JOURNAL OF CLINICAL MICROBIOLOGY, Feb. 1978, p. 180-185 Vol. 9, No. 20095-1 137/79/02-0180/06$02.00/0

Accuracy and Reproducibility of the Oxi/Ferm System inIdentifying a Select Group of Unusual Gram-Negative Bacilli

HARRIETTE NADLER,`* HARVEY GEORGE,' AND JUDITH BARR2Department of Clinical Laboratories and Division of Research, Lahey Clinic Foundation, Boston,Massachusetts 02215,' and Northeastern University, Medical Laboratory Science Program, Boston,

Massachusetts 021152

Received for publication 25 October 1978

The Oxi/Ferm (O/F) identification system was compared in a double-blindstudy to a conventional test battery for the characterization of 96 reference andclinical strains consisting of 83 nonfermentative and 13 oxidase-producing, fer-mentative gram-negative bacilli. The O/F tube and supplemental tests correctlyidentified 84% of the nonfermentative and 77% of the oxidase-producing, fermen-tative bacilli. However, when the supplemental tests were excluded and thebiochemical profiles generated by all nine O/F tube reactions were examined, theprofile accuracy reached 95% (79 of 83) for the nonfermentative and 93% (12 of13) for oxidase-producing, fermentative bacilli. Seven of the nine O/F substratereactions demonstrated -89% agreement with conventional reactions, whereasthe urea and arginine reactions provided 82 and 85% agreement, respectively.Replicate O/F tests with six selected organisms demonstrated 97% identificationreproducibility and 84% overall substrate reproducibility. The mean O/F identi-fication time was 2.6 days as compared to 3.3 days for the conventional system.Although this study suggests that the O/F system is a convenient, rapid, andaccurate alternative to conventional identification methods, several modificationsare recommended.

Nonfermentative and oxidase (OXI)-produc-ing, fermentative gram-negative rods have beenisolated from patients and the hospital environ-ment with increasing frequency, and their clini-cal significance has been established (3, 5, 6, 12,16, 19, 20). Despite the low degree of virulenceand limited invasiveness of these organisms,they have been isolated from genitourinary andrespiratory tracts, wounds, blood, and otherbody fluids of hospitalized patients and impli-cated as causative agents of sepsis, extensivesuppurative lesions, or septic shock (1, 17). Inaddition, these organisms may demonstrate mul-tiple drug resistance necessitating prompt iden-tification and susceptibility testing for adequatetherapy.

Simple, convenient, rapid, and standardizedidentification systems for these organisms havebeen developed recently. One of these, the Oxi/Ferm (O/F) system (Roche Diagnostics, Nutley,N.J.), proposes to provide identification of thisbacterial group in 48 to 72 h by the use ofminiaturized substrate quantities.

Earlier studies have evaluated certain aspectsof this system (4, 7, 8, 14, 15, 18). However, ourevaluation is unique in that it incorporates adouble-blind methodology and challenges thesystem with a broad spectrum of 37 species of

atypical, nonfermentative and OXI-positive fer-mentative bacilli, some rarely encountered andsome commonly encountered. This study inves-tigates identification and substrate accuracy, aswell as reproducibility and time and supplemen-tal test requirements of the O/F system.

MATERIALS AND METHODSBacteria. The 96 organisms selected for this study

reflect a comprehensive distribution of 83 nonfer-mentative bacilli, some commonly encountered andsome less frequently encountered, and 13 OXI-posi-tive, fermentative rods (Table 1). Forty organisms(42%) were stock cultures obtained from proficiencytesting agencies (College of American Pathologists,American Society of Clinical Pathologists, and Mas-sachusetts Department of Public Health), from anAmerican Society for Microbiology workshop (con-ducted by Rudolph Hugh), and from the Center forDisease Control (Robert Weaver). The remaining 56organisms (58%) were clinical isolates from urine, spu-tum, ear, eye, and wound cultures. Since Acinetobac-ter sp. and pyocyanogenic Pseudomonas aeruginosamay be readily identified by colonial and microscopicmorphology or by a limited biochemical profile, few ofthese strains were included. Atypical strains includedtwo nonfluorescent apyocyanogenic and six apyocyan-ogenic P. aeruginosa as well as one nonfluorescent P.fluorescens strain. All bacterial strains were main-tained at -80°C on glass beads (13).

