1969, 18(2):207. Appl. Microbiol. 

Cleeland Jr. and W. F. DelorenzoE. Grunberg, E. Titsworth, G. Beskid, R. EnterobacteriaceaeIdentification of System (Enterotube) for Rapid Efficiency of a Multitest

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APPLIED MICROBIOLOGY, Aug. 1969, p. 207-213Copyright © 1969 American Society for Microbiology

Vol. 18, No. 2Printed in U.S.A.

Effi.iency of a Multitest System (Enterotube) forRapid Identification of Enterobacteriaceae

E. GRUNBERG, E. TITSWORTH, G. BESKID, R. CLEELAND, JR., AND W. F. DELORENZO

Department ofChemotherapy, Hoffmann-La Roche, Inc., Nutley, New Jersey 07110

Received for publication 6 June 1969

Enterotube, a multiple-test system which combines nine biochemical tests useful inthe identification of members of the family Enterobacteriaceae, was tested and com-pared with the PathoTec test system in the identification of gram-negative bacilliisc ated from clinical specimens. It was found that both the Enterotube and thePathoTec systems rapidly and accurately defined the position of the organisms in themnujor groups of the family Enterobacteriaceae. Discrepancies were noted between theresults of citrate tests on Simmons' citrate-agar and in the Enterotube, as well asbetween Simmons' citrate-agar and the PathoTec citrate test. It was concluded thatthe Enterotube system provides a simple, reliable, and rapid method for the pre-sumptive identification of Enterobacteriaceae. The major advantage of the Entero-tube is that all tests are done simultaneously by inoculation from a single isolatedcolony.

Rapid idsntification of gram-negative bacilli of impregnated paper discs, provides a simplethe family j nterobacteriaceae isolated from clini- system for the detection of carbohydrate fermen-cal specim ns has long posed a problem for the tation and hydrogen sulfide formation.clinical laboratory. "The group to which an The use of the PathoTec test reagent-impreg-organism belongs must be determined by a com- nated paper strips (General Diagnostics Div.,bination of biochemical tests, not by a single Warner-Chilcott Laboratories, Morris Plains,property" [e.g., lactose fermentation (4) ]. Various N.J.) for the determination of certain biochemicalschema based on selected biochemical tests have tests-cytochrome oxidase, phenylalanine deami-been advocated to simplify the identification of nase, urease, and lysine decarboxylase activity,Enterobacteriaceae (4, 5, 7, 8). Edwards and indole production, and acetylmethylcarbinolEwing (4) list eight biochemical reactions which production (Voges-Proskauer test)-has beenserve to separate members of the family into reported (1, 6, 10). The test-paper strips eliminatefour principle divisions: Shigella-Escherichia; the need for special media and can be storedSalmonella - Arizona - Citrobacter (Escherichia indefinitely. However, since a heavy growth offreundii); Klebsiella-Aerobacter-Serratia; and the organism is needed for the performance ofProteus-Providencia (Proteus inconstans). Seven- the tests, a 24-hr pure culture, preferably onteen additional tests for further categorizing the solid medium free of indicators, is required.organisms are listed by these authors. Isenberg A method which would reduce the number ofand Berkman (7) recommend a battery of 12 inoculations and the equipment needed to per-biochemical reactions which characterize the form a series of biochemical tests for the identifi-broad subdivisions within the Enterobacteriaceae. cation of Enterobacteriaceae would be of greatIt is evident that the preparation of 12 to 25 benefit to the diagnostic bacteriologist. Recentlyindividual cultures is costly not only in terms of a multiple-test system, Enterotube, which com-time but also in terms of media preparation and bines in a single unit nine of the biochemicalequipment. tests most useful for the identification of members

Sanders et al. (11) devised an enrichment of the family Enterobacteriaceae, has beenbroth which permits rapid detection of lactose developed. This paper describes a study carriedand sucrose fermentation, indole production, and out to test the efficiency and accuracy of themotility, as well as a phenol-red-iron-agar Enterotube system in comparison with testsmedium which, when used with carbohydrate- routinely used in this laboratory.

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MATERUILS AND METHODS

Strains tested. Nine laboratory strains of represent-ative gram-negative bacilli and 57 gram-negativestrains isolated from specimens submitted for diag-nostic bacteriology by the special treatment unit ofNewark City Hospital and the Hoffmann-La Rochedispensary were studied. The clinical strains compriseisolates from 29 urines, 4 sputa, and 2 throat swabs.

