Synthesis of a selective agar medium for Yersinia enterocolitica Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by UNIVERSITY OF ARIZONA LIBRARY on 08/05/12 For personal use only.
D. A. S C . H ~ ~ M A N N Otrtorio Mitristty cJ'Hctrltlr. E r r ~ ~ i r o r r t t ~ eBtrc.trt~iology ~~t~rl Ltrhot~trror:~. BO.Y9000, T t ~ r t t ~ i t ~ At,r Tortirrto, l 0 1 1 t . C'trrrtrtltr , I M ~ WI R 5
Accepted July 30. 1979 S C H I E M A ND. N . A. 1979. Synthesisofa selective~tgarmediumforYc,r..sitritr c~rrrt~roco1itic.o. Can. J . Microbiol. 25: 1298-1304. A new agar medium for isolation of Yc,rsitritr c,trtrroc.olitic.o was formulated based on growth studies which defined an optimum basal. and the evaluation of selective chernic;ll agents, dyes, and antibiotics. The final formulation. designated cefsulodin-irgasan-novobiocin (CIN) agar, provided quantitative recovery of 40 different strains of Y. rtrrc,roc~olitic.oin 24 h usingincubation at 32°C o r with 48 h of incubation at 22°C. The medium was highly selective. especially ;rgzrinst P.sc,rrtlottrot~tr.(trc,r.rrgitloso. E.sc~lrc~t.ic~l~itr c,oli. Klrh.sirll~tptrrrrrtlotlitrr. and Protcrrs 1~1irt111ili.s. Colony morphology coupled with a differential reaction resulting from mannitol fermentation permitted discrimination of Y. rtltc,t.oc.olitic.o from most of those Cram-negative bacteria that were able togrow on the medium. Recove1.y and selective chal-acteristics of CIN agar were stable during stol-age at room temperature for 9 days. C I N agar gave a higher recovery of Y . errtrroc~olitic~trfrom feces both direct and with cold enrichment (0.4/1.5%) than Strlt~ro~rc~lItr-Sl~igc,lltr (0.0/0.7%) and MacConkey (0.0/0.9%) agars while significantly reducing the level of background organisms. S C . H I ~ . M , Z D. N NA. . 1979. Synthesis o f s selective agar medium for Yc,rsitlio c,trtc,roc.olitic.o.Can. J . Microbiol. 25: 1298- 1304. Une nouvellc gklose pour I'isolement de Yc,rsirritr c~trter.ot~o1itic.o a C-tl mise au point d';tprks des etudes de croissance qui ont permis de definir unc base optimale e t d'nprtls I'evalu:rtion d e I'effet d';tgents chirniques sdectifs. dc cololxnts et d'antibiotiques. La formule definitive. nppelee gelose cefsulodin-i~gasan-novobiocin (CIN), a perrnis une I-ecuplrntion quantitative de 40 ;tpres 24 h d'incubation I32"C ou 48 h h 22°C. C e milieu souches differentes de Y. c~t~io.oc.olitic.o oc~rtrgit~o.vo. E.sc~l~c~ric.l~io c.oli. Kle.11est fortement selectif pal-ticulitremcnt contre P.vclrt1ot~rotrtr.s .siclltr ptrc,rtrriotlitrc et Proterrs ttlir.ohi1i.s. La morphologie des colonies nssociee i une reaction differ-entiellc d e fermentation du mannitol pelmet une bonne discrimination entre Y . elltc,roc.olitic,tr et la plupart dcs batteries Gram-negatives capables de pousser sur ce milieu. Les proprietes de selection et de ricuperation de 1;)gelose CIN demeurent stables pendant 9jours b la temperature ambiantc. La gelose CIN utilisie pour isoler Y . rtr/c~r.oc.olitic.odes matieres fecales soit directemcnt OLI aprks enrichissement au froid donne un meilleur rendement (O.4/1.5%) que les gelose Snlrt~ot~~llo-SIIigrlltr (0.0/0.7%) et MacConkey (0.0/0.9%) tout en diminuant d e f21fon significative la qu;rntite de microo~,ganismesassocies. [ T n d u i t par le.journnl]
Bacteriological media presently used for the iso- e n t c r o t ~ o l i t i c ~such r, as the addition of Tween 80 to lation of Yt)i.sinirr c i l f e ~ ~ o c o l i t i crepresent u formu- MacConkey and deoxyribonuclease agars delations previously developed for general isolation scribed by Lee ( 1977), or the supplementation of S S of enteric bacteria. such as Srrli~~oi~clltr-Shigcllrragar with sodium desoxycholate as described by (SS) and MacConkey (Asakawa ct trl. 1973; Cap- Wauters (1973). The pe~formanceof media presrioli c.1 t r l . 1978; Kaneko er trl. 1978; Morris cf trl. ently used for recovery of Y. ci~tci.oc.oliti~.tr is f~-e1977; Toma 1973; Toma and Deidl-ick 1975; quently improved by incubation at lower temperaTsubokul-a c t t r l . 1973; Zen-Yoji cr t r l . 1974), tures (e.g. 22"C), which then requires 2 days or Drigalski (Lassen 1972), lactose-sucrose-UI-ea longer for colony development. (LSU) (Ahvonen 1972; Eiss 1975; Kapperud 1977). We undertook the development of a new selecdesoxycholate citrate (Szita and Svidro 1976). and tive agar medium specifically for isolation of Y. bismuth sulfite (Hanna cf t r l . 1977) agars. Saari and cntci.ocolirictr. beginning with growth studies Quan (T. N . Saari, and T . J . Quan. 1976. Abstr. which defined an optimum basal fol-mulation (in Annu. Meet. Am. Soc. Microbiol., p. 45) described preparation). Dyes (Schiemann 1979) and chemimedium, and cals (in preparation) I-epresenting potential seleca deoxycholate-citlate-mannitol Highsmith er t r l . (1977) reported the use of m-Endo tive agents were evaluated in another agar formubroth for isolation of Y. c~nferocolifit~n from water. lation for their effect on the quantitative recovery Fifty-seven antibiotics were There have been a few attempts to modify available of Y. enfe~~ocolifit~cr. media for improved selection or differen tat ion of Y. studied as potential selective agents and various 0008-4 16617911 1 1298-07$0 1 .00/0 @ I979 National Research Council of Canada/Conseil national d e recherches du Canada
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SCHIEMANN
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differential systems were examined in the final agalformulation. ~h~ evaluation of the agar medium which evolved from these separate studies is described in this paper. Materials and Methods
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Tcjst 0tgo11isttr.s The sources and types of 68 Y. c,~rrc,roc~olirico cultures used in thcse studies are identified in Table I. Stock cultures were maintained in refrigerated trypticase soy agar stabs and working c ~ r l t l ~ r eon s slants of the same medium. Inocula for test plates consisted of suspensions prepared in phosphate-buffered saline (pH 7.2) from overnight blood agar cultures incubated a t 30°C. Suspensions were diluted s o that 50-100 colonies were usually obtained on each of duplicate test plates inoculated by the spreading of 100 ILLdelivered by an automatic pipette. Recovery of test organisms was expressed as a percentage of the mean count obtained on duplicate blood agar plates prepared and incubated under the same conditions. Cultures of 69 other Gram-negative bacteria representing 10 different genera used to evaluate the selectivity of the formulated medium were maintained on refrigemted tl-ypticase soy agar slants and prepared for use in trypticase soy broth incubated overnight at 35'C. Inoculation of the selective medium. and MacConkcy ag:rrforcomparntive purposes. was by streaktng. Agor Mctlio Thc final medium formulated from gr-owth and selective agent studies is called cefsulodin-irgasnn-novobiocin (CIN) agar after important selective substances which i t contains. CIN agar is prcpared as follows: the basal is first prepared by suspending the following substances in 748 mL of high-purity distilled water: special peptone (Oxoid), 20 g: yeast extract (Difco). 