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ACCURACY AND REPRODUCIBILITY OF O/F

A double-blind evaluation was conducted with thecooperation of the quality control laboratory of theLahey Clinic Foundation, which assigned randomly toeach organism both an O/F system and a conventionalmethod code number.Unknown organisms were placed into tryptic soy

broth (TSB; Grand Island Biological Co. [GIBCO],Lawrence, Mass.) and incubated for 24 h at 35°C (O/F) and at 30°C (conventional). Broths were sequen-tially subcultured onto tryptic soy agar plates with 5%sheep blood (sTSA; GIBCO) and to TSA (GIBCO)without blood. After an incubation period of 24 h, theorganisms on TSA were used to inoculate O/F andconventional systems.O/F identification system. The nine biochemical

substrates of the O/F tube (lot numbers 0046 and0051) were inoculated by using an isolated colonyaccording to the manufacturer's instructions. Simul-taneously, a 16- to 18-h TSB motility (MOT) test, aKirby-Bauer (2) susceptibility battery (penicillin, am-picillin, carbenicillin, kanamycin, colistin, novobiocin,and nalidixic acid; Difco Laboratories, Detroit, Mich.),and a Kovacs OXI test (Marion Scientific, KansasCity, Mo.) were conducted. All O/F tubes and othersubcultures were incubated at 35°C while cultures forflagella staining remained at room temperature. Allmedia and corresponding reagents used in this evalu-ation were prepared commercially (GIBCO).TSA cultures were observed for purity and colonial

characteristics. As the manufacturer instructed, theO/F tube reactions were interpreted after 48 h ofincubation. The second edition of the O/F computermanual was used for organism identification or selec-tion of supplemental tests for differentiation of similarorganisms.Standard biochemical battery (conventional).

Isolated colonies from 24-h-old TSA cultures weresubcultured with an inoculating wire to a conventionalbiochemical battery consisting of: O/F-dextrose, lac-tose, maltose, and xylose (open and closed); Moeller'sdecarboxylase-lysine, ornithine, and control; argininedihydrolase; triple sugar iron; Simmon's citrate (CIT);Christensen's urea (URE); nitrate broth with Dur-ham's tube; TSB (xylene extraction for indole); and asemisolid MOT agar plate (M. Bennett, H. Nadler,and H. George, Am. J. Med. Technol., in press). AKovacs OXI test (tetramethyl reagent) and a Kirby-Bauer antibiogram (2) were also performed. All screwcaps of tubes were loosened, except tubes with oiloverlay, and were incubated with plates at 30°C. MOTagars, brain heart infusion cultures for flagella stain-ing, and other subcultures held for pigment productionwere incubated at room temperature. All conventionalmedia and corresponding reagents were obtained froma commercial source (GIBCO). Of all media utilized inthe evaluation 5% were tested for sterility at 30°C for48 h, and samples from each lot of medium were testedfor quality of performance by using reference strainsof Pseudomonas species, A. calcoaceticus var. Iwoffi,and A. anitratum.

All inoculated biochemicals were incubated and ex-amined for 4 consecutive days. Organisms were iden-tified according to standard criteria (9, 21; Identifica-tion of Glucose Non-Fermenting Gram-NegativeRods, American Society for Microbiology, 1975). If

adequate criteria for identification were not providedby data from the initial biochemical battery, supple-mental tests were performed. These tests were selectedfrom the following: flagella stain (Chroma-GesellschaftSchmid and Co., West Germany) (Identification ofGlucose Non-Fermenting Gram-Negative Rods,1975); Gram stain (Hucker method); catalase; Mac-Conkey; eosin-methylene blue; Salmonella shigella;sTSA; Seller's differential agar; cetrimide agar; deox-yribonuclease agar; o-nitrophenyl-,8-D-galactopyrano-side test; Mueller-Hinton agar (amylase test); phen-ylalanine deaminase agar; nutrient gelatin; nutrientbroth; TSB (42°C growth, 6.5% NaCl tolerance); andphenol red broth with carbohydrate disks (Difco).

