Vol. 16, No. 5JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 1982, p. 909-9190095-1137/82/110909-11$02.00/0Copyright © 1982, American Society for Microbiology

Rapid Method for Simultaneous Detection of the ArginineDihydrolase System and Amino Acid Decarboxylases in

MicroorganismsKIRK C. S. CHEN, 2* NANCY J. CULBERTSON,1 JOAN S. KNAPP,3'4 GEORGE E. KENNY,2 AND

KING K. HOLMES4Biochemistry Laboratory' and Neisseria Reference Laboratory,3 Division of Infectious Diseases, SeattlePublic Health Hospital, Seattle, Washington 98114, and Departments of Pathobiology2 and Medicine,4

University of Washington, Seattle, Washington 98195

Received 8 April 1982/Accepted 3 August 1982

A specific procedure has been developed for the detection of the first twoenzymes involved in the arginine dihydrolase system and the detection of thedecarboxylases of arginine, glutamic acid, histidine, lysine, ornithine, phenylala-nine, tryptophan, and tyrosine. A loopful of growth of each organism fromdihydrolase-decarboxylase induction agar medium (or broth) was washed andincubated separately with 0.2-ml samples of three test media supplemented withdifferent amino acids. Each spent test medium was dansylated, and the dansylderivatives were separated by two-dimensional thin-layer chromatography onpolyamide sheets. The end products (citrulline, ornithine, y-amino-n-butyric acid,and amines) produced during incubation were estimated by comparing thefluorescent intensities of end products from the spent test media and of thecorresponding parent amino acids from test medium controls after thin-layerchromatography. The method is reproducible, requiring incubation of an organismin three test media for 1 h for simultaneous detection of the first two enzymesinvolved in the arginine dihydrolase system and of eight amino acid decarboxyl-ases. This method has been successfully applied to gram-positive and gram-negative microorganisms and also to Mycoplasmatales. It could simplify andimprove the accuracy of the corresponding biochemical tests performed in clinicallaboratories for the identification and differentiation of microorganisms, and itmay prove particularly useful for the differentiation of species of Pseudomonasand Mycoplasma.

Enzymes involved in the arginine dihydrolasesystem and the amino acid decarboxylases arewidely distributed in microorganisms (1, 2, 8).The microbial arginine dihydrolase systemwhich converts arginine to ornithine via citrul-line consists of three enzyme reactions: arginine+ H20 -* citrulline + NH3 by arginine deimin-ase; citrulline + Pi ornithine + carbamylphosphate by catabolic ornithine carbamoyl-transferase; and carbamyl phosphate + ADP ,

ATP + CO2 + NH3 by carbamate kinase (1).Detection of the arginine dihydrolase systemand decarboxylases of lysine and ornithine hasproven valuable for differentiation within thefamily Enterobacteriaceae and is useful forcharacterizing certain genera within other fam-ilies (7). Tests for the arginine dihydrolase sys-tem are of value for differentiating aerobic Pseu-domona.s spp. from other nonfermentative gram-negative bacilli (9, 27) as well as within their owngenus (22, 25, 27, 29). The methods devised byMoller (17) for identifying Enterobacteriaceae

by detection of the dihydrolase and decarboxyl-ase systems still serve as the standard methods(7, 12, 16, 20, 23). Procedures for the detectionof the bacterial arginine dihydrolase system arebased on the elevation of pH (17, 27), on thedecrease of arginine concentration (25) aftergrowth of bacteria in a medium containing a highconcentration of arginine, or on the identifica-tion of the end products (ornithine or citrulline)by one-dimensional thin-layer chromatography(TLC) on cellulose layers (21, 29, 31). Proce-dures based on the alkalization of a medium canbe obfuscated by NH3 production, by decarbox-ylation of arginine or other amino acids, or byacid production from sugar fermentation; proce-dures based on the decrease of arginine concen-tration in the spent medium can be confused byother catabolic pathways for arginine (1). Al-though the procedures for identifying end prod-ucts by one-dimensional TLC provide more reli-able results, the arginine dihydrolase systemcannot be distinguished from arginase if citrul-