2 g; mannitol. 20 g ; pyi.uvic acid (sodium salt). 2 g ; sodi~inlchloride. I g: magnesium sulfnte.7HZ0, 10 mg (10 m L of a 0.1% stock solution); agar (Oxoid No. 4). 12 g. This suspension is brought to a boil to dissolve the agar completely. The solution is cooled to about 80-85°C and 10 m L of isgasan DP300 (2.4.4'-trichloro-2'-hydroxy diphenyl ether) (Ciba-Geigy) solution (0.04% in 95% ethanol) is added followed by vigorous shaking to volatilize the ethanol. The solution is cooled in a water bath to about 47°C. Bile salts (Difco) ( 2 g) are suspended in 200 mL of high-purity distilled water and dissolved by heating to boiling with stirring. T h e solution is cooled to about 47°C and added to the previous mixture. One miliilitre of 5 N NaOH is added followed by 10 m L of each of the following stock solutions: neutral red. 3 mglmL; crystal violet. 0.1 mg/mL; cefsulodin (Takeda Chemical Industries Ltd.). 1.5 mglmL; novobiocin (The U p ~ o h nCo.), 1 .5 mg/mL. Stock solutions ofantibiotics are stored at -70°C and thawed at room temperature just before use. The medium is then placed on a magnetic stirrer and 10 mL of a 10% solution of strontium chloride (tiltel--sterilized)is added slowly with constant mixing. T h e final pH is ad.justed to 7.4 with 5 N NaOH. Two modifications of the above fol-mula were also prepared for- evaluation: CIN(1) agar containing 0.5% insteed of 0.2% bile salts; and CIN(I1) agar containing 0.5% bile salts and 0.02% sodium citl-ate. All evaluations except the stability study were completed with CIN agar which was I day old and MacConkey agar which was no mol-e than 4 days old. I.soltrtiot~f,.otnFccc,.s CIN agar was compared with S S and MacConkey agars for
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recovery of Y. etltc,roc~olific~c( from fecal specimens reccived by the O n t a ~ i oCentral Public Health Laboratory in buffered glycerol saline tlxnspol-t medium. Plates were streakeddirectly after enrichment ofappr.oximatelyI of feces i n m L of M/ 15 phosphate-buffered sirline, pH 7.6, incubated at 4"Cfor 14 days..^^' agar was incubated a t 32°C for 24 h and SS and MacConkey agars at room tempel'ature fol- 48 h. Every attempt was made to eliminate any bias when we compared the three media. All tishingsofpl-esumptive colonies were done by one individual who was very familiar with the appearance and v;rriability of Y. pt~tproc.olitic,tron these media. T w o colonies only were selected for confirmation from each plate heal-ing organisms which resembled Y. ('tlt~t.ocoIiti(.(~most closely.
Results CIN(1) agar was evaluated with 30 different strains of Y. c)nter.oc.olitic.cl selected from those shown in Table 1. MacConkey agar was included for comparative purposes. Two incubation conditions were used. 32°C for 24 h and 22°C for 48 h. Overall recovery based on duplicate plates when compared with blood agar under the same conditions averaged 94 and 1005%for CIN as compared to 87 and 94% with MacConkey agar for 32 and 22"C, respectively. Because the formulation for CIN(1) agar developed a fine precipitate during storage. a modification was prepared containing 0.02% sodium citrate and a higher concentration of bile salts. This medium, CIN(I1) agar-, was compared with MacConkey agar for quantitative recovery of 33 strains of Y. cnter.ocoliticc1 selected from those shown in Table 1 . Overall recovery on CIN(I1) agar was 92 and 96% when compared with 93 and 88% on MacConkey agal- for 32 and 22°C. respectively. One strain did not grow on either medium at 32°C but was recovered quantitatively at 22°C. Three strains showed lower recovery at 32°C on CIN(I1) agar, suggesting inhibition by citrate. Furthermore, overall colony size and pigmentation were more restricted in the presence of citrate. The inclusion of sodium citrate in CIN(I1) agar did not prevent precipitation during storage, which produced a fine cloudiness in the otherwise transparent medium. Therefore, a third and final modification, designated simply CIN agar, was prepared in which the bile salts concentration was reduced to 0.2%. Precipitation was now minimal if care was taken in reducing the temperature of the basal medium and bile salts solution before mixing together, if the strontium chloride solution was added slowly with good stirring, and if room instead of refrigeration temperature was used for storage. Quantitative recovery of 40 strains of Y. enterocoliticcl on this medium is shown in Table 2.
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C A N . J . MICROBIOL. VOL. 75, 1979
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TABLE1. Yersinia ctzterocolitica cultures used to evaluate selective agar media Culture No."
Serotype 0
Biotype (Wauters)
Rhamnose fermentationb
Culture No.
Serotype
EM1 E002 E004 E006 E007 E008 E009 E012 E013 E014 E015 E016 E017 E020 E021 E022 E023 E025 E026 E028 E030 E03 1 E032 E040 E041 E042 E043 E044 E045 E046 E047 E050 E051 E052
9 9 9 8 8 8 8 3 3 3 6,30 6, 30 6,30 5,27 5,27 5,27 5,27 16 17 4,33 2,3 NTd 5 6 6 13,7 13,7 13, 7 16 16 16 12 5 6,31
2 2 2 1 1 1 1 4 4 4
-
E053 E055 E057 E059 E060 E06 1 E062 E063 E064 E066 E067 E068 E069 E070 E070 E076 E077 E083 E089 E093 E096 E097 El00 El05 El06 El29 El31 El32 El33 El34 El63 E l 64 El68 El72
6, 31 13,7 14 NT 13,7 6, 30 21 18 7,8 17 NT 4,33 5,27 5, 27 7,8 8 8 5,27 5,27 4 4,32 11 4 8 21 8 8 8 5,27 5,27 13, 7 NT NT 17
1
1 1 2 2 2 2 NAc
-
-
-
-
-
NA
'"
"
"
0
Biotype (Wauters)
Rhamnose fermentation
NA
NA
'6
4'
1 1 1 1 1 1 1 1 1 1 2 2 1 1 1 2 2 1 1 1 2 1 3 NA ' 6
+ +
-
+ + + + +
-
+ +
-
NA cc
‘G
" "
1 Atypical 1 1
-
+ + +
aCultures E001-E028 and E069-El06 are human and animal isolates obtained from Dr. Sandu Toma Canadian National Reference Center for Ycrsij~ia Toronto, Ontarlo. Cullures E030-E055 are human isolates obtained from Mrs. Eleanor Christenson, State Laboratory of Hygiene, Madison, Wisconsin. Cult(~resE129-El34 are human isolates obtained from D r . Marjorie Bissett, California State Department of Health, Berkeley, California. Cultures E057-E068 and E163-El72 are milk and food isolates obtained from own laborarory. bAll cultures with a rhaninosc reaction shown are sucrose-positive. =NA, not available. *NT, nontypable.