If inconclusive biochemical profiles were obtained,OXI, indole (IND), or nitrate tests were repeated,using different substrates (1% aqueous tetramethylparaphenylenediamine dihydrochloride [Sigma Chem-ical Co., St. Louis, Mo.] and IND-nitrate broth).

Substrate reproducibility. To determine sub-strate variability of the O/F system, 11 coded replicatecultures of each of 6 reference strains (P. aeruginosa-pyocyanogenic, Aeromonas hydrophila, Flavobacter-ium meningosepticum, Bordetella bronchiseptica, P.putrefaciens, and Alcaligenes denitrificans) were in-serted randomly into the study.

RESULTSIdentification accuracy. Accurate identifi-

cations were obtained with 69 of 82 (84%) non-fermentative bacilli and 10 of 13 (77%) OXI-positive, fermentative bacilli for an overall ac-curacy of 83%. One misidentified strain of Eike-nella corrodens was excluded from calculationof the O/F system's identification accuracy sincethe computer coding manual did not incorporatethis organism into its data base. Similar identi-fication accuracies were obtained with clinicalisolates (88% accuracy) and reference strains(75% accuracy).

All strains of P. cepacia, P. putrefaciens, Aci-netobacter, Flavobacterium, Achromobacterxylosoxidans, Vibrio, and Chromobacteriumviolaceum were correctly identified.The 16 misidentified bacteria and the corre-

sponding discrepant test reactions responsiblefor their misidentifications are listed in Table 1.Of the 16 misidentifications by the O/F system,only 5 resulted from inaccurate (false-negative)reactions within the O/F tube. The remaining11 resulted from inaccurate MOT tests, flagellastain misinterpretations, coding system inade-quacies, or from discrepancies in other test pro-cedures that were supplemental to the O/F tube.Substrate agreement. The overall agree-

ment of all O/F tube substrates plus the supple-mental MOT tests with conventional substrateswas 92% for nonfermentative and 93% for OXI-producing, fermentative gram-negative bacilli.The percent agreement of individual O/F sub-strates and the composite O/F system with con-

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182 NADLER, GEORGE, AND BARR

TABLE 1. O/F system identificationsNo. of

Organism straint Misidentification Discrepant reaction(s)fied/total

P. aeruginosa (pigmented) 0/2P. aeruginosa (nonpigmented) 1/8 Achromobacter spp. No code for ADH (-) strainsP. fluorescens, P. putida 2/11 P. putida, P. fluorescens Gelatin liquefactionP. maltophilia 1/10 Alcaligenes spp. OXI, weak (+)P. cepacia 0/3P. stutzeri (mendocina variety) 2/6 Achromobacter spp. XYL (-), inadequate code

P. vesicularis ADH and N2 (-)P. putrefaciens 0/1Nonsaccharolytic Pseudomonas 1/5 Alcaligenes spp. Flagella stain

Spp.A. anitratum 0/5A. Iwoffi 0/2Flavobacterium spp. 0/7Moraxella spp. 1/7 Alcaligenes spp. MOT (+)Alcaligenes spp. 2/5 Nonsaccharolytic Pseu- Flagella stain

domonas spp.Moraxella-like, CDC 4f MOT (-)

B. bronchiseptica 2/4 Alcaligenes spp. URE (-)CDC IVe No growth (nutrient broth)

A. xylosoxidans 0/3CDC Ve, CDC Va 0/2CDC IIj 1/1 CDC IVe IND (-), inadequate codeE. corrodens 1/1 P. haemolytica No codeP. multocida 0/3P. haemolytica 1/1 P. ureae No growth (MacConkey)P. ureae 1/1 P. haemolytica URE and OXI (-), inadequate

codeA. hydrophilia 0/4Plesiomonas shigelloides 1/1 V. parahaemolyticus Lysine (-)Vibrio cholerae, C. violaceum, V. 0/3parahaemolyticus

ventional biochemical reactions for nonferment-ative and OXI-positive, fermentative bacilli isillustrated in Table 2. Six of the 10 tests-anaer-obic glucose (AN-GLU), nitrogen gas (N2), hy-drogen sulfide (H2S), IND, aerobic xylose(XYL), and aerobic glucose (GLU)-showed-95% agreement between O/F and conventionalbiochemical reactions.