Broth cultures of the known laboratory strainswere first streaked on Trypticase Soy (TS; BBL) andEMB (Difco) agar to obtain isolated colonies. Clinicalspecimens were plated directly on TS, goat blood, andEMB agar for primary isolation of the organisms.

Enterotube. This is a prepared test system containedin a half-round, molded plastic tube which is dividedinto eight compartments, each containing a slant of astandard biochemical test medium. The flat side of thetube is sealed with a plastic film. A single wire, whichserves as an inoculating needle, extends lengthwisethrough the center of the tube and projects from eitherend. The ends are covered by plastic screwcaps.Small air holes in the side of three compartments(citrate, lysine, lactose) are sealed by a removable"activator" strip which is peeled off at the time ofinoculation to provide aerobic conditions duringincubation.To inoculate the Enterotube, the plastic caps are

removed and the inoculating wire is touched to thecenter of an isolated colony of the organism to betested. The wire is then withdrawn from the tube,thereby inoculating each of the media in the compart-ments. The wire may then be used to inoculate othertest media such as broth or motility test medium.After the wire has been withdrawn from the Entero-tube, the plastic caps are replaced and the activatorstrip removed. The test reactions are read after incu-bation for 18 to 24 hr at 37 C. After the reactions havebeen recorded, the tube may be reincubated to detectpossible delayed reactions.

Media in the compartments and the reaction of eachare listed below in the order in which they are con-tained in the tube starting from the first compartmentinoculated. (i) Citrate agar: a positive reaction isshown by a change of the indicator, bromothymol blue,from green to blue. (ii) Modified lysine-lactose me-dium: a positive reaction is shown by the change ofthe indicator, phenol red, from yellow to pink. (iii)Lactose fermentation: the fermentation of lactosechanges the indicator, phenol red, from pink to yellow.(iv) Dulcitol fermentation: the fermentation ofdulcitol changes the indicator, phenol red, from pinkto yellow. (v) Urea agar: the production of ammoniafrom urea is shown by a change of the indicator,phenol red, from yellow to pink. (vi) Phenylalaninedeaminase: to test for phenylalanine deamination,approximately 0.3 ml of 10% ferric chloride solutionis injected into the compartment through the plasticfilm by means of a syringe. This test must be per-formed after incubation for 18 to 24 hr. A positivereaction is indicated by the presence of a dark greencolor on the surface of the slant. (vii) Hydrogensulfide-indole agar: hydrogen sulfide production is

demonstrated by the formation of a black precipitatealong the line of inoculation.To detect the production of indole, approximately

0.3 ml of Kovac's reagent (9) is injected through theplastic film into the compartment by means of asyringe. In the presence of indole a cherry-red colordevelops. (The reagent used was prepared with p-dimethylaminobenzaldehyde, USP, obtained fromIndustrial Chemicals Division of Allied Chemical,Morristown, N.J.) (viii) Dextrose fermentation: thefermentation of dextrose changes the indicator,phenol red, from pink to yellow. AUl Enterobac-teriaceae ferment dextrose promptly. Therefore, thefailure of an organism to give a positive reaction inthis test indicates either that the inoculum was notcarried through all the compartments or that theorganism is not a member of the family Enterobac-teriaceae. The question of sufficient inoculum may beeasily resolved if the inoculating wire is used to inocu-late a broth or motility test medium when it is with-drawn from the Enterotube.

Paper strip tests (PathoTec). The growth of theorganisms on TS-agar plates (18 to 24 hr) was used inthe performance of the PathoTec tests according tothe manufacturers directions.

RESULTSLaboratory strains. The results of the tests

with known strains of representative gram-nega-tive bacilli are listed in Table 1. From the table, itmay be seen that both the Enterotube tests andthe PathoTec tests serve well in determining theposition of the organisms studied in the majorgroups of the family Enterobacteriaceae. Theclassification of Escherichia, Salmonella, andProteus groups by either method is clear cut. Thegeneral lack of biochemical activity of the indole-negative, dulcitol-negative Shigella strain testedis reflected in both test systems. In either case,motility studies would be necessary to classify theorganism. A study of fermentation reactions,especially of dextrose and lactose, would also benecessary with the PathoTec system. Klebsiellaare more readily classified by the Enterotubesystem than by the PathoTec system with whichancillary tests for motility and carbohydratefermentation would be required to determine towhich group the organism belonged. Nonpig-mented strains of Serratia, although definitelyfalling into the Klebsiella-Aerobacter-Serratiadivision when tested by either method, wouldprobably be classed as Aerobacter C by theEnterotube system. Motility, fermentation, andgelatin liquefaction studies would be required fora more definitive classification.The pseudomonads were tested because they

are clinically important gram-negative bacilliwhich might be confused with the Enterobac-teriaceae. Although Pseudomonas aeruginosa