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910 CHEN ET AL.

line is absent in the spent medium. Moreover, asuitable solvent system has not been reportedfor the separation of all possible components(arginine, citrulline, omithine, putrescine, andagmatine) in the test system (31). Gas-liquidchromatography has also been used for dem-onstrating ornithine and lysine decarboxylaseactivities (15) and for detecting arginine me-tabolites (19). However, the gas-liquid chroma-tography procedures described were not shownto be capable of detecting the decarboxylases ofglutamic acid, histidine, phenylalanine, trypto-phan, or tyrosine, and they were less efficientfor testing a large number of specimens. Themethods for detecting the arginine dihydrolasesystem and amino acid decarboxylases all haverequired incubation of the bacteria in mediacontaining high concentrations of each key ami-no acid tested for periods ranging from 1 to 24 hup to 4 to 10 days (15-17, 19-21, 23, 25, 27, 29,31).We previously described a rapid procedure

with only two separate incubations for the simul-taneous detection of eight bacterial amino aciddecarboxylases by identifying the dansyl deriva-tives of the decarboxylation products in thespent media after 1 h of incubation by TLC onpolyamide sheets (5). In this paper, we describea general procedure for the rapid and specificdetection of the arginine dihydrolase system(arginine deiminase and catabolic ornithine car-bamoyltransferase) and decarboxylases of argi-nine, glutamic acid, histidine, lysine, ornithine,phenylalanine, tryptophan, and tyrosine. Thismethod requires only three separate incubationsof the microorganisms for 1 h for simultaneousidentification of citrulline, ornithine, -y-amino-n-butyric acid (GABA), and seven amines, and ithas been successfully applied to species of En-terobacteriaceae, Pseudomonadaceae, Neisser-ia, Eikenella, Streptococcus, Staphylococcus,Bacillus, and Mycoplasmatales.

MATERIALS AND METHODSChemicals. L-Citrulline was obtained from Sigma

Chemical Co. (St. Louis, Mo.). Pyridine obtained fromMCB Manufacturing Chemists, Inc. (Cincinnati, Ohio)was redistilled and refrigerated. The sources of otherchemicals were described previously (5).

Dihydrolase-decarboxylase induction medium. Thestandard dihydrolase-decarboxylase induction agarmedium used in this study was prepared as follows.The ingredients of peptone-starch-dextrose (PSD) agarmedium (6) plus 0.1% each L-arginine hydrochloride,L-glutamic acid, L-histidine hydrochloride monohy-drate, L-lysine dihydrochloride, L-ornithine hydro-chloride, L-phenylalanine, and L-tryptophan and0.02% L-tyrosine were solubilized by heating at 100°Cfor 15 min. The pH of the medium was adjusted to 5.5with 6 N HCI (at about 70°C), and the medium wasautoclaved and dispensed in petri dishes (60 by 15 mm;

Falcon Plastics, Oxnard, Calif.). This induction agarmedium was used throughout this study unless other-wise described.

Dihydrolase-decarboxylase test. Three media wereused to test each microorganism for dihydrolase-de-carboxylase activity. Medium 1 consisted of PSDbroth supplemented with 0.1% L-arginine hydrochlo-ride. Medium 2 consisted of PSD broth supplementedwith the same amount of amino acid supplements usedfor the dihydrolase-decarboxylase induction medium,but without arginine. Medium 3 consisted of PSDbroth supplemented with 0.1% L-citrulline. The sup-plements were added to the autoclaved PSD broth.Each supplemented medium was sterilized by mem-brane filtration after the pH was adjusted to 5.5 with 6N HCI. Medium 1 was used only for tests of thearginine dihydrolase system and arginine decarboxyl-ase; medium 2 was used only for tests of decarboxyl-ases of glutamic acid, histidine, lysine, ornithine,phenylalanine, tryptophan, and tyrosine; and medium3 was used only for tests for catabolic omithinecarbamoyltransferase.