Less than 90% correlation was observed withURE (82%), arginine dihydrolase (ADH; 85%),and MOT tests (86%) (Table 3). Fifteen of 17URE test disagreements were due to false-neg-ative URE reactions in the O/F tube, especiallywith Pseudomonas spp. The majority of ADHtest discrepancies were attributable to false pos-itives in the conventional system. Ten of 13MOT test disagreements resulted from inaccur-ate MOT tests using the manufacturer's brothprocedure. In addition, agreement of the alkalin-ity reaction in O/F and conventional GLU or

XYL substrates occurred in only 38 of 70 (54%)instances.Substrate and identification reproduci-

bility. O/F substrate reproducibility was eval-

TABLE 2. Overall agreement between O/F andconventional substrates

O/F substrate Agreement'

AN-GLU ......... 81/83 (98)ADH ............. 79/93 (85)N2. 89/94 (95)H2S .................. ..... 93/93 (100)IND ......................... 87/90 (97)XYLb ................. ........ 87/92 (95)GLUb ........................ 88/93 (95)URE ............... ........ 75/92 (82)CIT ............. .... .... 84/94 (89)MOT .............. 80/93 (86)

aValues indicate number in agreement per totaltested; those in parentheses indicate percent. Averageagreement was 92%.

b Observation of alkalinization in carbohydrate com-partments was 38/70 (54%).

uated by replicate testing of six reference strains(Table 4). Fewer replicate tests were performedwith P. putrefaciens because of overgrowth ofseveral tests by another organism. Overall repro-ducibility for all positive O/F test responses and

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ACCURACY AND REPRODUCIBILITY OF O/F 183

TABLE 3. Organisms with less than 90% agreementbetween O/F and conventional substrates

Substrate Organism Amgeet

URE P. aeruginosa 6/9P. fluorescens, P. pu- 5/10

tidaP. cepacia 0/3P. stutzeri 5/6Moraxella Spp. 6/7Bordetella 3/4CDC Ve, Va, IIj 2/3Pasteurella Spp. 4/5

ADH P. fluorescens, P. pu- 7/10tida

P. stutzeri 5/6Nonsaccharolytic 2/4Pseudomonas spp.

Acinetobacter Spp. 5/6Flavobacterium Spp. 3/7Bordetella 3/4Vibrio Spp. 1/2

MOT (flagellated P. stutzeri 2/6organisms)

Nonsaccharolytic 4/5Pseudomonas spp.

Alcaligenes Spp. 4/5MOT (nonflagel- Acinetobacter Spp. 5/6

lated orga-nisms)

Flavobacterium Spp. 5/7Moraxella Spp. 5/7Pasteurella Spp. 4/5

a Values indicate number of strains in agreementper total number tested.

MOT tests was 84%, and overall reproducibilityfor all negative test reactions was 99.6%.

Substrate reproducibility for positive test re-actions ranged from 86 to 100% for ADH, H2S,IND, XYL, CIT, and MOT tests. Substrate re-producibilities of less than 86% were detectedwith N2, URE, AN-GLU, GLU, and ALK (al-kalinization of carbohydrates) reactions. Alka-linization of GLU and XYL compartments inthe O/F tube was a variable observation withonly 65% overall reproducibility for all organismstested. The reproducibility for a negative testreaction was -90% for all O/F substrates andMOT tests.The review of individual organism perform-

ance with the variable O/F substrates is shownin Table 5. Production of nitrogen gas was de-tectable in only 4 of 11 (36%) tests perfonnedwith A. denitrificans. Hydrolysis of the O/FURE was a variable demonstration with theweak urease-producing organisms; only 5 of 11(45%) tests with A. denitrificans and 1 of 11(9%) tests with P. aeruginosa were positive. TheO/F AN-GLU and GLU reactions were variablewith F. meningosepticum, a positive AN-GLU

TABLE 4. Overall O/F reproducibilityO/F substrate Agreement'

Positive reactionAN-GLU 14/21 (67)ADH 22/22 (100)N2 15/22 (68)H2S 6/7 (86)IND 21/21 (100)XYLb 11/11 (100)GLUb 26/32 (81)URE 16/33 (48)CIT 42/44 (95)MOT 48/51 (94)

Negative reactionAN-GLU, ADH, N2, H2S, IND, 336/336 (100)XYL, GLU, URE, and CIT

MOT 9/10 (90)

a Values indicate number in agreement per totaltested, and those in parentheses indicate percent. Av-erage reproducibility was 84 and 99.6% for positive andnegative reactions, respectively.

b Observation of alkalinization in carbohydrate com-partments was 51/79 (65%).