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IDENTIFICATION OF ENTEROBACTERIACEAE

TABLE 1. Comparison ofEnterotube and PathoTec systemsfor the identification ofgram-negative bacilli: representative laboratory strainsa

Proteus-Shigella-Escherichia Salmonella-Arizona- Klebsiella-Aerobacter- Provi-

division Citrobacter divisions Serralia division dencia Pseudomonasdivision5

System Biochemical test

Salmonella Klebsiella Serratia Proteus Pseudo- Pseudo-Shigella Escher- Salmonella Shl-Pem-Srai rlu monasmoaIflexnri ichia coli ityphos muelleri niae marcescens mirabilis aeruginosa Pseudo-

Entero- Dextrose + + + + + + + ±4-tube

Hydrogen - _ + + _ _ _ -

sulfideIndole - + _ _ _ _ - -|Phenylala- - - - - - - + - -nine

Urea - -4- ± + -Dulcitol - - _ + + - - _ |Lactose - + - - + - - (+Lysine - - (+) + (±) - | + (Citrate - + + + i +

S/higella or EierAerobacler Non- Non-Identification Aerobacter Escher- Salmonella Salmonella Klebsiella C or P. Entero- Entero-

C ichiaSerralia mirabilis bacteri- bacteri-aceae aceae

Patho- Cyto- - - - - - - - + +Tec chrome

oxidaseIndole +_ _ _ _ _

Phenylala- - - - - - - + _ -nine

Urea - - - - - + - -Voges- - _ _ + +_ _Proskauer

Lysine - - + + - ± - - -Citrate - - - + + + + ±+

Suigella or nifi Klebsiella- Klebsiella- P. Pseudo- Pseudo-Identification Aerobacler Es/e-Salmonella Salmonella Aerobacter- Aerobacter-miais mas mnac i/i Serratia, Serralia

aSymbols: +, positive; (+), delayed positive;-, negative.

b Escherichia freundii.c Proteus inconstans.

usually produces a characteristic pigment,nonpigmented strains are known to exist (12).P. pseudomallei may become slightly pigmentedafter several days of incubation, but the coloniesof many strains are colorless on TS agar for thefirst 24 to 48 hr and may be colorless on EMB aswell. The two pseudomonas strains tested gaveequivocal results by the Enterotube system sincethe fermentation of dextrose was weak or ques-tionable. The PathoTec CO test, on the otherhand, immediately demonstrated that theorganisms were pseudomonads.

±, weakly positive; (4), delayed weakly positive;

Identification of clinical isolates. A total of57 colonies of gram-negative bacilli suspectedof being Enterobacteriaceae were isolated fromclinical specimens and tested by the Enterotubesystem. The same isolates were tested by thePathoTec system after isolation in Sanders'Enrichment Medium (Difco) and plating on TSand EMB agar. Many strains were also tested onKligler's Iron Agar (Difco) and Simmons'Citrate Agar (Difco) (Table 2). Employing theschema of Edwards and Ewing (4), 30 strainswere classified as members of the Shigella-

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TABLE 2. Comparison of results with Enterotube and PathoTec tests in the identification ofclinical isolates"

Classification by Enterotube PathoTec results Other tests

30 Shigella-Escherichia division27 Escherichia: I +, PA -, Lact +

15 C-

4 C +, 8 C (+)

3 "Atypical" Escherichia: I +, PA-,Lact -, C -, Lys (+)

17 Klebsiella-Aerobacter-Serratia division3 Klebsiella: I -, PA -, Lact +,

C +, Lys +, H2S - [2 U +, IDulcitol +1

4 Aerobacter A (A. cloacae): I -,PA -, Lact +, C +, Lys -,U +, H2S-, Dulcitol -

1 Aerobacter B (A. aerogenes): I -,PA -, Lact +, C +, Lys (+)U -, H2S -, Dulcitol -

9 Aerobacter C (A. liquefaciens):I -, PA -, U -, H2S -, Dul-citol -

[ Lact -, Lys (+), C(+)][1 Lact - Lys -, C +]

[1 Lact (:±), Lys (+), C +j

[2 Lact +, Lys -, C-]