Preparation of inocula for the dihydrolase-decarbox-ylase test. The microorganisms used in these studieswere stock cultures from the American Type CultureCollection and from the Neisseria Reference Labora-tory. Pseudomonas spp. were generously provided byStephen A. Morse and Barbara Minshew. Citrobacterdiversus was obtained from Judith Hale. Ureaplasmaurealyticum type 8 (cloned 8 times) was obtained fromMaurice C. Shepard. Each microorganism wasstreaked on a dihydrolase-decarboxylase inductionagar medium plate and was grown aerobically at 37°Cfor 12 h unless otherwise described. One loopful(diameter, 2 mm) of growth from the microorganismgrown on an agar plate was suspended (by blending ina Vortex mixer) in each of three microfuge tubes (400,ul; Stockwell Scientific, Monterey Park, Calif.) con-taining 0.2 ml of 0.85% NaCI and centrifuged in amicrofuge (model 152; Beckman Instruments, Inc.,Fullerton, Calif.) for 1 min. The supernatant wasremoved. Washing of each pellet was repeated once toremove the metabolites from the spent induction medi-um. Then, 0.2 ml each of media 1, 2, and 3 was addedseparately to the three pellets and incubated aerobical-ly at 370C for 1 h after blending in a Vortex mixer,along with uninoculated test medium controls. Speciesof Enterobacteriaceae were also grown in the standardinduction broth and other induction agar media, in-cluding Trypticase soy agar (BBL Microbiology Sys-tems, Cockeysville, Md.) and brain heart infusion agarmedium (Difco Laboratories, Detroit, Mich.), with thesame pH and amino acid supplements as described forthe standard induction agar medium. Species of Neis-seria were grown as described (3). Species ofEikenellawere grown on chocolate agar without amino acidsupplements at pH 7.3 (no growth at pH 5.5) for 48 h ina CO2 incubator as described for Neisseria spp. Myco-plasmatales organisms were grown in soy peptonefresh yeast dialysate broth (13) supplemented with thefollowing additives for the indicated organisms: Myco-plasma hominis, 10% agamma horse serum; Achole-plasma laidlawii, 2% agamma horse serum; and Urea-plasma urealyticum, 2% agamma horse serum-30 mMurea-100 mM 2-(N-morpholino)ethanesulfonic acid,pH 6.3 (14). Organisms grown in broth were concen-trated by centrifugation at 12,000 x g for 10 min

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MICROBIAL DIHYDROLASE-DECARBOXYLASE DETECTION

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FIG. 1. Separation of dansyl amines, GABA, citrulline, and other amino acids by two-dimensional TLC onpolyamide sheets. Solvent systems used: first solvent system, water-formic acid (100:1.5); second solventsystem, benzene-glacial acetic acid (9:1); third solvent system, benzene-pyridine-glacial acetic acid (15:4:1). Thesolid spots are the positions of the dansyl derivatives after two-dimensional chromatography in the first andsecond solvent systems, and the open spots are those after chromatography in the third solvent system. Thedirection of each chromatographic system is indicated by an arrow with the solvent front at its tip.Abbreviations: Agma, agmatine; Cada, cadaverine; Cit, citrulline; Dns-OH, 1-dimethylaminonaphthalene-5-sulfonic acid; Hist, histamine; Om, ornithine; Phen, phenethylamine; Putr, putrescine; Trpt, tryptamine; Tyra,tyramine; Ala, alanine; Arg, arginine; Asn, asparagine; Asp, aspartic acid; Gln, glutamine; Glu, glutamic acid;Gly, glycine; His, histidine; lie, isoleucine; Leu, leucine; Lys, lysine; Met, methionine; Phe, phenylalanine; Pro,proline; Ser, serine; Thr, threonine; Trp, tryptophan; Tyr, tyrosine; Val, valine.

(except Mycoplasma spp., 8,000 x g for 30 min), andpellets were washed twice with 0.85% NaCl by blend-ing in a Vortex mixer and centrifuging to remove themetabolites from the spent induction media. A heavysuspension was made in 0.85% NaCl, and loopfuls ofeach suspension were separately incubated with thethree test media as described above.

Dansylation of the spent test media. After incubation,all inoculated tubes were centrifuged in a microfugefor 1 min to pellet the organisms. Supernatant (10 ,ul)from each tube (including test medium controls) wasremoved and placed separately in another microfugetube containing 40 p.l of 1 M NaHCO3. This mixturewas blended briefly in a Vortex mixer, and 50 IL1 ofdansyl chloride solution (5 mg per ml of acetone) wasadded to the mixture. The microfuge tubes werecapped tightly, blended in a Vortex mixer, and incu-bated at 37°C for 1 h.

Separation of dansyl derivatives by TLC on polya-mide sheets. After incubation, the reaction mixturewas centifuged for 1 min. The dansylated supernatant(4 p.l) from spent test medium 1, which had beenincubated with a test organism, was spotted on oneside (front side) of a Chen-Chin polyamide layer sheet(15 by 15 cm; Accurate Chemical & Scientific Corp.,Hicksville, N.Y.), and the dansylated supernatant (4p.1) from spent test medium 1 which had been incubat-ed with another test organism was spotted at the sameposition on the opposite side (back side) of the samesheet. Note that each 4-ptl sample of the dansylated

supernatant after centrifugation contained 0.4 p.l ofspent test medium. TLC was carried out by theprocedure described by Woods and Wang (30), exceptthat the second-dimension chromatography was run toonly about two-thirds the length of the sheet. Thepositions of ornithine and agmatine were then identi-fied. Dansyl citrulline could only be separated fromdansyl derivatives of threonine, serine, asparagine,and glutamine after chromatography in a third solventsystem (benzene-pyridine-glacial acetic acid, 15:4:1) inthe same direction as the second solvent system toabout half the length of the sheet. Dansyl agmatinewas slightly further separated from dansyl arginineafter chromatography in the third solvent system (Rfofdansyl agmatine at the third solvent system was about0.05, and the position of dansyl arginine remainedunchanged). However, better separation could beachieved by using the fourth solvent system (ethylace-tate-glacial acetic acid-methanol, 20:1:1) as describedpreviously (5; Rf of dansyl agmatine in the fourthsolvent system was about 0.2, and the position ofdansyl arginine remained unchanged).

Dansyl derivatives of spent test medium 2 and ofspent test medium 3, incubated with the same orga-nisms, were applied at the same position on oppositesides of the same sheet as described above and werechromatographed similarly, except that chromatogra-phy on the third and fourth solvent systems was notrequired because these two test media were not usedto detect arginine deiminase or arginine decarboxylase

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J. CLIN. MICROBIOL.912 CHEN ET AL.

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activity. The positions of ornithine, GABA, and theamines were then identified as described previously(5). Each dansylated uninoculated test medium controlwas spotted on the sheet and chromatographed as

described for its corresponding dansylated spent testmedium. Dansyl amino acids were identified as de-scribed previously (5). Only a set of controls for theexperiments of each day was required.

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FIG. 3. Separation of dansyl derivatives of 0.4 ,ul each of test medium 1 control (A, A'), test medium 2 control(C), spent test medium 1 (B, B'), and spent test medium 2 (D) incubated with Enterobacter hafniae for 1 h at37°C, by two-dimensional TLC on polyamide sheets by the system shown in Fig. 1 and described in the legend.After TLC in the first and second solvent systems, further TLC in the fourth solvent system, ethyl acetate-glacialacetic acid-methanol (20:1:1), was used for further separation of dansyl agmatine from dansyl arginine (A', B').See legend to Fig. 1 for abbreviations.