TABLE 5. Problems ofO/F substratereproducibility'

O/F substrate Organisim AgreementbURE P. aeruginosa 1/11

A. denitrificans 5/11B. bronchiseptica 10/11

N2 A. denitrificans 4/11AN-GLU F. meningosepticum 3/10GLU F. meningosepticum 4/10aSubstrates with <86% agreement in replicate teats.b Values indicate number of replicates in agreement

per total tested.

reaction was demonstrated in only 3 of 10 tests(30%), and a positive GLU reaction was observedin only 4 of 10 tests (40%).

Variability in the O/F URE test of one B.bronchiseptica and the O/F H2S test of one P.putrefaciens was responsible for their misiden-tification as Alcaligenes species. All other or-ganisms were identified correctly in the 63 rep-licate inoculations.Time required for O/F system identifi-

cations. A comparison of the time required forO/F and conventional system identifications re-vealed that 47% of the organisms were morerapidly identified by the O/F system with amean identification time of 2.6 days, in contrastto 3.3 days for the conventional system. Forty-five other organisms required the same identifi-cation time using both methods, and 42% of theorganisms required an average of 1.7 supplemen-tal tests to provide a final O/F identification.Ease in interpretation of substrates. The

O/F system. was convenient to use, and the

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184 NADLER, GEORGE, AND BARR

reaction end points were easily discernible byusing manufacturer's color charts in 89% (136of 153) of all O/F tubes inoculated for eitherorganism identification or reproducibility test-ing. Ambiguous end points in several compart-ments (GLU, XYL, and CIT) and URE colorchanges not described by the manufacturer didnot influence final identifications.

DISCUSSIONAs of this report, we are aware of six earlier

evaluations of the O/F system. Isenberg et al.(7), using the first edition of the computer man-ual, compared O/F and conventional substratereactions and identification accuracy for 21 spe-cies of commonly encountered, nonfermentativerods and Aeromonas, and reported an identifi-cation accuracy of 97%. Nord et al. (14), alsousing the first edition of the computer manual,conducted a similar evaluation with coded or-

ganisms of three bacterial genera (Pseudo-monas, Aeromonas, and Alcaligenes) and re-

ported an identification accuracy of 92%. Ober-hofer et al. (15), employing the second edition ofthe computer manual, evaluated the system inits identification of 33 species of common andoccasionally encountered, nonfermentative, andOXI-producing fermentative rods and found an

overall identification accuracy of 89%. Unlikepreviously described studies, that of Dowda (4)compared the identification accuracy of the O/Fsystem with the API system, rather than withconventional methods. This comparison used 17species of common nonfermentative and OXI-positive fermentative rods and yielded a 94%identification accuracy. The most recent inves-tigators, Shayegani et al. (18), evaluated 48 spe-cies of common and of less frequently encoun-tered nonfermentative bacilli with the O/F andconventional systems and indicated that 27.2%were misidentified and 16.7% were unidentified.

Unlike these earlier reports, this compositestudy was designed to combine a number ofcriteria 'which previously had been evaluatedindividually. We compared O/F and conven-tional systems by using a double-blind method-ology, identified a comprehensive and selectivedistribution of reference and clinical strains, anddetermined O/F system reproducibility, identi-fication time, and supplemental test require-ments, using the second edition of the computermanual.

In this evaluation, the O/F system, includingsupplemental tests, identified accurately 83% ofall bacteria tested. This finding falls within therange of accuracy (70 to 82%) estimated by La-page et al. for conventional and computer-as-sisted identification methods for nonfermenta-

tive bacilli (10). If the supplemental test reac-tions recommended by the computer manualhad provided appropriate test responses, the O/F system's identification accuracy would haveincreased to 95% (90 of 95).O/F substrate agreements with conventional

biochemicals of .-95% for AN-GLU, N2, H2S,IND, XYL, GLU, and 89% for CIT are consistentwith other studies (8, 14, 15). Our findings of an18% disagreement for URE and 15% disagree-ment for O/F MOT suggest that the manufac-turer should consider improving these specifictests.The less reproducible reactions noted in the

O/F tube were N2, URE, AN-GLU, and GLU.Our observation of marked variability of therapid N2 test with A. denitrificans is in agree-ment with the work of Isenberg et al. (7), andsubstitution of the N2 test by a more reliableprocedure may be advisable. Our finding thaturease production by Alcaligenes demonstratedonly 48% reproducibility is in agreement withother reports (7, 8, 14) of variability and inac-curacy of the URE reaction. In contrast to ear-lier reports (7, 8), our evaluation did not detectproblems with the CIT reaction. We did experi-ence problems with the AN-GLU and GLU re-actions of Flavobacterium spp., as did otherworkers (15).An increase in overall substrate reproducibil-

ity from 84 to 91% would be achieved by im-proving the URE and N2 substrates in the O/Ftest battery. The utility of the ALK test inorganism identification should be reevaluated,since we observed an agreement of only 54%between O/F and conventional GLU and XYLalkalinization and an average reproducibility ofonly 65% for the O/F ALK test.As a result of the problems experienced while

performing this study, we recommend the fol-lowing changes to improve overall accuracy ofO/F system identification. Improvement of thesupplemental test battery would be accom-plished by providing detailed instructions foreach supplemental test, e.g., inoculation of in-hibitory media or determination of growth at42°C, to ensure standardized results. Semisolidagar plates may provide a more reliable MOTtest than the manufacturer's recommendedbroth procedure. Replacement of gelatin lique-faction by a lecithinase test could provide a lessvariable and more rapid result. Increased use ofthe o-nitrophenyl-/8-D-galactopyranoside andthe 6.5% NaCl tolerance tests could improve thesystem's overall performance. Incorporation ofamylase (11), deoxyribonuclease and fluorescein(Flo and Tech agars; BBL) tests into the supple-mental test battery could provide additional

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ACCURACY AND REPRODUCIBILITY OF O/F 185

identification guidelines. Uniqueness of flagellamorphology could be used to differentiate non-

saccharolytic pseudomonads, e.g., P. diminutaand P. acidovorans.To facilitate more rapid identification, we rec-

ommend the simultaneous inoculation of the O/F tube and semisolid MOT plate and perform-ance of a Kirby-Bauer antibiogram incorporat-ing the special drugs indicated in the O/F codingmanual.

Suggested revisions of the computer manualinclude addition of organisms and correspondingdifferentiation criteria to six coding manualnumbers: 0000 Pasteurella spp. (OXI negative);0001 E. corrodens and P. putrefaciens (H2Snegative); 0021 CDC IIj (IND negative); 1071 P.stutzeri; 1171 P. aeruginosa (ADH negative);and 4271 V. parahaemolyticus. The computermanual should also print each test result of thebiochemical profile next to the code number tominimize misidentifications arising from clericalerrors in computing the four-digit code.The code numbers of three organisms were

not incorporated into the O/F coding manual:1041 P. stutzeri, 0200 P. multocida, and 4271 V.parahaemolyticus.

Overall evaluation and conclusion. Thisdouble-blind evaluation suggests that the O/Fsystem is a convenient, rapid, and acceptablealternative to conventional methods for identi-fication of both commonly and rarely encoun-

tered nonfermentative and OXI-positive fermen-tative, gram-negative bacilli in only 48 to 72 h.Until revisions are made, we suggest each O/Fnumerical identification be evaluated against themanufacturer's bacterial identification chartbased on expected biochemical reactions.

ACKNOWLEDGMENTS

We wish to thank Colleen Dolan, Maureen Gorham, Mer-edith Bennett, George Szabo, and Diane Unger for theirtechnical assistance; Donna Devinney for her technical assist-ance and continued support; and Robert Weaver (Center forDisease Control), Silva Tekeian (Massachusetts Departmentof Public Health), Steve Weinstein (Faulkner Hospital-for-merly with Boston Veterans Administration Hospital), Ther-esa Emery (Boston Lying-In Hospital), Janet Verna (Fra-mingham Union Hospital), Donna Dembrowski (Carney Hos-pital), and Evelyn Mello (Leonard Morse Hospital) for theircontribution of test organisms.