The results show that recovery on CIN was comparable to that on blood agar for all strains using either 32°C for 24 h or 22°C for 48 h. The colony size of Y. entrroc,oliticcr was larger on CIN than on MacConkey agar. The predominant colony size for 40 strains of Y. ent~rocoliticu was measured to the nearest 0.05 mm on both media and found to average 1.35 mm ( s = 0.185) versus 0.72 mm ( s = 0.149) at 32"C, and 1.61 mm (s = 0.176) versus 1.23 mm (s = 0.217) at 22°C for CIN and MacConkey agars, respectively. The colony appearance of Y. ente~.ocoliticaon CIN agar was quite distinctive when compared with growth on MacConkey agar. Colonies on CIN agar had a deep red center which had a rather sharp border, and was surrounded by an outer zone which was usually translucent. Even very small colonies,
which were more likely to be seen at 32 than at 22OC, had a red center when viewed with a stereomicroscope, and transmitted light. Pigmentation, the result of mannitol fermentation, was stronger and more complete at 22°C with 48 h of incubation than at 32°C with 24 h of incubation. The edge of the colony was either entire or irregular depending upon the strain. The selectivity of CIN agar is shown by the results presented in Table 3. All of the 69 strains of Gram-negative bacteria used to evaluate selectivity, representing 10 different genera, grew well on MacConkey agar at both temperatures, while only 17 strains grew on CIN agar at 32°C and 20 at 22°C. CIN agar was especially inhibitory to Pseucio~no~~as a e r ~ ~ g i ~ z oEscheric.lzia sa, coli, Klebsiellrl przeunzonirle, and Proteus mirabili.~,but did
SCHIEMANN
TABLE 2. Quantitative recovery of 40 strains of Yersirrin er~terocoliticnon CIN agar
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% recovery"
% recovery
Culture No.
32°C 24 h
22°C 48 h
Culture No.
EOO 1 E002 E004 E008 E012 E013 E014 E015 E017 E020 E021 E022 E023 E025 E030 E032 E040 E042 E044 E045
101 140 98 94 112 71 88 77 118 107 109 121 88 80 78 77 137 319b 114 86
91 139 89 99 84 89 94 97 108 99 98 131 102 100 103 107 79 290h 96 95
E046 E050 E05 1 E052 E053 E055 E073 E077 E083 E089 E093 E096 E097 El00 El05 El06 El 29 El31 El32 El33
1
X
32°C 24 h
22°C 48 h
106 94 105 97 95 89 107 125 98 100 77 99 105 95 90 110 107 94 100 91 99.5
88 109 96 123 115 103 93 113 92 148 71 99 103 86 97 96 106 105 103 104 101.3
"Based o n mean of duplicate plates conlpared to blood agar under same incubation conditions. bRepeated three times with same low recovery on blood agar. Later balches of blood agar gave eqoivalent recovery. Results not included in means.
TABLE 3. Selectivity of CIN agar against Gram-negative bacteria
No. strains recovereda Organism
No. strains tested
32°C 24 h
Acitrerobrrcter cnlconceric~rsvnr. ot~irrn~rrttr A . calcoace~icrrsunr. hvojji Aerornorlrrs Irydroplriln Cilrobncler cliuersus Leuirlen C . jierrrldii Enlerobncler. nerogerles E. crgglotr~ernr~s E. cloncne E. saknznkii Eschericlriir coli Flnvobnclerirrrr~orlorn~rrrr~ KlebsieNn osylocn K. prlerrtrro?~ine Prolerrs rrrir.nbilis P. rrlorgnnii Pseudololrronas nerriginosn P . cepncin P . j7uorescet1s P . maltophilin P . pulida P . slulzeri Serralin liquefaciens S. morcescens
Total OAll test strains grew well o n MacConkey agar at both incubation lemperatures.
22°C 48 h
C A N . J . MICROBIOL. VOL. 2 5 , 1979
TABLE 4. Stability of recovery and selectivity characteristics of CIN agar
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Storage time," days Test organism
Pserrrlot~~o~~r~s oer~rgitiosn(2 strains) Etrte~.obacteraeroge>les(2 strains) Escheric/~iacoli (2 strains) Serratia ~~~rrrcescet~s (1 strain)' Protelrs ~~lirnbilis (1 strain)
NGd NG NG NG NG
Growth on streak plates NG NG NG NG NG NG NG NG NG NG
NG NG NG NG NG
'At room temperature. bBascd o n mean of duplicate plates compared with recovery o n blood agar. ( N D , n o data. WG, n o ~ r o w t l i . .'Different strain than shown in Table 3.
TABLE 5. Relative performance of CIN, SS, and MacConkey agars for isolation of Yersinicr enterocoliticn from 527 fecal specimens
% specimens SS
CIN Direct
Cold"
Directb
MacConkey Coldc
Direct
Cold
Total growth present 3+ to4+ I + to 2+ 0 to few Presumptive colonies
present requiring confirmation" Y. e~lterocolitico isolated OAfter cold enrichnient in PBS. pH 7.6, at 4'C for 14 days. bFor 503 specimens. 'For 443 specimens. "'Based o n observations for 423 specimens.
not inhibit Set.rtrtirt. Recovery of Strlnzonelltr and Shigcllcr on CIN agar was not attempted because. fit-st, the primary interest was in a specific medium for Y. enro.oc~o1itic.nand not all potential enteric pathogens; and. secondly, available information on it-gasan indicated that these organisms are inhibited by concentrations lower than that used in CIN agar. Most of the other Gram-negative bacteria that were able to grow on CIN agar were easily differentiated from Y. entcrocoliticrr. Ento.obnctet colonies were mucoid. raised, and had a diffuse pink coloration in contrast to flat Y. entcroc~oliticacolonies with a confined deep red center. Pseudonzotzns colonies were colorless. and Acint~tobricfer colonies were pale yellow. Scuritia colonies re-
sembled E17terobcrctrr with a diffuse pink colol- and raised appearance; however, occasionally they could be confused with Y. cntcrocolitictr. Citt.ohcic.rer colonies came the closest in appearance to Y. ento.oc.olitien and could not always be reliably differentiated by morphology alone. The stability of CIN agar during storage at room temperature was demonstrated by the results shown in Table 4. There was no change in the quantitative recovery of five different serotypes of Y. et7tt~rocoliticcr..nor in the ability to inhibit completely eight strains representing five genera of other Gram-negative bacteria over 9 days' storage. The comparative pelformance of CIN with SS and MacConkey agars for recovery of Y. en-
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to.oc.olitic.rt from fecal specimens is shown in Table
5 . The background flora o n all media is reduced and the recovery of Y. c~17rci.oc.oliric.rt considerably improved by the use of cold enrichment. C I N agar showed a selectivity similar to S S but much greater than MacConkey agar. Differentiation of Y. cntpi.oc~olitic~rt on CIN was improved over that on SS agar, especially after cold enrichment when only 7% of the plates showed presumptive colonies on CIN agar compal-ed with 23% on S S agar. Isolation rates were highel- on CIN agar both with direct inoculation and after cold enrichment. C I N agar from every specimen recovered Y. c~ilrc~i~oc~olirit.cl f o ~ ~ npositive d by either S S and (or) MacConkey agars. Ten of I I colonies resembling Y. cnrci~o~.olitic~n on CIN agar and 9 of 1 I colonies which were did not appeal- to be Y. c~~~rci.oc~oliric.~r identified a s Cirrobcrc~rci.. Discussion Scrli~~o~~cllcr-Sl7igc~lltr (SS) and MacConkey agars have fi-equently been used for the isolation of Y. c17tc,l. oc.olit;c.n. we found, as have other workers ( ~ i ~ h c! ~ ~ 1977; i ~ L~~ h 1977; ~ ~ c[ 1978). that many stlxins of Y. c n f c l ~ o c ~ ~ ) l dj [ oj cnot ~cl grow well on ss agal-. ~h~ higher isolation I-ate I-ecently ,.eported by shmilovitz and K~~~~~~(1978) for Y , c,,rcroc.olitic.nfrom stool specimens with ss compared with MacConkey agar probably resulted from the greater selectivity of S S rather than its ability to support the growth of more strains. Our experiences with bismuth sulfite agar were similar in that certain strains of Y. ciltci.oc.o/i!ic~acould not be recovered on this medium. MacConkey agar, a s demonstrated in the work I-epol-ted here and observed by others (Mehlman cJt a / . 1978). grows all strains of Y. c i ~ t c ~ r o c ~ o l i tbut i c ~ its ( ~ .weakness lies in a low selectivity and in the absence o f a differential reaction. We found these shortcomings t o be pal-ticularly tr-oublesome with food enrichments which frequently contain large numbers of organisms that will grow and resemble Y. ciltc~roc.olitic.cron MacConke y agar. The medium developed in our laboratory has several distinct advantages over other entel-ic agar fol-mulations, including MacConkey agar. for the isolation of Y. ci7fc1.ocoliticn. It provides quantitative recovery with only 24 h of incubation at 32"C, which can be attributed in large part to a basal formulation synthesized specifically for optimum growth of Y. cnrcrocolitic~el.C I N agar is at the same time highly selective, especially against P. crclugii7o.sa. E. c.oli. K . p17c~rino17iae,and P. inirnhilis. which represent troublesome competitors in the isolation of Y. ~~nterocoliricer from
both clinical and environmental matel-ials. A color reaction I-esulting from fermentation of mannitol presents a characteristic colony appearance which can be used to differentiate Y. ci7rci.oc~olitic~crfrom most other Gram-negative bacteria able to grow on the medium. C I N agar significantly reduces the amount of fishing required for detection of Y. ci~rci.oc~olitiln among presumptive colonies compared with S S and MacConkey agars. This was demonstl-ated by observing that while only 7% of the CIN plates streaked from cold enrichment of fecal specimens contained presumptive colonies, the highest isolation I-ate was obtained by this combination of enrichment and medium. Other bacteria isolated on CIN agar from fecal specimens and sometimes rein colonial morphology sembling Y. c~ilrci~oc~oliticn are primarily species of Cirr.obac~rci~, which can easily be biochemically separated from Y. ~ n tcl.oc.olitic'cr.
