lbortory will be extended, since specimens cn be held for longer periods of time nd through greter temperture rnge with certinty tht the recovery of enteric pthogens will be within permissible limits when nlyzed t the lbortory subsequently. The corollry is tht n expeditious choice of medi my mke it possible to detect stool pthogens without direct recourse to the lbortory. If, for exmple, we compre the most effective combintion of medi, GN streked to XLD pltes, t 35 C nd t mbient tempertures, the mbient temperture produced more isoltes thn 35 C, 159 versus 157 slmonelle nd 90 versus 86 shigelle. With performnce like tht it would seem to be prejudicil to even refer to mbient temperture incubtion s disdvntged. But in direct streking, mbient temperture is t disdvntge becuse of colony size t 24 h. The direct method of slmonelle on XLD pltes t 35 C nd mbient temperture produced 118 nd 97 positive pltes, respectively, over the 7- dy incubtion period. Although these differences were not significnt (P > 0.1) if the first dy were to be omitted, during which the mbient pltes did not produce identifible colonies, the dt for dys 2 through 7 were 85 positives t 35 C to 80 t mbient temperture, truly insignificnt difference. Thus mbient temperture is shown to be equl in ccurcy of nlysis, but 1 dy slower in time for nlysis for the slmonelle. In fct, s dy 7 pproches, mbient temperture retins its usefulness better becuse pltes t 35 C become unredble becuse of overgrowth of coliforms nd protei, etc. Ambient is not stisfctory for shigelle by direct streking, however; 35 C is superior by 98 to 70 mrgin (P < 0.05). The shigelle in generl pose problems not found in slmonelle detection. Their filure to survive in mixed flor hs been documented by Hentges (4) nd Isenberg et l. (5) s well s by ourselves. The die-off of shigelle in ll of the broths t ny period other thn 1-dy incubtions mkes it necessry to redefine cceptble. Is the 20% decrese in positives seen in GN nd sline in Tble 2 cceptble? Certinly the 36% decrese seen with selenite is of dubious cceptbility. But then selenite is not n cceptble medium for shigelle under the best of circumstnces, with only 77 positives versus 227 for GN nd 223 for sline t 24-h incubtion. By dy 4 the positives from ll broths hve decresed by 49% nd t seven dys by 60%. With the combintion of GN nd XLD the shigelle

6 286 TAYLOR AND SCHELHART recoveries on dy 2 re comprble with dy 1 t both 35 C nd mbient temperture, 56 versus 61, respectively, nd therefore cceptble. After dy 2, the nlytic ccurcy decreses, with 7-dy incubtion producing only 24/61 positives (60.7% loss) even with the most fvored medi combintion. The need for better holding-trnsport medium for shigelle still exists (3, 7). Slmonelle re obviously robust orgnisms quite competent to survive in their ecologicl society nd to be detectble under lmost ny circumstnces. CB medium nd direct streking showed losses of 24 nd 22%, respectively, by dy 7 of incubtion (Tble 1). Considering tht these figures include the worst tempertures nd worst medi combintions, cceptble becomes n ll-encompssing definition. The observtion of only 14 to 18.8% decrese in isoltions for GN nd selenite for 7-dy incubtion, nd with mbient temperture fluctutions of 4 C to 52 C, indictes the fesibility of using these medi s trnsport-enrichment broths under field conditions when lbortory fcilities re not immeditely vilble. The projected recovery of 84% positives on the verge is n cceptble degree of efficcy especilly since, were the incubtion periods to be less thn 7 dys nd the tempertures less thn the extremes recorded here, even higher recovery rtes would ccrue. Of course, missing slmonelle when there re 192 pltes resulting from single stool is not s esy s when only three pltes re streked. Actully, however, there re reltively few times in these nlyses tht GN broth, XLD pltes, nd 24 h t either mbient temperture or 35 C did not find the pthogen, so tht the 192 replictions were not tht much more effective thn three pltes ech by direct nd indirect methods. In Tble 5, the theoreticl totls, ny positive found by ny medium or ny method, compred to the XLDdetected positives showed XLD to be 91% s effective s ll combintions for shigelle, nd 94 nd 98% for direct nd indirect detection of slmonelle, respectively. Our experiences with the medi nd timetemperture reltionships under considertion led us to conclude tht if we were to hve to do stool nlyses under field conditions, tht is, crrying wht we could in vehicle from villge to villge, whether it be in the tropics or ny wrm re of the globe, nd with no opportunity to rech n estblished lbortory within week, our procedure would be s follows. We would crry tubes of sterile sline nd GN broths, cotton-tipped swbs, sterile or nonsterile, nd XLD pltes. We would inoculte one tube ech of sline nd GN broth from the J. CLIN. MICROBIOL. stool. At 24 h we would strek n XLD plte from ech, sving the broths. At 24 nd 48 h we would exmine nd pick the pltes, the 48-h plte being used only if the 24-h plte were negtive, but negtives would be retined nd inspected dily until overgrown. As for identifiction, the two-tube systems, r/b 1 nd 2, or dulcitol lctose sucrose iron medium (8) nd modified lysine broth (1) would sufflice for preliminry biochemicl chrcteriztion, nd the slnts would be dequte for mintennce of the isoltes until serotyping could be performed t the lbortory. This gretly simplified protocol, lcking in sophisticted instrumenttion, should produce cceptble results for surveying the sttus nd identity of dirrhel disese in geogrphicl res deficient in lbortory fcilities. Since trnsport by rough-terrin vehicles, light ircrft, or helicopters is not uncommon to even reltively remote res, lbortory tems cn move into nd out of such res more esily thn they cn find fcilities competent to do enteric nlyses. It is hoped tht the study herein presented will prove of vlue in prgmtic clinicl microbiology. ACKNOWLEDGMENT This reserch ws supported by the U.S. Army Reserch Office, Durhm, N.C. LITERATURE CITED 1. Bonev, S. I., Z. Zkhriev, nd P. Gentchev Comprtive study of medi for determintion of lysine decrboxylse ctivity. Appl. Microbiol. 27: Cry, S. G., nd E. B. Blir New trnsport medium for shipment ofclinicl specimens. I. Fecl specimens. J. Bcteriol. 88: Dunn, C., nd W. J. Mrtin Comprison of medi for isoltion of slmonelle nd shigelle from fecl specimens. Appl. Microbiol. 22: Hentges, D. J Inhibition of Shigell flexneri by the norml intestinl flor. II. Mechnisms of inhibition by coliform orgnisms. J. Bcteriol. 97: Isenberg, H. D., S. Kominos, nd M. Siegel Isoltion of slmonelle nd shigelle from n rtificil mixture of fecl bcteri. Appl. Microbiol. 18: Lmnn, C., P. R. Argon, nd A. P. de Rod Isoltion in the tropics of intestinl pthogens on SS medi without controlled incubtion. Am. J. Hyg. 64: Morris, G. K., J. A. Koehler, E. J. Gngros, nd R. G. Shrrr Comprison of medi for direct isoltion nd trnsport of shigelle from fecl specimens. Appl. Microbiol. 19: Tylor, W. I Nouveu millieu pour l'identifiction rpide des Slmonell et Shigell. Ann. Inst. Psteur (Pris) 103: Tylor, W. I., nd D. Schelhrt Effect of temperture of incubtion on performnce of medi in the detection of enteric pthogens. Appl. Microbiol. 25: Tylor, W. I., J. H. Silliker, nd H. P. Andrews Isoltion of slmonelle from food smples. I. Fctors ffecting the choice of medi for the detection nd enumertion of Slmonell. Appl. Microbiol. 6: