JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 1978, p. 313-3190095-1137/78/0008-0313$02.00/0Copyright © 1978 American Society for Microbiology

Vol. 8, No. 3

Printed in U.S.A.

Carbon Substrate Utilization Studies of Some Cultures ofAlcaligenes denitrificans, Alcaligenes faecalis, and

Alcaligenes odorans Isolated from Clinical SpecimensH. R. RARICK, P. S. RILEY,`* AND R. MARTIN'

Department ofParasitology and Laboratory Practice, University ofNorth Carolina, Chapel Hill, NorthCarolina 27514,1 and Center for Disease Control, Atlanta, Georgia 303332

Received for publication 19 June 1978

One hundred and sixty-two cultures of Alcaligenes species (A. denitrificans,A. faecalis, and A. odorans) of clinical origin were characterized by routinediagnostic and carbon substrate utilization techniques. The microorganisms weretested for their ability to utilize a total of 188 substrates. Substrate utilization wasassayed by (i) growth stimulation and (üi) substrate alkalinization. The A. deni-trificans and A. odorans cultures had unique substrate utilization profiles foreach species. The A. faecalis isolates were redefined by colonial morphology intotwo biotypes: (i) biotype I, morphologically and biochemically similar to the A.denitrificans cultures and (ii) biotype II, morphologically similar to the A.odorans cultures.

Recognition and identification of nonfermen-tative, gram-negative bacterial rods are promi-nent problems in the clinical microbiology lab-oratory, primarily because very few positive re-actions are obtained when these microorganismsare examined by conventional diagnostic proce-dures. In particular, some of these bacteria donot oxidatively acidify carbohydrate substrates,and thus the phenotypic markers which form anintegral part of dichotomous keys used for theidentification of these bacteria are ineffective.

Nutritional characterization, by the use of car-bon substrate utilization (CSU) tests, has beenused in a number of laboratories as a character-ization tool. Stanier et al. (15), Ralston et al (12),Baumann et al. (1, 2), Otto and Pickett (11), andGilardi (5, 6) have used this technique to char-acterize bacterial species. Although useful infor-mation has been obtained with this procedure,it is not easily adapted for use in the clinicaldiagnostic laboratory. Problems with inoculumstandardization, consumption of large amountsof media, and growth end point evaluation makethis procedure somewhat suspect. Riley et al.(13) were able to control some of these factorsby incorporating the Steers replicator (16) intotheir CSU experiments. With this replicator,they found that a relatively large number ofcultures could be processed accurately and rap-idly.

Recently, Oberhofer and Rowen (9) and Ottoand Pickett (11) devised new media for the studyof CSU profiles. In both instances, the alkalini-zation of the growth medium was used as a

reaction end point. This alkalinization resultedin a color change which is much simpler tointerpret than relative differences in colonialgrowth.This report concerns the determination of

CSU profiles for the gram-negative, glucose-non-oxidative microorganisms Alcaligenes denitrifi-cans, A. odorans, and A. faecalis. The modifiedStanier technique (13) and the media of Ober-hofer and Rowen (9) and Otto and Pickett (11)were investigated.

MATERLALS AND METHODSBacteria. The bacterial cultures were obtained

from the stock culture collection maintained by theSpecial Bacteriology Section at the Center for DiseaseControl. They were identified from clinical isolatesthat were submitted to this laboratory for furtherinvestigation (Table 1).Growth stimulation studies. The technique of

Stanier et al. (15), as previously described (13), wasused. The bacterial cultures were grown on tryptone-glucose-yeast extract agar slants (7) for 18 to 24 h. Thecell mass was washed off the slants with sterile basalsalts solution. After the cellular growth was suspended,the optical density of the suspensions was adjusted to0.35 to 0.40 at a wavelength of 550 um (4.8 to 5.2 ,ug ofdry weight per ml). These suspensions were thendiluted 1/10 in sterile basal salts solution before beingused to fMi the replicator seed plate. After drying, thesubstrate plates were inoculated in duplicate with thereplicator. The inoculum on each plate's surface wasallowed to dry before the culture was incubated at35°C for 48 h.The final concentrations of the substrates were 0.2%

for carbohydrates and 0.1% for all other substrates.313

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314 RARICK, RILEY, AND MARTIN