LITERATURE CITED

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2. Bauer, A. W., W. M. Kirby, J. C. Shems, and M.Turek. 1966. Antibiotic susceptibility testing by astandardized single disk method. Am. J. Clin. Pathol.45:493-496.

3. Cabrera, H. A., and M. A. Drake. 1975. An epidemic ina coronary care unit caused by Pseudomonas species.Am. J. Clin. Pathol. 64:700-704.

4. Dowda, H. 1977. Evaluation of two rapid methods foridentification of commonly encountered nonfermentingor oxidase-positive, gram-negative rods. J. Clin. Micro-biol. 6:605-609.

5. Gilardi, G. L. 1972. Infrequently encountered Pseudo-monas species causing infection in humans. Ann. Intern.Med. 77:211-215.

6. Henriksen, S. 1973. Moraxella, Acinetobacter, and theMimeae. Bacteriol. Rev. 37:522-561.

7. Isenberg, H. D., and J. Sampson-Scherer. 1977. Clin-ical laboratory evaluation of a system approach to therecognition of nonfermentative or oxidase-producinggram-negative, rod-shaped bacteria. J. Clin. Microbiol.5:336-340.

8. Kilbourn, J. P. 1977. Oxi-Ferm system: letter to theeditor. Am. J. Med. Technol. 43:1167-1168.

9. King, E. 0. 1972. The identification of unusual pathogenicgram negative bacteria. United States Department ofHealth, Education, and Welfare, Public Health Service,Center for Disease Control, Atlanta, Ga.

10. Lapage, S. P., S. Bascomb, W. R. Willcox, and M.Curtis. 1973. Identification of bacteria by computer:general aspects and perspective. J. Gen. Microbiol. 77:273-290.

11. Lee, W. S. 1976. Use of Mueller-Hinton agar as amylasetesting medium. J. Clin. Microbiol. 4:312.

12. Louria, D. B. 1974. Superinfection: a partial overview, p.1-18. In J. E. Prier and H. Friedman (ed.), Opportun-istic pathogens. University Park Press, Baltimore.

13. Nagel, J. G., and L. J. Kunz. 1972. Simplified storageand retrieval of stock cultures. Appl. Microbiol. 23:837-838.

14. Nord, C. E., B. Wretlind, and A. Dahlback. 1977.Evaluation of two test-kits-API and Oxi/Ferm tube-for identification of oxidative-fermentative gram-nega-tive rods. Med. Microbiol. Immunol. 163:93-97.

15. Oberhofer, T. R., J. W. Rowen, G. F. Cunningham,and J. W. Higbee. 1977. Evaluation of the Oxi/Fermtube system with selected gram-negative bacteria. J.Clin. Microbiol. 6:559-566.

16. Rosenthal, S. L. 1974. Sources of pseudomonas and aci-netobacter species found in human culture materials.Am. J. Clin. Pathol. 62:807-811.

17. Sanford, J. P. 1973. Approach to the evaluation andprevention of aerobic gram-negative bacillary nosocom-ial infections, p. 43-55. In Seminar on gram-negativeinfections. Schering Corp., Kenilworth, N.J.

18. Shayegani, M., A. M. Lee, and D. M. McGlynn. 1978.Evaluation of the Oxi/Ferm tube system for identifica-tion of nonfermentative gram-negative bacilli. J. Clin.Microbiol. 7:533-538.

19. United States Public Health Service. 1974. NosocomialPseudomonas cepacia bacteremia caused by contami-nated pressure transducers. Morbid. Mortal. WeeklyRep. 23:423.

20. von Graevenitz, A., and J. Weinstein. 1971. Pathogenicsignificance of Pseudomonas fluorescens and Pseudo-monas putida. Yale J. Biol. Med. 44:265-273.

21. Weaver, R. 1974. Gram-negative organisms: an approachto identification. Center for Disease Control, Atlanta,Ga.

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