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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 1976, p. 668-679Copyright © 1976 American Society for Microbiology

Vol. 31, No. 5Printed in U.S.A.

Use of a Quantitative Oxidase Test for CharacterizingOxidative Metabolism in BacteriaPETER JURTSHUK, JR.* AND DONALD N. McQUITTY

Department of Biology, University of Houston, Houston, Texas 77004

Received for publication 12 December 1975

It was possible to quantitate the terminal oxidase(s) reaction using bacterialresting-cell suspensions and demonstrate the usefulness of this reaction fortaxonomic purposes. Resting-cell suspensions of physiologically diverse bacteriawere examined for their capabilities of oxidizing N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) using a manometric assay. For organisms havingthis capability, it was possible to calculate the conventional TMPD oxidaseQ(02) value (microliters of 02 consumed per hour per milligram [dry weight]).All cultures were grown heterotrophically at 30 C, under identical nutritionalconditions, and were harvested at the late-logarithmic growth phase. TheTMPD oxidase Q(02) values showed perfect correlation with the Kovacs oxidasetest and, in addition, it was possible to define quantitatively that point which-separated oxidase-positive from oxidase-negative bacteria. Oxidase-negativebacteria exhibited a TMPD oxidase Q(02) value (after correcting for the endoge-nous by substraction) of -33 and had an uncorrected TMPD/endogenous ratio of-5. The TMPD oxidase Q(02) values were also correlated with the data obtained

for the Hugh-Leifson Oxferm test. In general, bacteria that exhibited a respira-tory mechanism had high TMPD oxidase values, whereas fermentative orga-nisms had low TMPD oxidase activity. All exceptions to this are noted. Thisquantitative study also demonstrated that organisms that (i) lack a type ccytochrome, or (ii) lack a cytochrome-containing electron transport system, likethe lactic acid bacteria, exhibited low or negligible TMPD oxidase Q(02) values.From the 79 bacterial species (36 genera) examined, it appears that this quanti-tative oxidase test has taxonomic value that can differentiate the oxidativerelationships between bacteria at the subspecies, species, and genera levels.

The para-phenylenediamines (PPDs), partic-ularly the tetramethyl- and dimethyl- deriva-tives (TMPD and DMPD, respectively), arebasic dyes that can function as biological elec-tron donors. Both derivatives have been usedextensively in microbiological studies as quali-tative indicators of oxidase activity (10, 14, 15,29). Originally Gordon and McLeod (15) foundthe oxidase test to be particularly useful for therapid identification ofNeisseria gonorrhea andVibrio cholerae. Kovacs (29) standardized theoxidase test procedure by using the more sensi-tive TMPD derivative and defined the time in-terval (10 s) during which a bacterial colonymust turn blue in color to be considered oxidasepositive. Gaby and Hadley (14), using a testtube cytochrome oxidase test, monitored theoxidation of DMPD-oxalate, in the presence ofa-naphthol, and obtained results comparable tothe Kovacs oxidase test (13).The taxonomic importance of the qualitative

oxidase test has become apparent from thestudies of several investigators (3, 11, 14, 39).

Ewing and Johnson (11) were able to differ-entiate Aeromonas and Plesiomonas from orga-nisms of the family Enterobacteriaceae usingthe oxidase test. Steel (38) examined 1,660 orga-nisms of various genera and showed the Kovacsoxidase reaction to be a useful taxonomic tool.The oxidase test also has been used in studieswith Staphylococcus spp. (39), and this test,together with the glucose fermentation reaction(1), was found to be useful in classifying orga-nisms of the family Micrococcaceae (3).

It also has been possible to quantitate thePPD a-naphthol oxidation reaction in tissues;in this instance, it is more commonly referredto as the indophenol oxidase reaction (27, 40,41). The early attempts at quantitating theTMPD oxidase reaction in bacteria werehindered by the complexity of the reaction andits uncertain relationship to catalase and per-oxidase activity (12, 31). Yamagutchi (43) im-proved upon the quantitation procedure andmeasured manometrically both the PPD andDMPD oxidation reactions, which he related to

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the dry weight of the bacterial cells. Oxidase-positive bacteria exhibited higher activities forPPD and DMPD oxidation than did oxidase-negative bacteria. Later, Richardson (34) stud-ied this same manometric DMPD oxidase reac-

tion, using Brucella whole cells, and he at-tempted to correlate oxidase activity to viablecell count. Most recently the quantitative man-ometric TMPD oxidase analysis has been ex-

tended to measure the potent terminal oxidaseactivity in Azotobacter vinelandii (25), where ithas shown that the TMPD oxidation kinetics bywhole cells were remarkably similar to thoseobtained for the electron transport particle (19).Reduced TMPD readily penetrates the intact A.vinelandii cell, and its oxidation by the electrontransport system is comparable to the terminaloxidase reaction measured by cytochrome c oxi-dation (20, 24). This same quantitative TMPDoxidation reaction was also used successfullyfor measuring the highly potent oxidase reac-

tion in Neisseria spp. (23).The survey study reported herein was de-

signed to determine whether or not this TMPDoxidase reaction could be used as a quantitativeindex for establishing the extent that terminaloxidase activity occurred in a large variety ofoxidase-positive and -negative bacteria. Sev-

QUANTITATIVE OXIDASE TEST 669

enty-nine bacterial strains, representing 36genera, were examined quantitatively for theircapabilities of oxidizing TMPD; the resultswere then correlated to the data obtained forboth the Kovacs oxidase test and the Hugh-Leifson Oxferm (H-L O/F) test. For the lattertest, an attempt was made to establish whetheror not TMPD oxidation could be correlated tothe different oxidative and/or fermentativemetabolic patterns exhibited by bacteria (17).

MATERIALS AND METHODS

Microorganisms and cultural conditions. Thebacteria used for this study are listed in Tables 1through 4; the sources of all cultures are identifiedin Acknowledgments.

Resting-cell suspensions were prepared from bac-teria grown at 30 C in nutrient broth (Difco), whichcontained 1% (wt/vol) sucrose supplemented with0.5% (wt/vol) yeast extract (Difco). The marine bac-teria Pseudomonas bathycetes and Vibrio parahae-molyticus were grown on the medium to which 1.5%(wt/vol) NaCl was added. Vitreoscilla stercorariawas grown on this same medium, which was supple-mented with 0.25% (wt/vol) K2HPO4, and glucosewas substituted for sucrose. All seed cultures were

allowed adequate time to adapt on the above me-

dium, and batch cultures were prepared in 400-mlquantities, grown in 1-liter flasks placed on a rotary

TABLE 1. Quantitative study on the TMPD oxidase reaction using whole cells of bacteria that are commonlyoxidase positive

Q(02) valuerAbbrevi-

Microorganism0 Sourceb ation in TMPD TMPD/Fig. 1 Endogd TMPD minus Endog

Endog o

Branhamella (Neisseria) catarrhalis (25238) M C Wisconsin Nc 16 1,752 1,736 110B. catarrhalis Gp4 U Maryland Nc 14 1,612 1,598 115B. catarrhalis NC31 U Maryland Nc 17 1,630 1,613 96

Neisseria elongata (25295) M C Wisconsin Ne 5 640 635 128N. flava (14221) M C Wisconsin Nf 7 1,329 1,322 190N. mucosa Houston H D Nm 9 764 755 85N. sicca Houston H D Ns 8 865 857 108

Pseudomonas acidovorans (15668) ATCC Psac 18 1,140 1,122 63P. aeruginosa (15442) UTHMS Psa 17 1,760 1,743 104P. bathycetes (23597) U Maryland Psb 13 528 515 41P. fluorescens (13525) ATCC Psf 26 1,487 1,461 57P. stutzeri 320 UTHGSBS Pss 22 1,209 1,187 55P. maltophiliae (13637) ATCC Psm 18 43 25 2

Vibrio parahaemolyticus biotype alginolyti- U Maryland Va 2 91 89 46cus 156-70

V. parahaemolyticus FC1011 U Maryland Vp 1 35 34 35V. parahaemolyticus SAK3 U Maryland Vp 1 28 27 28V. (metschnikovii) cholerae biotype proteuse ATCC Vm 2 9 7 4

(7708)

a Names in parentheses indicate the nomenclature used in Bergey's Manual, 7th ed. All numbers in parentheses are theATCC designation; all other numbers (or letters) represent strain designations.

b Abbreviations for sources are identified in Acknowledgments.c Expressed as microliters of 02 per hour per milligram (dry weight) at 30 C.d Endogenous value; this represents the cellular respiration rate obtained in the absence of ascorbate-TMPD.e Oxidase-negative bacteria.


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TABLE 2. Quantitative study on the TMPD oxidase reaction using whole cells of oxidase-positive bacteria

Q(02) valuecAbbrevi-

Microorganisma Sourceb ation in TMPD TMPD/Fig. 1 Endogd TMPD minus Endog

Endog og

Achromobacter xerosis (14780) ATCC Ax 14 407 393 29Aeromonas hydrophila (9071) UTHGSBS Ah 12 151 139 13A. liquefaciens (14715) UTHGSBS Al 23 534 511 23Agrobacterium tumefaciens (15955) ATCC Agt 12 1,686 1,674 141Alcaligenes faecalis (8750) ATCC Alf 16 1,083 1,067 68Azotobacter vinelandii 0 (12518) U Houston Av 19 1,221 1,202 64Azotomonas insolita (12412) ATCC Ai 11 2,165 2,154 197Flavobacterium capsulatum (14666) ATCC Fc 38 318 280 8Micrococcus (Sarcina) luteus U Houston Si 16 80 64 5M. (S. flava) luteus UTHGSBS Sf 9 114 105 13Moraxella osloensis U Maryland Mo 8 1,001 993 125Rhizobium meliloti F-28 U Houston Rm 7 1,080 1,073 154R. meliloti 3DOal U Houston Rm 9 693 684 77Spirillum itersonii (12639) ATCC Si 6 791 785 132Sporosarcina (Sarcina) ureae UTHGSBS Su 7 657 650 94

a. b. . dSee Table 1.

TABLE 3. Quantitative study on the TMPD oxidase reaction using whole cells ofoxidase-negative bacteria ofthe family Enterobacteriaceae

Q(WO) valuerAbbrevi-

Microorganism" Sourceb ations in TMPD TMPD/Fig. 1 Endogd TMPD minus Endog

Endog og

Enterobacter aerogenes U Houston Ea 6 12 6 2

Escherichia coli B U Houston Ec 13 26 13 2E. coli B UTHGSBS Ec 7 21 14 3E. coli B/r UTHGSBS Ec 9 29 20 3E. coli C UTHGSBS Ec 10 43 33 4E. coli Crookes U Houston Ec 6 27 21 5E. coli K12 UTHGSBS Ec 8 39 31 5E. coli K12S UTH 2593r UTHGSBS Ec 7 31 24 4E. coli K12S UTH 3672 UTHGSBS Ec 6 14 8 2E. coli F UTHGSBS Ec 5 12 7 2E. coli Ollla, UTHGSBS Ec 4 9 5 2

Klebsiella pneumoniae (13882) UTHMS Kp 4 6 2 2K. pneumoniae M5al U Sussex Kp 2 6 4 3

Proteus morganii U Houston Pm 3 8 5 3P. vulgaris U Houston Pv 3 8 5 3

Salmonella typhimurium (6444) UTHMS St 6 8 2 1

Serratia marcescens U Houston Sm 8 8 0 1

b. d See Table 1.Stock culture numbers of the University of Texas at Houston Graduate School of Biomedical Sciences (UTHGSBS).

shaker (72 cycles/min). Bacterial growth was moni-tored turbidimetrically so that all batch cultureswere harvested (by centrifugation) at the late-loga-rithmic growth phase, i.e., two-thirds of the maxi-mal growth concentration. The pelleted cells weresuspended in 0.02 M potassium phosphate buffer, pH7.5, homogenized, and stored overnight at 4 C. Onthe following day, the resting-cells suspension wascentrifuged, and the pellet was resuspended, ho-mogenized, and standardized turbidimetrically sothat a 1/100 dilution of this suspension (in 0.02 M

phosphate buffer) gave a reading of 0.700 to 0.800optical density units at 420 nm. The concentration ofresting cells used for the estimation of TMPD oxi-dase activity ranged from 7.8 to 67.2 mg (dry weight)per ml.

Chemical and enzyme assays. The sources andthe preparation of the various chemical reagentsneeded for the quantitative oxidase assay have beendescribed previously (19, 21, 23, 25, 26). Manometricassays were performed with Warburg flasks, and allTMPD oxidase values are reported using the con-

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TABLE 4. Quantitative study on the TMPD oxidase reaction using whole cells of other diverse oxidase-negative bacteria

Q(02) valuerAbbrevi-

Microorganisma Sourceb ation in TMPD TMPD/Fig. 1 Endogd TMPD minus EndogEndog Eno