RESULTS

Detection of the arginine dihydrolase systemand of amino acid decarboxylases by identifica-tion of dansyl end products from the spent testmedia on polyamide sheets after two-dimensionalchromatography. The relative positions of dan-syl derivatives of citrulline, ornithine, GABA,amines, and other amino acids from the spenttest media after two-dimensional chromatogra-phy (Fig. 1) were determined as described previ-ously (5). Dansyl citrulline in the dansylatedspent test medium 1 could be detected afterfurther chromatography in the third solvent sys-tem (Fig. 1), whereas dansyl agmatine in thedansylated spent test medium 1 could be identi-

fied clearly after further chromatography in thethird and fourth solvent systems.TLC methods were applied for the detection

of the first two enzymes involved in the argininedihydrolase system (arginine deiminase and cat-abolic ornithine carbamoyltransferase) and ofdecarboxylases of phenylalanine and tyrosine ofStreptococcus faiecalis (Fig. 2). Citrulline wasproduced by Streptococcusfaecalis during incu-bation in medium 1 for 1 h at 37°C (compare 2A'and B'), and ornithine was produced duringincubation in both media 1 and 3 for 1 h at 37°C(compare 2A and B, E and F), showing thepresence of arginine deiminase (resulting in pro-duction of citrulline from arginine) and of cata-bolic ornithine carbamoyltransferase (resulting

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MICROBIAL DIHYDROLASE-DECARBOXYLASE DETECTION 917

FIG. 4. Production of decarboxylases of glutamicacid, lysine, and ornithine by Enterobacter hafniaeunder different growth conditions. (A) Same condi-tions used for Fig. 3 (optimum, PSD agar supplement-ed with eight amino acids as described and pH adjust-ed to 5.5). (B) pH 7.0 with amino acid supplements;same strong glutamic acid decarboxylase activity as

shown in (A), but no omithine decarboxylase activityor weak lysine decarboxylase activity. (C) pH 7.0without amino acid supplement; weak activities ofdecarboxylases of glutamic acid, lysine, and ornithine.The incubation conditions for the detection of aminoacid decarboxylases were the same as for Fig. 3. Seelegend to Fig. 1 for abbreviations.

in production of ornithine from citrulline) inStreptococcus faecalis. Phenethylamine and ty-ramine were produced during incubation in me-dium 2 for 1 h at 37°C (compare 2C and D),indicating the presence of both phenylalanineand tyrosine decarboxylases in the same orga-nism.The same methods were applied for the detec-

tion offour decarboxylases produced by Entero-bacter hafniae (Fig. 3). Agmatine was producedby Enterobacter hafniae during incubation inmedium 1 for 1 h at 37°C (compare 3A and B, A'and B'), indicating the presence of argininedecarboxylase. Cadaverine, GABA, and putres-

cine were produced by the same organism dur-ing incubation in medium 2 for 1 h at 37°C(compare Fig. 3C and D), demonstrating thepresence of decarboxylases of lysine (resultingin production of cadaverine), glutamic acid (re-sulting in production of GABA), and ornithine(resulting in production of putrescine). Note thatdansyl derivatives of all test medium controlsand spent test media were spotted on the sameside of sheets shown in Fig. 2 and 3 for easyillustration and comparison with Fig. 1, whereasthose spotted as described above were for theroutine tests for dihydrolase-decarboxylase ac-tivity.

Activities of enzymes involved in the argininedihydrolase system and amino acid decarboxyl-ases in selected microorganisms. To determinethe dihydrolase-decarboxylase activity in eachmicroorganism, the fluorescent intensities (de-tected on the polyamide sheets after two-dimen-sional chromatography) of the dansyl deriva-tives of the end products in the spent test mediawere compared with those of the correspondingparent amino acids in the test medium controls.Although dansyl glutamic acid could not sepa-rate from dansyl aspartic acid after two-dimen-sional chromatography in the first two solventsystems, the amount of aspartic acid presentwas negligible compared with the amount ofglutamic acid (parent amino acid of GABA)present in the uninoculated test medium 2 con-trol, which was supplemented with 0.1% glutam-ic acid (4). If necessary, dansyl glutamic acidcould be easily separated from dansyl asparticacid with the fourth solvent system (28). Theactivity of each enzyme was rated as strong if itsend product produced during incubation hadabout the same fluorescent intensity under UVlight as its parent amino acid in the test mediumcontrol; weak if its end product was easilyidentified but its fluorescent intensity was muchweaker than that of its parent amino acid in thecontrol; or moderate if the fluorescent intensityof the end product was intermediate betweenthose described for strong and weak (Table 1).The results obtained for species of Enterobac-teriaceae grown in the PSD induction agar (Ta-ble 1) were similar to those obtained with growthin PSD induction broth or in other inductionmedia, as described above (data not shown).