[4 Lact Lys + or (+), C (+) or

8 Proteus-Providencia division7 Proteus mirabilis: I -, H2S +,

PA +, U + Lact -, C + or (+)

1 P. rettgeri: I +, H2S i, PA +,U +, C +

2 Indeterminant: dextrose +[1 Lact (+), all other tests-][1 I +, H2S +, PA +, Lact +,Lys -, C +1

13 1 +, PA -, C -, VP -1 I +, PA-, C +, VP -1 I +, PA -, C-, VP +7 1 +, PA -, C -,VP -1 I-, PA -,C +, VP -3 1 +, PA-, C +, VP +1 +3 I +, PA-, C-, Lys +,VP -

1 I - PA -, C +, Lys -,VP +

1 C +, VP +1 I +, C +, VP +3 I -, PA -, C +, Lys -,VP +, U +

1 I-, PA -, C +, Lys -,VP +, U -

1 I -, PA -, C -, Lys -,VP +

1 I - VP Lys +, C +1 -, PA -, VP -, Lys +,C -, U-, CO -

11I- PA VP +, Lys +,C +, U :, CO -

I1I- PA-, VP Lys-,C-, U -, CO -

1 -, VP +, C , CO -4 CO +

21-, PA +, U +, C +

1I-, PA +, U +, C-3 I-, PA +1 I-1 I -, PA -, U -, C +,CO +

1 CO +1 Lys +

8 Simmons'-, 1 ±1 Simmons'-1 Simmons'-7 Simmons'-1 Simmons' +3 Simmons' +1 Simmons' -3 Simmons' - KIA Lact-,2 EMB Lact +, 1 motile1 EMB Lact -, motile

1 Simmons' +, not motile

1 Simmons' +, not motile

3 Simmons' +,

1 Simmons' +

1 Simmons' +, motile

1 Simmons' +, motile1 Simmons' +, motile

1 Motile

1 Simmons' (±)

1 Simmons' +,1 -

Mixed cultureMixed culture

a Abbreviations: I, indole; PA, phenylalanine deaminase; U, urease; Lact, Lactose; Lys, lysine decar:boxylase; C. citrate; VP, Voges-Proskauer; CO, cytochrome oxidase; KIA. Kliger's Iron Agar. Symbols-+, positive; (+), delayed positive; 4, weakly positive; (±t), delayed weakly positive; -, negative

Escherichia division since they were indole- fermented lactose in the Enterotube. This waspositive and phenylalanine deaminase-negative confirmed by the demonstration of lactose fer-when tested by Enterotube. Of these strains, 27 mentation on EMB agar. Of the 27 strains, 15were found to be Escherichia since they also were citrate-negative in the Enterotube.

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IDENTIFICATION OF ENTEROBACTERIACEAE

TABLE 3. Correlation of the Enterotube andPatho Tec testsa

Entero- Entero- TotalTest tube tubeegeenpositive negative agreeing

Indole ................... 29/31 18/19 47/50Phenylalanine deami-nase ................... 6/7 38/38 44/45

Urease................... 6/8 19/21 25/29Lysine decarboxylase..... 7/10 22/26 29/36Citrate ................... 16/26 18/20 34/46

a Values are expressed as the number of Patho-Tec test in agreement/number tested.

The PathoTec test results with these 15 strainswere in agreement with the Enterotube resultsexcept for one strain which was citrate-positiveby the PathoTec test. Another strain, althoughPathoTec citrate-negative was Voges-Proskauer(VP)-positive. One of 11 strains tested grew onSimmons' citrate agar, although it was citrate-negative by both the Enterotube and PathoTeccitrate tests.The remaining 12 indole-positive, lactose-

fermenting strains were found to be citrate-positive in the Enterotube. These characteristicsindicated that the organisms were E. intermedia(2). However, seven of these strains were citrate-negative and VP-negative in PathoTec tests andfailed to grow on Simmons' Citrate Agar. Onestrain was PathoTec and Simmons' citrate-posi-tive but indole- and VP-negative by the PathoTectest. Three strains which were citrate- and VP-positive in the PathoTec tests grew on Simmons'Citrate Agar. The twelfth strain did not grow onSimmons' Citrate Agar. It was indole-positive inthe PathoTec test but was not otherwise tested inthis system.Three indole-positive, phenylalanine deaminase-

negative strains did not ferment lactosein the Enterotube or in Kligler's Iron agar.These strains were lysine decarboxylase-positiveboth in the Enterotube and the PathoTec test,indicating that the organisms were not Shigella.Lactose fermentation or motility was demon-strated by conventional methods and theorganisms were classed as "atypical" Escherichia.