Acknowledgments T h e author acknowledges the invaluable techniand Jean hcal assistance l ~of Irena~ Rybakowski ~ Bryce, and the generous provision of Y. ellrcl.oc.oliric.rr cultures by Dr. S. T o m a . Dr. M. Bissett, and Mrs. E. Christenson. This work was SLIPported in part by Research grant PR-742 fi-om the Ontario Ministryof Health. A H V O N E NP.. 1972. Human yersiniosis in Finland. I. Bacteriolserology, A n n , Clin, Res, 4: 30-38, ogy As,\KAM,,.\. Y..S. A ~ ~ ~N.,KAGA.IA. \ ~ ~M . N ~ R. S A K A Z A Kand I . K. T A M U R A1973. . T w o community outbreaks of human infection with Ye,~..~i/ritr r/lte/.ocolitic.(~. J. Hyg. 71: 7 15-723. C A P R I ~ LT I. . A. J . D R A P E A Uand . S. KASATIYA. 1978. Yc,rsi/litr c~t,rc,~.ocolitic.tr: serotypes and biotypes isolated from humans and the environment in Quebec. Canada. J . Clin. Microbiol. 8 : 7-11. Elss. J . 1975. Selective culturing of Yc,~,.si/lier o11rrocoIitic.tr at a low temperature. Scand. J . Infect. Dis. 7: 249-251. H A N N A M. . 0.. J . C. SI.EWAKT,Z. L. C A R P E N I E R and . C. V A N D L R Z A N1977. ~ - . Development of Yc,r.si~licre/ltc,~.oc.olitic,(/-likeorganisms in pure and mixed cultures on different bismuth sulfite agars. J . Food Prot. 40: 676-677. H I G H S M I T HA.. K.. J. C . F E E L E Yand , G. K. MORRIS.1977. Yc,/..si/lio c,/~rc,roc,olitic.tr: a review of the bacterium and recommended laboratory methodology. Health Lab. Sci. 14: 253-260. HIGHSMITH. A. K.. J . C . F E E L C YP.. S K A L I YJ .. G. WELLS.and from B. T . W o o o . 1977. Isolation of Yc,,:c.itritr c~t~terocolifictr well water and growth in distilled water. Appl. Environ. Microbiol. 34: 745-750. K A N E K ~K.. . S . H A M A D AY. , KASAI.and E. KATO. 1978. Occurrence of Yer.c.itlier o~to.ocolitictr in house rats. Appl. Environ. Microbiol. 36: 314-318. KAPPERUD. G. 1977. Yc,,sit~io~ , t ~ t ' r o c o l i t iand ~ . ~ Yr~vitlirrlike microbes isolated from mammals and water in Norway and Denmark. Acta Pathol. Microbiol. Scand. Sect. B, 85: 129-135.
~
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CAN. J. MICROBIOL. VOL. 2 5 , 1979
Yc,t,.sitritr orrr~t.oc~olitic~tr infections in Hungary. Acta MiLASSEN,J . 1972. Yc,~sitlioc,t~tc,roc.olitic.trin drinking water. crobiol. Acad. Sci. Hung. 23: 191-203. Scand. J . Infect. Dis. 4: 125- 127. L E E ,W. H . 1977. Two plating media modified with Tween 80for T O M A , S. 1973. Survey on the incidence of Yc~t.sirlitrc,t2trt~oc~olitico in the Province of Ontario. Can. J . Public Health, isolating Y~,.;itzitrc~t~to.oc~olitictr.. Appl. Environ. Microbiol. 64: 477-487. 33: 215-216. . Isolation of Yc,t..sit~itr M E H L ~ I AI.N J. . , C . C. G. A U L I S I Oand , A. C . SANDERS.1978. T O M A .S.. and V. R. D E I D K I C K1975. o~tc~t.ocolitic.tr from swine. J . Clin. Microbiol. 2: 478-48 I. Problems in the recovery and identification of Yc,rsitrio from T S U ~ O K U KM.. A , K. OI'SUKI.and K. I T A G A K1973. ~ . Studies o n food. J . Assoc. Off. Anal. Chem. 61: 761-771. Yc,,:sitlicrrt~toac~olitictr.. I. Isolation of Y. orto.ocolitic.rr from MORRIS. G. K., J . C. F E E L ~ Y W.. T . M A R T I Nand , J . G. WELL-S. swine. Jpn. J . Vet. Sci. 35: 419-423. 1977. Isolation and identification of Yo..sitri(r i,t~to.oc.olitic.tr. WAUTERS. G. 1973. Improved methods for the isolation and the Public Health Lob. 35: 217-232. Contrib. Microbiol. recognition of Yrt..sitrio rtrteroc~olitic~o. S C H I E M A N D. N . A. 1979. Influence of dyes on the quantitative Immunol. 2: 68-70. recovery of Y. c,trtc~roc.olitic.tr. Appl. Environ. Microbiol. (In A . Y. Y A N A G A W A . ZEN-YOJI.H.. S. S A K A IT. . M A R U Y A M and press). and Yc,r.sitlitr SHhllrovl-rz. M.. and B. K K E . I . Z ~ R 1978. . Isolates of Yc~~sitlier 1974. Isolation of Yersitlitr o~tet~oc~olitic~ei p.sc~rttlot~rboc~rtIc).si.s from swine. Jpn. J . Microbiol. 18: c~trtc~r~oc~oliti~ from clinical cases in northern Israel. Israel J. 103-105. Med. Sci. 14: 1048- 105.5. SZI.IA.J.. and A. SVIDRO.1976. A five-year study of human
D. A. S C . H ~ ~ M A N N Otrtorio Mitristty cJ'Hctrltlr. E r r ~ ~ i r o r r t t ~ eBtrc.trt~iology ~~t~rl Ltrhot~trror:~. BO.Y9000, T t ~ r t t ~ i t ~ At,r Tortirrto, l 0 1 1 t . C'trrrtrtltr , I M ~ WI R 5
Accepted July 30. 1979 S C H I E M A ND. N . A. 1979. Synthesisofa selective~tgarmediumforYc,r..sitritr c~rrrt~roco1itic.o. Can. J . Microbiol. 25: 1298-1304. A new agar medium for isolation of Yc,rsitritr c,trtrroc.olitic.o was formulated based on growth studies which defined an optimum basal. and the evaluation of selective chernic;ll agents, dyes, and antibiotics. The final formulation. designated cefsulodin-irgasan-novobiocin (CIN) agar, provided quantitative recovery of 40 different strains of Y. rtrrc,roc~olitic.oin 24 h usingincubation at 32°C o r with 48 h of incubation at 22°C. The medium was highly selective. especially ;rgzrinst P.sc,rrtlottrot~tr.(trc,r.rrgitloso. E.sc~lrc~t.ic~l~itr c,oli. Klrh.sirll~tptrrrrrtlotlitrr. and Protcrrs 1~1irt111ili.s. Colony morphology coupled with a differential reaction resulting from mannitol fermentation permitted discrimination of Y. rtltc,t.oc.olitic.o from most of those Cram-negative bacteria that were able togrow on the medium. Recove1.y and selective chal-acteristics of CIN agar were stable during stol-age at room temperature for 9 days. C I N agar gave a higher recovery of Y . errtrroc~olitic~trfrom feces both direct and with cold enrichment (0.4/1.5%) than Strlt~ro~rc~lItr-Sl~igc,lltr (0.0/0.7%) and MacConkey (0.0/0.9%) agars while significantly reducing the level of background organisms. S C . H I ~ . M , Z D. N NA. . 1979. Synthesis o f s selective agar medium for Yc,rsitlio c,trtc,roc.olitic.o.Can. J . Microbiol. 25: 1298- 1304. Une nouvellc gklose pour I'isolement de Yc,rsirritr c~trter.ot~o1itic.o a C-tl mise au point d';tprks des etudes de croissance qui ont permis de definir unc base optimale e t d'nprtls I'evalu:rtion d e I'effet d';tgents chirniques sdectifs. dc cololxnts et d'antibiotiques. La formule definitive. nppelee gelose cefsulodin-i~gasan-novobiocin (CIN), a perrnis une I-ecuplrntion quantitative de 40 ;tpres 24 h d'incubation I32"C ou 48 h h 22°C. C e milieu souches differentes de Y. c~t~io.oc.olitic.o oc~rtrgit~o.vo. E.sc~l~c~ric.l~io c.oli. Kle.11est fortement selectif pal-ticulitremcnt contre P.vclrt1ot~rotrtr.s .siclltr ptrc,rtrriotlitrc et Proterrs ttlir.ohi1i.s. La morphologie des colonies nssociee i une reaction differ-entiellc d e fermentation du mannitol pelmet une bonne discrimination entre Y . elltc,roc.olitic,tr et la plupart dcs batteries Gram-negatives capables de pousser sur ce milieu. Les proprietes de selection et de ricuperation de 1;)gelose CIN demeurent stables pendant 9jours b la temperature ambiantc. La gelose CIN utilisie pour isoler Y . rtr/c~r.oc.olitic.odes matieres fecales soit directemcnt OLI aprks enrichissement au froid donne un meilleur rendement (O.4/1.5%) que les gelose Snlrt~ot~~llo-SIIigrlltr (0.0/0.7%) et MacConkey (0.0/0.9%) tout en diminuant d e f21fon significative la qu;rntite de microo~,ganismesassocies. [ T n d u i t par le.journnl]
Bacteriological media presently used for the iso- e n t c r o t ~ o l i t i c ~such r, as the addition of Tween 80 to lation of Yt)i.sinirr c i l f e ~ ~ o c o l i t i crepresent u formu- MacConkey and deoxyribonuclease agars delations previously developed for general isolation scribed by Lee ( 1977), or the supplementation of S S of enteric bacteria. such as Srrli~~oi~clltr-Shigcllrragar with sodium desoxycholate as described by (SS) and MacConkey (Asakawa ct trl. 1973; Cap- Wauters (1973). The pe~formanceof media presrioli c.1 t r l . 1978; Kaneko er trl. 1978; Morris cf trl. ently used for recovery of Y. ci~tci.oc.oliti~.tr is f~-e1977; Toma 1973; Toma and Deidl-ick 1975; quently improved by incubation at lower temperaTsubokul-a c t t r l . 1973; Zen-Yoji cr t r l . 1974), tures (e.g. 22"C), which then requires 2 days or Drigalski (Lassen 1972), lactose-sucrose-UI-ea longer for colony development. (LSU) (Ahvonen 1972; Eiss 1975; Kapperud 1977). We undertook the development of a new selecdesoxycholate citrate (Szita and Svidro 1976). and tive agar medium specifically for isolation of Y. bismuth sulfite (Hanna cf t r l . 1977) agars. Saari and cntci.ocolirictr. beginning with growth studies Quan (T. N . Saari, and T . J . Quan. 1976. Abstr. which defined an optimum basal fol-mulation (in Annu. Meet. Am. Soc. Microbiol., p. 45) described preparation). Dyes (Schiemann 1979) and chemimedium, and cals (in preparation) I-epresenting potential seleca deoxycholate-citlate-mannitol Highsmith er t r l . (1977) reported the use of m-Endo tive agents were evaluated in another agar formubroth for isolation of Y. c~nferocolifit~n from water. lation for their effect on the quantitative recovery Fifty-seven antibiotics were There have been a few attempts to modify available of Y. enfe~~ocolifit~cr. media for improved selection or differen tat ion of Y. studied as potential selective agents and various 0008-4 16617911 1 1298-07$0 1 .00/0 @ I979 National Research Council of Canada/Conseil national d e recherches du Canada
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SCHIEMANN
,
differential systems were examined in the final agalformulation. ~h~ evaluation of the agar medium which evolved from these separate studies is described in this paper. Materials and Methods
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Tcjst 0tgo11isttr.s The sources and types of 68 Y. c,~rrc,roc~olirico cultures used in thcse studies are identified in Table I. Stock cultures were maintained in refrigerated trypticase soy agar stabs and working c ~ r l t l ~ r eon s slants of the same medium. Inocula for test plates consisted of suspensions prepared in phosphate-buffered saline (pH 7.2) from overnight blood agar cultures incubated a t 30°C. Suspensions were diluted s o that 50-100 colonies were usually obtained on each of duplicate test plates inoculated by the spreading of 100 ILLdelivered by an automatic pipette. Recovery of test organisms was expressed as a percentage of the mean count obtained on duplicate blood agar plates prepared and incubated under the same conditions. Cultures of 69 other Gram-negative bacteria representing 10 different genera used to evaluate the selectivity of the formulated medium were maintained on refrigemted tl-ypticase soy agar slants and prepared for use in trypticase soy broth incubated overnight at 35'C. Inoculation of the selective medium. and MacConkcy ag:rrforcomparntive purposes. was by streaktng. Agor Mctlio Thc final medium formulated from gr-owth and selective agent studies is called cefsulodin-irgasnn-novobiocin (CIN) agar after important selective substances which i t contains. CIN agar is prcpared as follows: the basal is first prepared by suspending the following substances in 748 mL of high-purity distilled water: special peptone (Oxoid), 20 g: yeast extract (Difco). 2 g; mannitol. 20 g ; pyi.uvic acid (sodium salt). 2 g ; sodi~inlchloride. I g: magnesium sulfnte.7HZ0, 10 mg (10 m L of a 0.1% stock solution); agar (Oxoid No. 4). 