TABLE 1. Sources of isolation of the Alcaligenescultures

A. deni- A. fae- A. odor-Source trificans calis (63 ans (60

strains) strains) strains)

Abscesses, ulcers i 3Blood 4 7Cerebrospinal fluid 3 1 1Ear 1 13 9Environment 4 3Eye 3 1Feces 1 2 1Finger 2Mastoid 2Mouthwash 5Peritoneal fluid 3Pleural fluid 2Sputum 3 6 1Urine 3 5 24Wounds 4 7Miscellaneous 9 7 6Unknown 4 6 5

The substrates used were acetamide, acetate, cis-acon-itate, trans-aconitate, adipate, adonitol,"-alanine, D-alanine, L-alanine, allantoin, m-aminobenzoate, p-ami-nobenzoate, -y-aminobutyrate, DL-2-aminobutyrate, 2-aminoethanol, ô-aminovalerate, a-amylamine, andine,anthranilate, D-arabinose, L-arabinose, arginine, as-

paragine, aspartate, azelate, benzoate, benzylamine,benzylformate, betaine, borate, 1,4-butanediol, bu-tanol, butylamine, butyramide, butyrate, caprate, cap-roate, caprylate, catechol, cellobiose, cetrimide, cit-rate, DL-citrulline, creatine, creatinine, cysteine, cys-tine, dextrin, 1,3-dihydroxy-2-propanone, docosanoate,dulcitol, erythritol, ethanol, DL-ethionine, ethyleneglycol, ethylene glycol monomethyl ether, folate, for-mate, fructose, fucose, galactose, gentisate, glucosa-mine, gluconate, glucose, glutamate, glutamine, glutar-ate, DL-glycerate, glycerol, glycine, glycolate, glyoxyl-ate, heptanoate, 1,6-hexanediol, hippurate, histamine,histidine, m-hydroxybenzoate,p-hydroxybenzoate, DL-

/3-hydroxybutyrate,"/-hydroxy-,6-methylglutarate, hy-droxyproline, indoleacetate, inositol, inulin, isobu-tanol, isobutyrate, isoleucine, isophthalate, isopropa-nol, isovalerate, isovaline, itaconate, 2-ketogluconate,a-ketoglutarate, kynurenate, DL-lactate, lactose, lae-vulinate, laurate, DL-leucine, linoleate, linolenate, ly-sine, D-malate, L-malate, maleate, malonamide, malo-nate, maltose, mandelate, mannitol, mannose, melezi-tose, melibiose, mesaconate, methanol, methionine,methylamine, mucate, myristate, naphthalene, nico-tinamide, nicotinate, DL-norleucine, DL-norvaline, oc-tadecanoate, DL-ornithine, oxalate, pantothenate, pe-largonate, pentadecanoate, pentadecanol, phenol,phenylacetate, phenylalanine, phthalate, pimelate, po-tassium hydrogen phthalate, proline, propanol, pro-pionamide, propionate, propylene glycol, protocate-chuate, putrescine, pyruvate, quinate, raffinose, ribose,L-rhamnose, salicin, salicylate, sarcosine, sebacate, ser-

ine, sodium thiocynate, sorbitol, sorbose, spermine,suberate, succinamide, succinate, sucrose, tartarate,m-tartarate, terephthalate, testosterone, trehalose,

threonine, tributyrin, tryptamine, D-tryptophan, DL-trytophan, Tween 20, Tween 40, Tween 60, Tween 80,tyrosine, undecane, undecanoate, urea, uric acid, val-eramide, valerate, DL-valine, and xylose.

Substrate alkalinization. The alkalinization tech-nique was performed by the procedure of Oberhoferand Rowen (9). The substrates were added to thebasal medium, Simmons citrate base (Difco Labora-tories), to a final concentration of 0.5% (the finalconcentration of mucate was 0.1%). The filter-steri-lized substrates were aseptically added to the sterilemelted agar base (mucate and saccharate were steri-lized by autoclaving at 15 pounds [ca. 680.228 g] and121°C for 15 min). The completed media (pH 6.8)were dispensed in 3-ml aliquots into screw-cappedtubes (13 by 100 mm) and allowed to cool on slantboards. The slants were inoculated with one drop ofan 18- to 24-h heart infusion broth (Difco) culture andincubated at 35°C. The incubated slants were exam-ined at 1, 2, and 7 days. A positive result (alkalinizationof the substrate) was recorded when the color of themedium changed to blue.The Otto and Pickett medium was prepared and

tested as described (11). Subsequently, some modifi-cations were made: (i) the substrates were filter-steri-lized; (ii) the agar content was raised to 1.5% to facii-tate slant preparation; and (iii) the medium was dis-pensed in 3-ml aliquots into screw-capped tubes (13by 100 mm).

Diagnostic studies. The biochemical tests werethose routinely used in the Special Bacteriology Sec-tion for the characterization of microorganisms sub-mitted for identification. The methods for preparingthe media and performing the tests have been previ-ously described (4, 8, 14). Ail agar slant culture mediawere inoculated with one drop of an 18- to 24-h heartinfusion broth culture. Flagella stains were performedby using Clark's modification (3) of the Leifson tech-nique.

RESULTS

Some of the biochemical characteristics deter-mined for each of the bacterial cultures are listedin Table 2. Each of the peritrichously flagellatedcultures was oxidase positive and grew onMacConkey agar. None of the carbohydrate ox-idative-fermentative media was acidified by anyof the Alcaligenes species. Denitrification wasobserved in all of the A. denitrificans culturesand 43% of the A. faecalis cultures. However,only the A. denitrificans bacteria were able toreduce nitrate beyond the oxidation level ofnitrite. Nitrite, but not nitrate, was reduced byeach of the A. odorans cultures. Most of theAlcaligenes organisms displayed no proteolyticactivity and demonstrated motility in semisolidagar tubes.One hundred and eighty-eight substrates were

examined for growth stimulation of the Alcali-genes microorganisms (Table 3). Fourteen ofthese substrates (acetate, D-alanine, L-alanine,aspartate, citrate, glutamate, histidine, DL-lac-

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CARBON SUBSTRATE UTILIZATION IN ALCALIGENES 315

TABLE 2. Some characteristics of the AlcaligenesspeciesA. deni- A. odor- A. fae-

Test or substratea tca ans (60 calis (63s ns) strains) strains)stramns)

Acidification of:Glucose OF ob o 0Other sugars O O O

Growth on:MacConkey agar 100 100 100SS agar 65 100 79TGY at 25°C 100 100 100TGY at 350C 100 100 100TGY at 420C 52 20 75

Oxidase 100 100 100Catalase 100 98 100Citrate alkalinization 90 100 95Urea hydrolysis 15 3 6Nitrite reduction 100 100 0Nitrate reduction 100 0 43Gas from nitrate 100 0 0Motility 100 97 97Gelatin hydrolysis 0 20 0Litmus milk peptoniza- 0 0 0

tionChromogenesis 0 0 0Lysine decarboxylase 0 0 0Ornithine decarboxyl- 0 0 0

aseArginine dehydrolase 0 0 0Esculin hydrolysis 0 0 0

a OF, Oxidative-fermentative; SS, salmonella-shi-gella; TGY, tryptone-glucose-yeast extract.

b Percentage of cultures producing positive reaction.C Xylose, mannitol, lactose, sucrose, maltose, glyc-

erol, salicin, L-arabinose, adonitol, dulcitol, galactose,fructose, mannose, rhamnose, trehalose, raffinose, sor-bitol, inositol, cellobiose, inulin, dextrin, glycogen,erythritol, melibiose, melezitose, and starch.

tate, laurate, L-malate, phenylalanine, proline,pyruvate, and tyrosine) stimulated the growthof 90% of all of the Alcaligenes cultures tested.They consisted primarily of amino acids andshort-chain fatty acids. Ninety-six of the sub-strates did not enhance bacterial growth. More-over, seven of the substrates (p-aminobenzoate,catechol, cetrimide, indoleacetate, isophthalate,suberate, and terephthalate) either partially orcompletely inhibited cellular growth. When fourof the substrates (p-phenyl phenol, palmitate,stearate, and tridecanoate) were incorporatedinto the basal medium, the plates were tooopaque to provide accurate growth determina-tions. The remaining 78 substrates producedvarious reactions among the Alcaligenes species.