Seventeen isolates were placed in the Klebsiella-Aerobacter-Serratia division since they wereindole-, phenylalanine deaminase-, and hydrogensulfide-negative by the Enterotube tests. Eightstrains which were lactose- and citrate-positivewere classified on the basis of their reactions withlysine, dulcitol, and urease as Klebsiella (threestrains), Aerobacter A (A. cloacae, four strains),or Aerobacter B (A. aerogenes, one strain). ThePathoTec tests were in agreement except for one

citrate test which was negative, although thestrain was positive in the Enterotube and onSimmons' citrate agar. The remaining nine strainswere classified as Aerobacter C (A. liquefaciens)since they displayed negative or delayed reactionswith lactose, lysine, and urea or were citrate-negative. However, the PathoTec CO test demon-strated that four of the isolates were cytochromeoxidase-positive and thus were pseudomonads.The PathoTec test results with the remaining fivestrains identified as Aerobacter C by Enterotubealso indicated that the organisms were AerobacterC, although the citrate tests with two strainsdisagreed with the citrate tests in the Enterotube.

Eight isolates were placed in the Proteus-Providencia division since in the Enterotube theywere phenylalanine deaminase- and urease-positive. Seven were identified as Proteusmirabilis, being indole-negative and hydrogensulfide-positive. The PathoTec tests also identifiedthese organisms as P. mirabilis. One isolate whichappeared to be P. rettgeri from the Enterotubetests was found to be a Pseudomonas species whentested for oxidase activity with the PathoTec COstrip.Two colonies which gave confusing, therefore

indeterminant, results in Enterotube were foundto be mixed cultures upon examination of TSagar plates streaked from the broths inoculatedwith the Enterotube wires.When the results with individual tests repre-

sented in both systems were compared (Table 3),few discrepancies were noted in the indole,phenylalanine deaminase, and urease tests. In 3of 50 indole tests, 1 of 45 phenylalanine, and 4 of29 of the urease tests, the PathoTec results werecontrary to those obtained with the Enterotube.Mann and Gandelman (10) reported a numberof discrepancies between the PathoTec indole testand the conventional indole test with strains ofProteus vulgaris and P. rettgeri. The discrepan-cies noted here occurred with one strain each ofEscherichia, Klebsiella, and a pseudomonad. Theorganism which was phenylalanine-positive byEnterotube but -negative by PathoTec test wasone of the pseudomonads identified by the Patho-Tec CO test. This pseudomonad was also urease-positive in the Enterotube but -negative byPathoTec test. The two organisms that gavepositive urease reactions by PathoTec but werenegative in the Enterotube were Aerobacter Cstrains, organisms which are generally variablein urease activity. Of 10 strains which were lysinedecarboxylase-positive in the Enterotube, sevenwere also positive by the PathoTec test. ThePathoTec lysine decarboxylase-negative orga-nisms were one indole-positive, citrate-positivestrain classified as E. intermedia, a Klebsiella,

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TABLE 4. Comparison of the Enterotube and Patho-Tec tests for citrate utilization with tests on

Simmons' citrate agara

CitrateTest

Positive Negative

Enterotube ......... 14/23 13/15PathoTec ............ 11/12 19/23

a Values are expressed as the number ofSimmons' citrate in agreement to the number tested.

and an Aerobacter strain. There was agreementbetween the two test systems with 22 of the 26strains which were lysine decarboxylase-negativein the Enterotube. Two Escherichia, one P.mirabilis, and one Aerobacter C strain werepositive in the PathoTec test. Thus, a total of 29of the 36 tests were in agreement. Gandelman andMann (6) reported that 16 strains of P. mirabiliswhich were lysine decarboxylase-positive in thePathoTec test were -negative upon testing withlysine decarboxylase broth (Difco). No Escheri-chia or Aerobacter strains were tested in theirstudy. The PathoTec tests for citrate utilizationwere in agreement with the Enterotube with 34 ofthe 46 strains compared. One Aerobacter C andone Escherichia which were Enterotube citrate-negative were -positive by the PathoTec test,whereas seven Escherichia, the Aerobacter B, oneAerobacter C, and one P. mirabilis strain werepositive in the Enterotube but negative in thePathoTec test.When the results of the citrate tests with the