DISCUSSIONThe procedures described in this report allow

simultaneous detection of the first two enzymesinvolved in the arginine dihydrolase system andof eight amino acid decarboxylases by three 1-hincubations per organism, instead of one incuba-tion for each enzyme as tested by existing meth-ods. This method can also be applied for thedetection of the dihydrolase and decarboxylase

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918 CHEN ET AL.

systems in species of Enterobacteriaceae grownin induction broth or other induction agar media,and it may prove generally applicable for othermicroorganisms. Prolonged incubation of thetest media did not improve detection of theexisting enzymes involved in the arginine dihy-drolase system (data not shown) or of the aminoacid decarboxylases as previously described (5);instead, it increased the possibilities of detectionof the biosynthetic amino acid decarboxylasessuch as ornithine, lysine, and arginine decarbox-ylases (18).Use of test medium 3 for detecting the argi-

nine dihydrolase system in microorganisms isnecessary because the arginine dihydrolase sys-tem cannot be distinguished from arginase ifcitrulline is not detected in the spent test medi-um 1 (presumably owing to the high turnoverrate of the intermediate citrulline by catabolicornithine carbamoyltransferase). The produc-tion of ornithine in both spent test medium 1 andspent test medium 3 is a clear proof of thearginine dihydrolase system in the microorgan-ism tested. Less ornithine was usually found inthe spent test medium 3 than in the spent testmedium 1 (Table 1), presumably because of thelower permeability of citrulline into the microbi-al cells (24, 26).Amino acid decarboxylases in microorga-

nisms are substrate and acid inducible (8). Forexample, both amino acid supplements and lowpH are required for optimum production ofamino acid decarboxylases by Enterobacter haf-niae (Fig. 4). When we detected the argininedihydrolase system in the microorganisms listedin Table 1 by using the induction medium andtest media 1 and 3 at pH 7.0, the activities of thetwo enzymes (arginine deiminase and catabolicornithine carbamoyltransferase) were eitherslightly increased or remained the same, but in afew cases citrulline was not detected, presum-ably because catabolic ornithine carbamoyl-transferase has a higher activity at neutral pH(data not shown). Therefore, the induction medi-um and the three test media described above arerequired for the simultaneous detection of thedihydrolase and decarboxylase systems inmicroorganisms. The results listed in Table 1agree with those obtained by identifying the endproducts of the arginine dihydrolase enzymesand decarboxylases of arginine, lysine, and orni-thine with one-dimensional TLC on celluloseplates (10, 11, 21, 29, 31, 32) and with gas-liquidchromatography (15, 19).

This method may prove useful for differentiat-ing species of Pseudomonas and Mycoplasma-tales. As shown in Table 1, preliminary resultsindicate that Pseudomonas aeruginosa, Pseudo-monas fluorescens, and Pseudomonas putidashow only strong arginine dihydrolase enzyme

activity, whereas Pseudomonas maltophiliashows weak arginine dihydrolase enzyme activi-ty but strong lysine decarboxylase activity;Pseudomonas cepacia shows both strong lysineand ornithine decarboxylase activities but noarginine dihydrolase enzyme activity. Urea-plasma urealyticum shows no dihydrolase-de-carboxylase activity. Mycoplasma hominisshows only the arginine dihydrolase enzymeactivity, whereas Acholeplasma laidlawii showsonly ornithine decarboxylase activity.