Utilization results for 21 substrates provideda potential basis for differentiation among theAlcaligenes species (Table 4). More than 95% ofthe A. odorans cultures utilized butyramide,

glycolate, methionine, phenol, DL-ethionine, DL-leucine, and phenylacetate, but no more than33% of the other bacteria utilized these sub-strates. At least 80% of the A. denitrificans andA. faecalis cultures but less than 13% of the A.odorans cultures utilized -y-aminobutyrate and,B-alanine. Moreover, the A. denitrificans cul-tures utilized glutamate (100%), gluconate (80%),DL-citrulline (80%), and isoleucine (78%), butless than 33% of the A. faecalis organisms wereable to metabolize these substrates. The A. fae-calis organisms were characterized by stimula-tion levels below 90%.The substrates acetamide, 8-alanine, malo-

nate, nicotinamide, propionate, saccharate, andtartrate were used to determine which of thesubstrate alkalinization methods was most use-ful for our purposes. Each of the procedures wastested in screw-capped tubes (13 by 100 mm)because of the false positive reactions observedwhen compartmentalized petri dishes were used.These results were due to cross-contaminationof adjacent substrates by the ammonia releasedby the metabolizing microorganisms.The carbon substrate alkalinization profiles of

20 cultures each of A. denitrificans, A. faecalis,and A. odorans were determined (Table 5). Thebacterial cultures alkalinized each of the sub-strates tested. Only nicotinamide (A. denitrifi-cans), saccharate (A. odorans), and tartrate (A.odorans) were not utilized by cultures of eachspecies. Little difference was observed whenreactivity patterns of the bacteria on the twotypes of substrate media were compared. Incu-bating the slants of the Otto and Pickett (11)media for longer intervals resulted in a reactionreversion in some of the saccharate tubes inoc-ulated with A. denitrificans and A. faecaliscultures. After 6 days of incubation, the per-centage of positive reactions dropped from 90 to50 (A. denitrificans) and from 60 to 10 (A.faecalis). No reaction reversions were noted inthe Oberhofer and Rowen (9) media.The Oberhofer and Rowen medium was used

to determine substrate aLkalinization patterns of162 Alcaligenes cultures (Table 6). Twenty-onesubstrates which appeared to be capable of es-tablishing unique CSU profiles for each specieswere tested. The A. denitrificans cultures gavepositive reactions (280%) on nine substrates (bu-tyramide, formate, malate, mesaconate, m-tar-tarate, propionamide, saccharate, serine, andmucate), negative reactions on one (nicotina-mide), and variable reactions on the remainingsubstrates. Reactions of A. odorans cultureswere positive on 11 substrates (acetamide, bu-tyramide, butyrate, formamide, formate, malon-amide, malate, malonate, nicotinamide, propion-amide, and propionate), negative on 9, and var-

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J. CLIN. MICROBIOL.316 RARICK, RILEY, AND MARTIN

TABLE 3. Substrate utilization by the Alcaligenes species (modified Stanier technique)A. deni- A fae- A. odor- A. deni- A. fae- A. odor-

Substratea trificans calis (63 ans (60 Substratea trficans calis (63 ans (60t39 strains) strains) (39 strains) strains)

strains) strains)l~~ ~ ~ ~ ~~0100. n

AcetamideAcetatecis-Aconitatetrans-AconitateAdipate,B-AlanineD-AlanineL-Alaniney-AminobutyrateDL-2-AminobutyrateAnthranilateAsparagineAspartate1,4-ButanediolButanolButyramideCaprateCaproateCaprylateCitrateDL-CitrullineCysteineEthanolDL-EthionineFormateGentisateGlutamateGlutamineDL-GlycerateGlycolateGlyoxylateGluconateGlutarateGlycineHeptanoate1,6-HexanediolHippurateHistidinep-Hydroxybenzoatef3-Hydroxy-/3-methyl-

glutarateDL-/3-HydroxybutyrateIsobutyrateIsoleucineIsovalerateIsovalineItaconate

23b921004497809090821536100100645623363638908097592880369510010033778010049365174925497