two systems were compared with the results oftests on Simmons' Citrate Agar (Table 4), it wasfound that 27 of 38 (71 %) of the Enterotubetests and 30 of 35 (86%) of the PathoTec testswere in agreement with the Simmons' citratetests. Thus, it appears that discrepancies incitrate utilization may be expected with either theEnterotube or the PathoTec system when com-pared to Simmons' Citrate Agar. The results withthe PathoTec citrate tests reported here differsomewhat from those reported by Borchardt (1),who found no false positives and a low percentageof false negatives with the PathoTec test whencompared with Simmons' citrate. However, itwas pointed out that false negatives would occurif the inoculum for the test was not sufficientlyheavy. Eight of the Simmons' citrate-negativestrains which were citrate-positive in the Entero-tube were considered to be Escherichia intermedia.The citrate reactions of these strains were weakor delayed in the Enterotube.

DISCUSSIONIt was the purpose of this investigation to assess

the rapidity and accuracy with which gram-negative bacilli of the family Enterobacteriaceaecould be presumptively identified by the use of theEnterotube in comparison with the use of thereagent-impregnated paper strip tests of thePathoTec system. Previous experience had shownthat the PathoTec tests, when used in conjunctionwith conventional fermentation tests, were ofgreat help in providing information about thebiochemical characteristics of isolates of gram-negative bacilli. The tests for cytochrome oxidaseand phenylalanine deaminase activity and indoleproduction are easily and quickly performed andthey quickly detect pseudomonads and membersof the Proteus-Providence groups, especiallywhen the organisms are present in pure culture inthe original specimen. The preparation of theremaining tests for urease and lysine decar-boxylase activity, citrate utilization, and acetyl-methylcarbinol production (Voges-Proskauertest) are simple and require relatively shortincubation periods. Only one reagent, 40% KOHfor the VP test, is required. However, since alarge inoculum is necessary for the performanceof the tests, the organism, unless present originallyin pure culture, must be isolated and subculturedon solid medium. On the other hand, althoughovernight incubation is required before the testscan be read, the Enterotube may be inoculateddirectly from the suspect colony on the primaryisolation plate. After overnight incubation, tworeagents are added and the results of all the testscan be read immediately. Since a series of inocula-tions are required with the PathoTec tests, thenumber depending on the organisms beingstudied, whereas a single inoculation suffices forthe Enterotube, the latter takes considerably lesstime to perform.

LITERATURE CITED

1. Borchardt, K. A. 1969. A faster test for gram-negative bac-teria. Lab. Manage. 7:47.

2. Breed, R. S., E. G. D. Murray, and N. R. Smith. 1957.Bergey's manual of determinative bacteriology. 7th ed.The Williams & Wilkins Co., Baltimore.

3. Christensen, W. B. 1946. Urea decomposition as a means ofdifferentiating Proteus and paracolon cultures from eachother and from Salmonella and Shigella types. J. Bacteriol.52:461-466.

4. Edwards, P. R., and W. H. Ewing. 1962. Identification ofEnterobacteriaceae, 2nd ed. Burgess Publishing Co., Minne-apolis.

5. Eickhoff, T. C., B. W. Steinhauser, and M. Finland. 1966.The KlebsieUa-Enterobacter-Serratia division. Biochem-ical and serological characteristics and susceptibility toantibiotics. Ann. Intern. Med. 65:1163-1179.

6. Gandelman, A. L., and P. H. Mann. 1965. An evaluation ofreagent-impregnated paper strips for use in the process of

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VOL. 18, 1969 IDENTIFICATION OF El

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nosis in a general hospital. Ann. N.Y. Acad. Sci. 98:647-699.

8. Kauffmann, F. 1956. A simplified biochemical table of En-terobacteriaceae. Acta Pathol. Microbiol. Scand. 39:103-106.

9. Kovacs, N. 1928. Eine vereinfachte Methode zum Nachweisder Indolbildung durch Bakterien. Z. Immunitatetsforsch55:311-315.

VTEROBACTERIACEAE 213

10. Mann, P. H., and A. L. Gandelman. 1965. An evaluation ofreagent-impregnated paper strips for use in the process ofidentifying certain species of clinically important bac-teria. H. Testing for the production of indole. Curr. Therav.Res. 7:510-512.

11. Sanders, A. C., J. E. Faber, Jr., and T. M. Cook. 1957. Arapid method for the characterization of enteric pathogenusing paper discs. Appl. Microbiol. 5:36-40.

12. Yow, E. M.. and E. S. Townsend. 1953. A comparison of thesensitivity of Pseudomonas aeruginosa to various antibi-otics. Antibiot. Chemother. 3:709-717.

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