Conventional methods for detecting decar-boxylases of histidine, phenylalanine, and tyro-sine require tedious manometric assays (8). Ourmethod provides a simple procedure for thedetection of these three decarboxylases and mayprovide a biochemical test for identifying anddifferentiating microorganisms. For example,the lengthy tyrosine-clearing test (12) for somebacteria, such as Morganella morganii andProvidencia stuartii, may be replaced by thismethod since the tyrosine clearing may be due tothe conversion of water-insoluble tyrosine towater-soluble tyramine (solubility increasesover 20-fold after conversion) by tyrosine decar-boxylase (see Table 1).The method described here is rapid, sensitive,

and inexpensive, and thus it is potentially usefulfor clinical laboratories. Six polyamide sheets(12 specimens applied) can be chromatographedin a glass jar (13 by 16 by 26 cm) and air dried.Approximately 1 h is required to complete chro-matography for the first and second solventsystems and for the third and fourth solventsystems (2 h for the all solvent systems). Polya-mide sheets can be washed and reused at least 10times as described previously (5). Because of thehematoxic, hepatotoxic, and carcinogenic po-tential of benzene, a chemical exhaust hoodshould be used for working with the chromato-graphic jars and for hanging the polyamidesheets to dry. The third solvent system de-scribed in this report allows the separation ofdansyl derivatives of asparagine, citrulline, glu-tamine, serine, and threonine, and it may beuseful for rapid qualitative analyses of free ami-no acids in physiological fluids and in the exo-peptidase digests of proteins or peptides.

ACKNOWLEDGMENTSThis study was supported by Research Program Project

grant AI-12192 from the National Institutes of Health, and byPublic Health Service grant AI-06720 from the National Insti-tute of Allergy and Infectious Diseases.We gratefully acknowledge the help of Barbara Minshew,

who provided several of the species of organisms used in thisstudy.

LITERATURE CITED1. Abdelal, A. T. 1979. Arginine catabolism by microorga-

nisms. Annu. Rev. Microbiol. 33:139-168.

J. CLIN. MICROBIOL.

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2. Boeker, E. A., and E. E. Snell. 1972. Amino acid decar-boxylases, p. 217-253. In P. D. Boyer (ed.), The en-zymes, vol. 6. Academic Press, Inc., New York.

3. Chen, K. C. S., and T. M. Buchanan. 1980. Hydrolasesfrom Neisseria gonorrhoeae, the study of gonocosin, anaminopeptidase-p, a proline iminopeptidase, and an aspar-aginase. J. Biol. Chem. 255:1704-1710.

4. Chen, K. C. S., P. S. Forsyth, T. M. Buchanan, and K. K.Holmes. 1979. Amine content of vaginal fluid from un-treated and treated patients with nonspecific vaginitis. J.Clin. Invest. 63:828-835.

5. Chen, K. C. S., and K. K. Holmes. 1981. A rapid proce-dure for detection of bacterial amino acid decarboxylases.Anal. Biochem. 111:60-66.

6. Dunkelberg, W. E., Jr., R. Skaggs, and D. S. Kellog, Jr.1970. Method for isolation and identification of Coryne-bacterium vaginale (Haemophilus vaginalis). Appl. Mi-crobiol. 19:47-52.

7. Edwards, P. R., and W. H. Ewing. 1972. Identification ofEnterobacteriaceae, 3rd ed., p. 21-47. Burgess PublishingCo., Minneapolis, Minn.

8. Gale, E. F. 1946. The bacterial amino acid decarboxyl-ases. Adv. Enzymol. Relat. Subj. Biochem. 6:1-32.

9. Gilardi, G. L. 1969. Characterization of the oxidase-negative, gram-negative coccobacilli (the Achromobacter-Acinetobacter group). Antonie van Leeuwenhoek J. Mi-crobiol. Serol. 35:421-429.

10. Goldschmidt, M. C., and B. M. Lockhart. 1971. Rapidmethods for determining decarboxylase activity: argininedecarboxylase. Appl. Microbiol. 22:350-357.

11. Goldschmidt, M. C., B. M. Lockhart, and K. Perry. 1971.Rapid methods for determining decarboxylase activity:ornithine and lysine decarboxylases. AppI. Microbiol.22:344-349.

12. Hickman, F. W., J. J. Framer IIH, A. G. Steigerwalt, andD. J. Brenner. 1980. Unusual groups of Morganella("Proteus") morganii isolated from clinical specimens:lysine-positive and ornithine-negative biogroups. J. Clin.Microbiol. 12:88-94.