1003179416474

639495570879595804141521002122274071679525516Oo5

100846832203232404114361001751

100449

225648

10010067o3

12100100

265938390O7

100O7267100O

93839768O

100100100100

83

9310018o

88971710

832810078933

a-KetoglutarateKynurenateDL-LactateLaevulinateLaurateDL-LeucineLysineD-MalateL-MalateMaleateMalonateMesaconateMethionineMucateNicotinateDL-NorleucineDL-NorvahnePantothenatePelargonatePhenolPhenyl acetatePhenylalaninePimelateProlinePropanolPropionamidePropionateProtocatechuatePyruvateQuinateSerineSuccinateTartaratem-TartarateThreonineTributyrinTryptamineD-TryptophanDL-TryptophanTyrosineUndecanoateUric acidValeramideValerateDL-ValineDL-Ornithine

1002810038100305995100927982239721693346332333100909277368549100

O51O

33927251288585100413669315623

1005210016

100251960100958

48O673259211052333210057100226094631004138O1060il21377179

10041O

84226

21

835297o

10097122

10083903

100317

100937O

10097100O

10068100100

8100

3O

32o2oo83501001005070100632393

a Ninety-six of the substrates that were examined provided no growth enhancement for any of the Alcaligenesspecies.

b Percentage of cultures producing positive reactions after 2 days of incubation.

able on 1 (/3-alanine). The A. faecalis microor- substrates in each of the substrate utilizationganisms were positive on 4 substrates (/3-alanine, media. A. faecalis cultures, however, utilizedbutyrate, malate, and propionate) and gave var- substrates at levels between 40 and 60%. Obser-iable reactions on the other 17 substrates. vation of the colonial morphology of the A.A. denitrificans and A. odorans cultures had faecalis cultures on rabbit blood agar plates

high levels of activity (80 to 100%) on several indicated two distinct types of growth. The mor-

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CARBON SUBSTRATE UTILIZATION IN ALCALIGENES 317

phology ofA. faecalis type I colonies was similarto that of A. denitrificans. The colonies were

small in diameter (cO.5 mm), low convex, glis-tening, and had an entire edge. The morphologyofA. faecalis type II colonies was similar to thatof A. odorans, but did not produce the darkgreen color on rabbit blood agar. The type IIcolonies were larger in diameter (1.5 to 2.0 mm),umbonate with a spreading periphery, and gran-ular. Forty of the A. faecalis cultures were des-ignated as type I, and 23 culture were designatedas type II.

Reevaluation of the substrate utilization datain Tables 4 and 6 provided supporting evidence

for the separation of the A. faecalis cultures intotwo types (Table 7). The A. faecalis type Iorganisms utilized a greater number of sub-strates than did type II, and the activity percentlevels were higher in most cases. The A. faecalistype II organisms were more reactive only onpropionate, fi-alanine, and butyrate.The CSU profiles of the A. faecalis type I and

type II cultures were compared with those of A.denitrificans and A. odorans microorganisms(Table 7). The A. faecalis type I cultures had a

CSU profile quite similar to that of the A. den-itrificans cultures. However, there were notable

TABLE 4. Substratespotentially differential for theAlcaligenes species

Substrate

AdipateButyramidey-AminobutyrateGlycolateD-Malatem-TartarateMethionineMucatePhenolPropionamidePimelateDL-EthionineGlutarateIsoleucineDL-LeucinePhenylacetateDL-NorleucineB-AlanineCysteineDL-CitrullineGluconate

A. deni-trificans

(39strains)

97a

24823395922397233790281007830336980978080

A. fae-calis (63strains)

702780326160o

67336057o

32925325987512533

A. odor-ans (60strains)

4100

2100

22

1004

100100

O

979310097971001293O

3

a Percentage of cultures producing positive reac-tions.