12 g. This suspension is brought to a boil to dissolve the agar completely. The solution is cooled to about 80-85°C and 10 m L of isgasan DP300 (2.4.4'-trichloro-2'-hydroxy diphenyl ether) (Ciba-Geigy) solution (0.04% in 95% ethanol) is added followed by vigorous shaking to volatilize the ethanol. The solution is cooled in a water bath to about 47°C. Bile salts (Difco) ( 2 g) are suspended in 200 mL of high-purity distilled water and dissolved by heating to boiling with stirring. T h e solution is cooled to about 47°C and added to the previous mixture. One miliilitre of 5 N NaOH is added followed by 10 m L of each of the following stock solutions: neutral red. 3 mglmL; crystal violet. 0.1 mg/mL; cefsulodin (Takeda Chemical Industries Ltd.). 1.5 mglmL; novobiocin (The U p ~ o h nCo.), 1 .5 mg/mL. Stock solutions ofantibiotics are stored at -70°C and thawed at room temperature just before use. The medium is then placed on a magnetic stirrer and 10 mL of a 10% solution of strontium chloride (tiltel--sterilized)is added slowly with constant mixing. T h e final pH is ad.justed to 7.4 with 5 N NaOH. Two modifications of the above fol-mula were also prepared for- evaluation: CIN(1) agar containing 0.5% insteed of 0.2% bile salts; and CIN(I1) agar containing 0.5% bile salts and 0.02% sodium citl-ate. All evaluations except the stability study were completed with CIN agar which was I day old and MacConkey agar which was no mol-e than 4 days old. I.soltrtiot~f,.otnFccc,.s CIN agar was compared with S S and MacConkey agars for
1299
recovery of Y. etltc,roc~olific~c( from fecal specimens reccived by the O n t a ~ i oCentral Public Health Laboratory in buffered glycerol saline tlxnspol-t medium. Plates were streakeddirectly after enrichment ofappr.oximatelyI of feces i n m L of M/ 15 phosphate-buffered sirline, pH 7.6, incubated at 4"Cfor 14 days..^^' agar was incubated a t 32°C for 24 h and SS and MacConkey agars at room tempel'ature fol- 48 h. Every attempt was made to eliminate any bias when we compared the three media. All tishingsofpl-esumptive colonies were done by one individual who was very familiar with the appearance and v;rriability of Y. pt~tproc.olitic,tron these media. T w o colonies only were selected for confirmation from each plate heal-ing organisms which resembled Y. ('tlt~t.ocoIiti(.(~most closely.
Results CIN(1) agar was evaluated with 30 different strains of Y. c)nter.oc.olitic.cl selected from those shown in Table 1. MacConkey agar was included for comparative purposes. Two incubation conditions were used. 32°C for 24 h and 22°C for 48 h. Overall recovery based on duplicate plates when compared with blood agar under the same conditions averaged 94 and 1005%for CIN as compared to 87 and 94% with MacConkey agar for 32 and 22"C, respectively. Because the formulation for CIN(1) agar developed a fine precipitate during storage. a modification was prepared containing 0.02% sodium citrate and a higher concentration of bile salts. This medium, CIN(I1) agar-, was compared with MacConkey agar for quantitative recovery of 33 strains of Y. cnter.ocoliticc1 selected from those shown in Table 1 . Overall recovery on CIN(I1) agar was 92 and 96% when compared with 93 and 88% on MacConkey agal- for 32 and 22°C. respectively. One strain did not grow on either medium at 32°C but was recovered quantitatively at 22°C. Three strains showed lower recovery at 32°C on CIN(I1) agar, suggesting inhibition by citrate. Furthermore, overall colony size and pigmentation were more restricted in the presence of citrate. The inclusion of sodium citrate in CIN(I1) agar did not prevent precipitation during storage, which produced a fine cloudiness in the otherwise transparent medium. Therefore, a third and final modification, designated simply CIN agar, was prepared in which the bile salts concentration was reduced to 0.2%. Precipitation was now minimal if care was taken in reducing the temperature of the basal medium and bile salts solution before mixing together, if the strontium chloride solution was added slowly with good stirring, and if room instead of refrigeration temperature was used for storage. Quantitative recovery of 40 strains of Y. enterocoliticcl on this medium is shown in Table 2.
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C A N . J . MICROBIOL. VOL. 75, 1979
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TABLE1. Yersinia ctzterocolitica cultures used to evaluate selective agar media Culture No."
Serotype 0
Biotype (Wauters)
Rhamnose fermentationb
Culture No.
Serotype
EM1 E002 E004 E006 E007 E008 E009 E012 E013 E014 E015 E016 E017 E020 E021 E022 E023 E025 E026 E028 E030 E03 1 E032 E040 E041 E042 E043 E044 E045 E046 E047 E050 E051 E052
9 9 9 8 8 8 8 3 3 3 6,30 6, 30 6,30 5,27 5,27 5,27 5,27 16 17 4,33 2,3 NTd 5 6 6 13,7 13,7 13, 7 16 16 16 12 5 6,31
2 2 2 1 1 1 1 4 4 4
-
E053 E055 E057 E059 E060 E06 1 E062 E063 E064 E066 E067 E068 E069 E070 E070 E076 E077 E083 E089 E093 E096 E097 El00 El05 El06 El29 El31 El32 El33 El34 El63 E l 64 El68 El72
6, 31 13,7 14 NT 13,7 6, 30 21 18 7,8 17 NT 4,33 5,27 5, 27 7,8 8 8 5,27 5,27 4 4,32 11 4 8 21 8 8 8 5,27 5,27 13, 7 NT NT 17
1
1 1 2 2 2 2 NAc
-
-
-
-
-
NA
'"
"
"
0
Biotype (Wauters)
Rhamnose fermentation
NA
NA
'6
4'
1 1 1 1 1 1 1 1 1 1 2 2 1 1 1 2 2 1 1 1 2 1 3 NA ' 6
+ +
-
+ + + + +
-
+ +
-
NA cc
‘G
" "
1 Atypical 1 1
-
+ + +
aCultures E001-E028 and E069-El06 are human and animal isolates obtained from Dr. Sandu Toma Canadian National Reference Center for Ycrsij~ia Toronto, Ontarlo. Cullures E030-E055 are human isolates obtained from Mrs. Eleanor Christenson, State Laboratory of Hygiene, Madison, Wisconsin. Cult(~resE129-El34 are human isolates obtained from D r . Marjorie Bissett, California State Department of Health, Berkeley, California. Cultures E057-E068 and E163-El72 are milk and food isolates obtained from own laborarory. bAll cultures with a rhaninosc reaction shown are sucrose-positive. =NA, not available. *NT, nontypable.