13. Kenny, G. E. 1967. Heat-lability and organic solvent-solubility of Mycoplasma antigens. Ann. N.Y. Acad. Sci.143:676-681.

14. Kenny, G. E., and F. D. Cartwright. 1977. Effect of ureaconcentration on growth of Ureaplasma urealyticum (T-strain mycoplasma). J. Bacteriol. 132:144-150.

15. Lambert, M. A. S., and C. W. Moss. 1973. Use of gaschromatography for detecting ornithine and lysine decar-boxylase activity in bacteria. Appl. Microbiol. 26:517-520.

16. Maccani, J. E. 1979. Aerobically incubated medium fordecarboxylase testing of Enterobacteriaceae by replica-plating methods. J. Clin. Microbiol. 10:940-942.

17. MHlIer, V. 1955. Simplified tests for some amino aciddecarboxylases and for the arginine dihydrolase system.Acta Pathol. Microbiol. Scand. 36:158-172.

18. Morris, D. R., and R. H. Fillinme. 1974. Regulation ofamino acid decarboxylation. Annu. Rev. Biochem.43:303-325.

19. Moss, C. W., M. A. Lambert, and W. B. Cherry. 1972.Use of gas chromatography for determining catabolicproducts of arginine by bacteria. Appl. Microbiol. 23:889-893.

20. Nord, C.-E., A. A. Lindberg, and A. Dahlback. 1975. Fourhour-tests for the identification of Enterobacteriaceae.Med. Microbiol. Immunol. 161:231-238.

21. Ottow, J. C. G. 1974. Arginine dihydrolase activity inspecies of the genus Bacillus revealed by thin-layer chro-matography. J. Gen. Microbiol. 84:209-213.

22. Palleroni, N. J., M. Doudoroff, R. Y. Stanier, R. E. So-lanes, and M. Mandel. 1970. Taxonomy of the aerobicpseudomonads: the properties of the Pseudomonas stut-zeri group. J. Gen. Microbiol. 60:215-231.

23. Smith, H. L., Jr., and P. Bhat-Fernandes. 1973. Modifieddecarboxylase-dihydrolase medium. Appl. Microbiol.26:620-621.

24. Smith, P. F. 1960. Amino acid metabolism of PPLO. Ann.N.Y. Acad. Sci. 79:543-550.

25. Stanier, R. Y., N. J. PaDleroni, and M. Doudoroff. 1966.The aerobic Pseudomonads: a taxonomic study. J. Gen.Microbiol. 43:159-271.

26. Taylor, J. J., and J. L. Whitby. 1964. Pseudomonas pyo-cyanea and the arginine dihydrolase system. J. Clin.Pathol. 17:122-125.

27. Thornley, M. J. 1960. The differentiation ofPseudomonasfrom other gram-negative bacteria on the basis of argininemetabolism. J. Appl. Bacteriol. 23:37-52.

28. Weiner, A. M., T. Platt, and K. Weber. 1972. Amino-terminal sequence analysis of proteins purified on a nano-mole scale by gel electrophoresis. J. Biol. Chem.247:3242-3251.

29. Williams, G. A., D. J. Blazevic, and G. M. Ederer. 1971.Detection of arginine dihydrolase in nonfermentativegram-negative bacteria by use of thin-layer chromatogra-phy. Appl. Microbiol. 22:1135-1137.

30. Woods, K. R., and K. T. Wang. 1967. Separation ofdansyl-amino acids by polyamide layer chromatography.Biochim. Biophys. Acta 133:369-370.

31. Zoig, W., and J. C. G. Ottow. 1973. Improved thin-layertechnique for detection of arginine dihydrolase among thePseudomonas species. Appl. Microbiol. 26:1001-1003.

32. Zolg, W., and J. C. G. Ottow. 1974. Thin-layer chroma-tography of arginine, lysine and ornithine decarboxylaseactivity among Pseudomonas spp. and Enterobac-teriaceae. Microbios 10:225-231.

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