TABLE 6. Substrate alkalinization reactions of theAlcaligenes cultures on Oberhofer and Rowen

mediaA. deni-..deni-A. fae- A. odor-

Substrate tri cans calis (63 ans (64stra40s) strains) strains)stramns)

Acetamide 53a 33 100Adipate 78 54 0,8-Alanine 75 89 50Azelate 65 51 0Butyramide 81 51 100Formamide 58 32 100Formate 100 65 100Malate 100 100 88Malonamide 50 33 88Malonate 55 40 100Mesaconate 83 62 0Mucate 83 49 0Nicotinamide 0 32 100Pimelate 75 54 0Propionamide 83 33 98Propionate 55 87 100Saccharate 83 54 0Serine 100 63 9m-Tartarate 98 56 0Tartrate 68 21 0Butyrate 36 95 100

a Percentage of cultures producing positive reac-tions after 2 days of incubation.

TABLE 5. Comparison of substrate alkalinization by the Alcaligenes species on Oberhofer and Rowen andOtto and Pickett media

A. denitrificansa A. faecalis' A. odorans'Substrate

opb ORb OP OR OP ORAcetamide 40c 55 20 30 100 100fP-Alanine 80 75 90 90 50 50Malonate 60 55 55 40 100 100Nicotinamide 0 0 50 35 100 100Propionate 65 65 90 95 100 100Saccharate 90 90 55 60 0 0Tartrate 50 65 30 30 0 0

aTwenty cultures of each species were tested.b Op, Otto and Pickett media; OR, Oberhofer and Rowen media.C Percentage of cultures producing positive reactions.

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318 RARICK, RILEY, AND MARTIN

TABLE 7. Selected substrate utilization by A.faecalis type I and type IH, A. denitrificans, and A.

odorans cultures

A. dent- A. faecalistrificans A. odor-

Substrate t40 ans (60 Type I Type IIstrains) strains) (40 (23

strains) strains)

Modified StanierAdipate 97a 4 98 13m-Tartarate 92 2 93 4Mucate 97 4 98 13Pimelate 90 0 90 0Propionate 56 100 37 91Malate 100 78 100 100fP-Alanine 75 50 83 96

Substrate alkalini-zation

Adipate 78 0 88 0Butyramide 81 100 83 0Butyrate 36 100 70 100Formamide 58 100 50 0Fornate 100 100 100 4Mesaconate 83 0 98 0m-Tartarate 97 0 88 0Nicotinamide O 100 48 0Propionamide 81 99 53 0Saccharate 81 0 95 0Serine 100 9 100 4Tartrate 67 0 33 0Pimelate 75 0 85 0Mucate 83 0 78 0

aPercentage of cultures producing positive reactions after2 days of incubation.

differences in that the A. denitrificans orga-nisms were more reactive on propionamide andtartrate, and the A. faecalis type I cultures weremore reactive on butyrate and nicotinamide.CSU profiles of the A. faecalis type Il and A.odorans cultures were similar when determinedby the modified Stanier method, but little simi-larity was noted when they were determined bysubstrate alkalinization.

DISCUSSIONThe Alcaligenes species were able to utilize

the substrates in each of the systems tested.However, differentiation of these bacteria by theuse of the CSU profiles was not absolute. Rather,with the modified Stanier technique, the Alca-ligenes species produced identical growth stim-ulation profiles on 61% (14 positives at >90%,and 99 negative reactions) of the substratestested. Short-chain fatty acids, amino acids, andone amide, valeramide, were the compoundsthat provided growth stimulation for each of themicroorganisms. The unutiized substrates var-ied from carbohydrates to long-chain fatty acidsand ring structure-containing compounds.The CSU pattern of the A. odorans cultures

was quite different from those of the other Al-

caligenes species. At least 95% of the A. odoransmicroorganisms utiized butyramide, glycolate,methionine, phenol, DL-ethionine, DL-leucine,and phenylacetate, whereas most of the otherAlcaligenes organisms did not utilize these sub-strates. Cultures of A. faecalis and A. denitrifi-cans produced CSU patterns which were morenearly related to each other than to the A.odorans pattern. However, A. denitrificans cul-tures did utilize glutamate (100%) DL-citrulline(80%), gluconate (80%), and malonate (79%) athigher levels than did the A. faecalis microor-ganisms (s33%).The CSU profiles determined from the sub-