The results show that recovery on CIN was comparable to that on blood agar for all strains using either 32°C for 24 h or 22°C for 48 h. The colony size of Y. entrroc,oliticcr was larger on CIN than on MacConkey agar. The predominant colony size for 40 strains of Y. ent~rocoliticu was measured to the nearest 0.05 mm on both media and found to average 1.35 mm ( s = 0.185) versus 0.72 mm ( s = 0.149) at 32"C, and 1.61 mm (s = 0.176) versus 1.23 mm (s = 0.217) at 22°C for CIN and MacConkey agars, respectively. The colony appearance of Y. ente~.ocoliticaon CIN agar was quite distinctive when compared with growth on MacConkey agar. Colonies on CIN agar had a deep red center which had a rather sharp border, and was surrounded by an outer zone which was usually translucent. Even very small colonies,
which were more likely to be seen at 32 than at 22OC, had a red center when viewed with a stereomicroscope, and transmitted light. Pigmentation, the result of mannitol fermentation, was stronger and more complete at 22°C with 48 h of incubation than at 32°C with 24 h of incubation. The edge of the colony was either entire or irregular depending upon the strain. The selectivity of CIN agar is shown by the results presented in Table 3. All of the 69 strains of Gram-negative bacteria used to evaluate selectivity, representing 10 different genera, grew well on MacConkey agar at both temperatures, while only 17 strains grew on CIN agar at 32°C and 20 at 22°C. CIN agar was especially inhibitory to Pseucio~no~~as a e r ~ ~ g i ~ z oEscheric.lzia sa, coli, Klebsiellrl przeunzonirle, and Proteus mirabili.~,but did
SCHIEMANN
TABLE 2. Quantitative recovery of 40 strains of Yersirrin er~terocoliticnon CIN agar
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% recovery"
% recovery
Culture No.
32°C 24 h
22°C 48 h
Culture No.
EOO 1 E002 E004 E008 E012 E013 E014 E015 E017 E020 E021 E022 E023 E025 E030 E032 E040 E042 E044 E045
101 140 98 94 112 71 88 77 118 107 109 121 88 80 78 77 137 319b 114 86
91 139 89 99 84 89 94 97 108 99 98 131 102 100 103 107 79 290h 96 95
E046 E050 E05 1 E052 E053 E055 E073 E077 E083 E089 E093 E096 E097 El00 El05 El06 El 29 El31 El32 El33
1
X
32°C 24 h
22°C 48 h
106 94 105 97 95 89 107 125 98 100 77 99 105 95 90 110 107 94 100 91 99.5
88 109 96 123 115 103 93 113 92 148 71 99 103 86 97 96 106 105 103 104 101.3
"Based o n mean of duplicate plates conlpared to blood agar under same incubation conditions. bRepeated three times with same low recovery on blood agar. Later balches of blood agar gave eqoivalent recovery. Results not included in means.
TABLE 3. Selectivity of CIN agar against Gram-negative bacteria
No. strains recovereda Organism
No. strains tested
32°C 24 h
Acitrerobrrcter cnlconceric~rsvnr. ot~irrn~rrttr A . calcoace~icrrsunr. hvojji Aerornorlrrs Irydroplriln Cilrobncler cliuersus Leuirlen C . jierrrldii Enlerobncler. nerogerles E. crgglotr~ernr~s E. cloncne E. saknznkii Eschericlriir coli Flnvobnclerirrrr~orlorn~rrrr~ KlebsieNn osylocn K. prlerrtrro?~ine Prolerrs rrrir.nbilis P. rrlorgnnii Pseudololrronas nerriginosn P . cepncin P . j7uorescet1s P . maltophilin P . pulida P . slulzeri Serralin liquefaciens S. morcescens
Total OAll test strains grew well o n MacConkey agar at both incubation lemperatures.
22°C 48 h
C A N . J . MICROBIOL. VOL. 2 5 , 1979
TABLE 4. Stability of recovery and selectivity characteristics of CIN agar
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Storage time," days Test organism
Pserrrlot~~o~~r~s oer~rgitiosn(2 strains) Etrte~.obacteraeroge>les(2 strains) Escheric/~iacoli (2 strains) Serratia ~~~rrrcescet~s (1 strain)' Protelrs ~~lirnbilis (1 strain)
NGd NG NG NG NG
Growth on streak plates NG NG NG NG NG NG NG NG NG NG
NG NG NG NG NG
'At room temperature. bBascd o n mean of duplicate plates compared with recovery o n blood agar. ( N D , n o data. WG, n o ~ r o w t l i . .'Different strain than shown in Table 3.
TABLE 5. Relative performance of CIN, SS, and MacConkey agars for isolation of Yersinicr enterocoliticn from 527 fecal specimens
% specimens SS
CIN Direct
Cold"
Directb
MacConkey Coldc
Direct
Cold
Total growth present 3+ to4+ I + to 2+ 0 to few Presumptive colonies
present requiring confirmation" Y. e~lterocolitico isolated OAfter cold enrichnient in PBS. pH 7.6, at 4'C for 14 days. bFor 503 specimens. 'For 443 specimens. "'Based o n observations for 423 specimens.
not inhibit Set.rtrtirt. Recovery of Strlnzonelltr and Shigcllcr on CIN agar was not attempted because. fit-st, the primary interest was in a specific medium for Y. enro.oc~o1itic.nand not all potential enteric pathogens; and. secondly, available information on it-gasan indicated that these organisms are inhibited by concentrations lower than that used in CIN agar. Most of the other Gram-negative bacteria that were able to grow on CIN agar were easily differentiated from Y. entcrocoliticrr. Ento.obnctet colonies were mucoid. raised, and had a diffuse pink coloration in contrast to flat Y. entcroc~oliticacolonies with a confined deep red center. Pseudonzotzns colonies were colorless. and Acint~tobricfer colonies were pale yellow. Scuritia colonies re-
sembled E17terobcrctrr with a diffuse pink colol- and raised appearance; however, occasionally they could be confused with Y. cntcrocolitictr. Citt.ohcic.rer colonies came the closest in appearance to Y. ento.oc.olitien and could not always be reliably differentiated by morphology alone. The stability of CIN agar during storage at room temperature was demonstrated by the results shown in Table 4. There was no change in the quantitative recovery of five different serotypes of Y. et7tt~rocoliticcr..nor in the ability to inhibit completely eight strains representing five genera of other Gram-negative bacteria over 9 days' storage. The comparative pelformance of CIN with SS and MacConkey agars for recovery of Y. en-
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to.oc.olitic.rt from fecal specimens is shown in Table
5 . The background flora o n all media is reduced and the recovery of Y. c~17rci.oc.oliric.rt considerably improved by the use of cold enrichment. C I N agar showed a selectivity similar to S S but much greater than MacConkey agar. Differentiation of Y. cntpi.oc~olitic~rt on CIN was improved over that on SS agar, especially after cold enrichment when only 7% of the plates showed presumptive colonies on CIN agar compal-ed with 23% on S S agar. Isolation rates were highel- on CIN agar both with direct inoculation and after cold enrichment. C I N agar from every specimen recovered Y. c~ilrc~i~oc~olirit.cl f o ~ ~ npositive d by either S S and (or) MacConkey agars. Ten of I I colonies resembling Y. cnrci~o~.olitic~n on CIN agar and 9 of 1 I colonies which were did not appeal- to be Y. c~~~rci.oc~oliric.~r identified a s Cirrobcrc~rci.. Discussion Scrli~~o~~cllcr-Sl7igc~lltr (SS) and MacConkey agars have fi-equently been used for the isolation of Y. c17tc,l. oc.olit;c.n. we found, as have other workers ( ~ i ~ h c! ~ ~ 1977; i ~ L~~ h 1977; ~ ~ c[ 1978). that many stlxins of Y. c n f c l ~ o c ~ ~ ) l dj [ oj cnot ~cl grow well on ss agal-. ~h~ higher isolation I-ate I-ecently ,.eported by shmilovitz and K~~~~~~(1978) for Y , c,,rcroc.olitic.nfrom stool specimens with ss compared with MacConkey agar probably resulted from the greater selectivity of S S rather than its ability to support the growth of more strains. Our experiences with bismuth sulfite agar were similar in that certain strains of Y. ciltci.oc.o/i!ic~acould not be recovered on this medium. MacConkey agar, a s demonstrated in the work I-epol-ted here and observed by others (Mehlman cJt a / . 1978). grows all strains of Y. c i ~ t c ~ r o c ~ o l i tbut i c ~ its ( ~ .weakness lies in a low selectivity and in the absence o f a differential reaction. We found these shortcomings t o be pal-ticularly tr-oublesome with food enrichments which frequently contain large numbers of organisms that will grow and resemble Y. ciltc~roc.olitic.cron MacConke y agar. The medium developed in our laboratory has several distinct advantages over other entel-ic agar fol-mulations, including MacConkey agar. for the isolation of Y. ci7fc1.ocoliticn. It provides quantitative recovery with only 24 h of incubation at 32"C, which can be attributed in large part to a basal formulation synthesized specifically for optimum growth of Y. cnrcrocolitic~el.C I N agar is at the same time highly selective, especially against P. crclugii7o.sa. E. c.oli. K . p17c~rino17iae,and P. inirnhilis. which represent troublesome competitors in the isolation of Y. ~~nterocoliricer from
both clinical and environmental matel-ials. A color reaction I-esulting from fermentation of mannitol presents a characteristic colony appearance which can be used to differentiate Y. ci7rci.oc~olitic~crfrom most other Gram-negative bacteria able to grow on the medium. C I N agar significantly reduces the amount of fishing required for detection of Y. ci~rci.oc~olitiln among presumptive colonies compared with S S and MacConkey agars. This was demonstl-ated by observing that while only 7% of the CIN plates streaked from cold enrichment of fecal specimens contained presumptive colonies, the highest isolation I-ate was obtained by this combination of enrichment and medium. Other bacteria isolated on CIN agar from fecal specimens and sometimes rein colonial morphology sembling Y. c~ilrci~oc~oliticn are primarily species of Cirr.obac~rci~, which can easily be biochemically separated from Y. ~ n tcl.oc.olitic'cr.
Acknowledgments T h e author acknowledges the invaluable techniand Jean hcal assistance l ~of Irena~ Rybakowski ~ Bryce, and the generous provision of Y. ellrcl.oc.oliric.rr cultures by Dr. S. T o m a . Dr. M. Bissett, and Mrs. E. Christenson. This work was SLIPported in part by Research grant PR-742 fi-om the Ontario Ministryof Health. A H V O N E NP.. 1972. Human yersiniosis in Finland. I. Bacteriolserology, A n n , Clin, Res, 4: 30-38, ogy As,\KAM,,.\. Y..S. A ~ ~ ~N.,KAGA.IA. \ ~ ~M . N ~ R. S A K A Z A Kand I . K. T A M U R A1973. . T w o community outbreaks of human infection with Ye,~..~i/ritr r/lte/.ocolitic.(~. J. Hyg. 71: 7 15-723. C A P R I ~ LT I. . A. J . D R A P E A Uand . S. KASATIYA. 1978. Yc,rsi/litr c~t,rc,~.ocolitic.tr: serotypes and biotypes isolated from humans and the environment in Quebec. Canada. J . Clin. Microbiol. 8 : 7-11. Elss. J . 1975. Selective culturing of Yc,~,.si/lier o11rrocoIitic.tr at a low temperature. Scand. J . Infect. Dis. 7: 249-251. H A N N A M. . 0.. J . C. SI.EWAKT,Z. L. C A R P E N I E R and . C. V A N D L R Z A N1977. ~ - . Development of Yc,r.si~licre/ltc,~.oc.olitic,(/-likeorganisms in pure and mixed cultures on different bismuth sulfite agars. J . Food Prot. 40: 676-677. H I G H S M I T HA.. K.. J. C . F E E L E Yand , G. K. MORRIS.1977. Yc,/..si/lio c,/~rc,roc,olitic.tr: a review of the bacterium and recommended laboratory methodology. Health Lab. Sci. 14: 253-260. HIGHSMITH. A. K.. J . C . F E E L C YP.. S K A L I YJ .. G. WELLS.and from B. T . W o o o . 1977. Isolation of Yc,,:c.itritr c~t~terocolifictr well water and growth in distilled water. Appl. Environ. Microbiol. 34: 745-750. K A N E K ~K.. . S . H A M A D AY. , KASAI.and E. KATO. 1978. Occurrence of Yer.c.itlier o~to.ocolitictr in house rats. Appl. Environ. Microbiol. 36: 314-318. KAPPERUD. G. 1977. Yc,,sit~io~ , t ~ t ' r o c o l i t iand ~ . ~ Yr~vitlirrlike microbes isolated from mammals and water in Norway and Denmark. Acta Pathol. Microbiol. Scand. Sect. B, 85: 129-135.
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Yc,t,.sitritr orrr~t.oc~olitic~tr infections in Hungary. Acta MiLASSEN,J . 1972. Yc,~sitlioc,t~tc,roc.olitic.trin drinking water. crobiol. Acad. Sci. Hung. 23: 191-203. Scand. J . Infect. Dis. 4: 125- 127. L E E ,W. H . 1977. Two plating media modified with Tween 80for T O M A , S. 1973. Survey on the incidence of Yc~t.sirlitrc,t2trt~oc~olitico in the Province of Ontario. Can. J . Public Health, isolating Y~,.;itzitrc~t~to.oc~olitictr.. Appl. Environ. Microbiol. 64: 477-487. 33: 215-216. . Isolation of Yc,t..sit~itr M E H L ~ I AI.N J. . , C . C. G. A U L I S I Oand , A. C . SANDERS.1978. T O M A .S.. and V. R. D E I D K I C K1975. o~tc~t.ocolitic.tr from swine. J . Clin. Microbiol. 2: 478-48 I. Problems in the recovery and identification of Yc,rsitrio from T S U ~ O K U KM.. A , K. OI'SUKI.and K. I T A G A K1973. ~ . Studies o n food. J . Assoc. Off. Anal. Chem. 61: 761-771. Yc,,:sitlicrrt~toac~olitictr.. I. Isolation of Y. orto.ocolitic.rr from MORRIS. G. K., J . C. F E E L ~ Y W.. T . M A R T I Nand , J . G. WELL-S. swine. Jpn. J . Vet. Sci. 35: 419-423. 1977. Isolation and identification of Yo..sitri(r i,t~to.oc.olitic.tr. WAUTERS. G. 1973. Improved methods for the isolation and the Public Health Lob. 35: 217-232. Contrib. Microbiol. recognition of Yrt..sitrio rtrteroc~olitic~o. S C H I E M A N D. N . A. 1979. Influence of dyes on the quantitative Immunol. 2: 68-70. recovery of Y. c,trtc~roc.olitic.tr. Appl. Environ. Microbiol. (In A . Y. Y A N A G A W A . ZEN-YOJI.H.. S. S A K A IT. . M A R U Y A M and press). and Yc,r.sitlitr SHhllrovl-rz. M.. and B. K K E . I . Z ~ R 1978. . Isolates of Yc~~sitlier 1974. Isolation of Yersitlitr o~tet~oc~olitic~ei p.sc~rttlot~rboc~rtIc).si.s from swine. Jpn. J . Microbiol. 18: c~trtc~r~oc~oliti~ from clinical cases in northern Israel. Israel J. 103-105. Med. Sci. 14: 1048- 105.5. SZI.IA.J.. and A. SVIDRO.1976. A five-year study of human