strate alkalinization studies were no more effec-tive in differentiating the Alcaligenes speciesthan those obtained with the modified Staniersystem. The A. denitrificans cultures utilizedmesaconate (83%), m-tartarate (97%), saccharate(81%), and mucate (83%), but none of thesesubstrates was utilized by any of the A. odoranscultures. The A. odorans cultures utilized nico-tinamide (100%; A. denitrificans, 0%), malon-amide (87%), propionamide (99%), and formam-ide (100%), but no more than 33% of the A.faecalis organisms utilized these substrates. TheA. faecalis microorganisms utilized butyrate(95%), but only 36% of the A. denitrificans cul-tures utilized this substrate.The A. faecalis cultures consisted of two dis-

tinct groups as determined by (i) colonial mor-phology, and (ii) CSU profiles. The profile of thegroup I organisms was quite similar to that ofthe A. denitrificans cultures. With the modifiedStanier technique, the group II organisms had aprofile quite similar to that of the A. odoranscultures. However, the results obtained for thegroup II cultures with the substrate alkaliniza-tion technique were unlike those of any of theother organisms used in this study.Although use of CSU profiles did not allow

absolute differentiation of the organisms, thesepatterns did serve to separate the Alcaligenesspecies from phenotypically closely relatedgroups (Table 8). When the substrate alkalini-zation method is used, the Group IVc(2) andGroup IVe cultures can be differentiated fromthe Alcaligenes organisms by testing with thesubstrates butyramide and formate. Cultures ofBordetella bronchiseptica can be separated bytesting for utilization of saccharate and formate.Pseudomonas diminuta and P. alcaligenes cul-tures can be differentiated from the Alcaligenesorganisms by testing with the substrates butyr-amide and D-malate.Reading and recording the CSU results were

much easier with the alkalinization procedurethan with the modified Stanier technique, be-

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CARBON SUBSTRATE UTILIZATION IN ALCALIGENES 319

TABLE 8. Comparison of substrate alkalinization patterns of the Alcaligenes species and other similarorganisms which are isolated from clinical specimens

Substrate alkalinization of:aBacteria

Butyramide Formate Nicotinamide Saccharate Pimelate D-Malate

A. denitrificans + + - + +A odorans + + + - -+(-A. faecalis I + + - + + +A. faecalis II - - - - - +B. bronchiseptica +(-) - - + - +Group IVc(2) - + + + + +Group IVe - +P. diminutaP. alcaligenes - - - _ +

a +, 80 to 100% alkalinization; +(-), 70 to 80% aLkalinization; -, 50% or less aLkalinization.

cause it was simpler to observe a color changethan to compare colonial growth. Moreover, thepreparation, storage, and inoculation of the me-dia were simplified.Both Otto and Pickett (11) and Oberhofer and

Rowen (9) substrate media provided satisfactoryresults in recording substrate utilization. How-ever, the Otto and Pickett medium is more dif-ficult to prepare than the Oberhofer and Rowenmedium, and we have stored the latter for up to4 months at 4°C with no apparent loss in reac-tion sensitivity.The results we obtained with both substrate

alkalinization media agreed with most publishedresults (10, 11). However, observations of culturereactions on tartrate and saccharate were quitedifferent from previously recorded results. Otherinvestigators (11) have reported that these sub-strates provide different results for each of theAlcaligenes species, but experimental observa-tions in our laboratory did not support thesefindings. This variance in results is probably dueto the small number of cultures which the otherinvestigators used in their study.

LITERATURE CITED

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2. Baumann, P., M. Doudoroff, and R. Y. Stanier. 1968.A study of the Morasella group. II. Oxidase-negativespecies (genus Acinetobacter). J. Bacteriol. 9501520-1541.

3. Clark, W. A. 1976. A simplified Leifson flagella stain. J.Clin. Microbiol. 3:632-634.

4. Ewing, W. H., and B. R. Davis. 1970. Media and tests

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