Acinetobacter calcoaceticus 208 UTHGSBS Ac 7 14 7 2A. calcoaceticus (19606) ATCC Ac 4 21 17 5Arthrobacter globiformis (8010) ATCC Ag 5 21 16 4Bacillus cereus U Houston Bc 56 66 10 1B. firmus (14575) UTHGSBS Bf 8 14 6 2B. megaterium U Houston Bm 2 9 7 5B. pumilus (70) UTHGSBS Bp 3 3 0 1B. subtilis W-23 U Houston Bs 2 9 7 5B. (vulgatus) subtilis UTHGSBS Bv 2 4 2 2Corynebacterium 7E1C (19067) UT Austin 7E1C 10 23 13 2C. diphtheriae (11913) ATCC Cd 4 18 14 5Gafftya tetragena U Houston Gt 10 12 2 1G. tetragena (10875) UTHMS Gt 14 15 1 1Lactobacillus casei (7469) UT Galveston Lc 1 1 0 1Micrococcus (lysodeikticus) luteus (4698) ATCC Ml 30 46 16 2Mycobacterium fortuitum UTHMS Mf 10 13 3 1M. phlei U Houston Mp 16 33 17 2M. smegmatis U Houston Ms 13 17 4 1Nocardia asteroides (3308) ATCC Na 5 12 7 2Pediococcus cerevisiae (8081) UT Galveston Pc 2 4 2 2Staphylococcus (albus) aureus U Houston Sa 3 4 1 1S. aureus U Houston Sau 4 12 8 3S. aureus (6538) UTHMS Sau 5 11 6 2Streptococcus faecalis (8043) UT Galveston Sf 1 1 0 1S. faecalis (9790) UT Galveston Sf 1 1 0 1S. pneumoniae (6360) ATCC Spn 1 1 0 1S. pyogenes (10389) UTHMS Spy 2 2 0 1Streptomyces griseus (10137) ATCC Sg 12 36 24 3Vitreoscilla stercoraria (15218) NTSU Vs 21 18 0 1Xanthomonas phaseoli (9563) ATCC Xp 22 23 1 1

a. b. c. d See Table 1.

vential Q(02) value (see footnote c, Table 1). Theendogenous respiration represents the Q(02) valueobtained in the absence of ascorbate and TMPD inthe assay system. Suitable controls were alwaysemployed to ensure that: (i) the assay measured theascorbate-reduced TMPD oxidation by the bacterialcell suspension (23, 25); (ii) no oxidation reactionoccurred in the absence ofTMPD; and (iii) no chemi-cal (or nonenzymatic) auto-oxidation of TMPD oc-curred during the assay interval (19). TMPD oxidaseassays of the marine organisms were performed inthe presence and absence of 1.5% (wt/vol) NaCl.Slightly higher TMPD oxidase Q(02) values wereobtained from assays in which the NaCl was omit-ted, and these are the values reported herein.H-L O/F test. Isolated bacterial colonies, grown

on Trypticase soy broth (BBL) containing 0.8% yeastextract (Difco) and 1.5% agar, were used to inoculatethe H-L O/F semisolid carbohydrate medium. Acidand gas production, from the aerobic and anaerobicdissimilation of the test carbohydrates, were re-corded in accordance with the scheme proposed byHugh and Leifson (17).

Oxidase test. The test was performed in the man-ner described by Kovacs (29). The bacterial colonyused as the inoculum was a mate of that used for theO/F test.

RESULTSThe results of the quantitative TMPD oxi-

dase survey study are presented in Tables 1 to4. The corrected TMPD oxidase value (TMPDminus endogenous) and the TMPD/endogenousratio were the two parameters that were mostvaluable for interpreting the data presented.Table 1 shows the TMPD oxidation Q(02) val-ues for organisms of the genera Branhamella,Neisseria, Pseudomonas, and Vibrio. Classi-cally, these organisms are predominantly oxi-dase positive and are known to possess activeterminal oxidases. Noting the two exceptions(see footnote e, Table 1), bacteria in these gen-era possessed very high TMPD oxidase Q(02)values. Three strains of Branhamella catar-rhalis had corrected TMPD oxidation valuesranging from 1,598 to 1,736, which were sub-stantially higher than the values reported fororganisms of the genus Neisseria, with which itwas formerly classified. Neisseria spp. also ex-

hibited high corrected TMPD oxidase Q(02)values, but these were slightly lower andranged from 635 to 1,322. The TMPD/endoge-

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672 JURTSHUK AND McQUITTY

nous ratios showed very little difference be-tween these two genera of microorganisms.The oxidase-positive Pseudomonas spp. had

corrected TMPD oxidase Q(02) values rangingfrom 515 to 1,743 and TMPD/endogenous ratiosthat ranged from 41 to 104. As expected, theoxidase-negative Pseudomonas maltophilia ex-hibited a low corrected TMPD oxidation Q(02)value of 25 and a TMPD/endogenous ratio of 2.The corrected TMPD oxidase rates for all theother Pseudomonas spp. that were oxidase pos-itive were at least 20-fold higher than the valueobtained for P. maltophilia.Organisms of the genus Vibrio, which pre-

dominantly contains oxidase-positive species,had somewhat lower corrected TMPD oxidaseQ(02) values. The three V. parahaemolyticusstrains had TMPD oxidase values ranging from27 to 89 and TMPD/endogenous ratios rangingfrom 28 to 46. Both these values were high incontrast to that noted for the oxidase-negativeV. (metschnikovii) cholerae biotype proteus,whose comparable values were 7 and 4, respec-tively. The oxidase-positive V. parahaemolyti-cus strains had TMPD oxidation values thatwere 7 to 11 times more active than the oxidase-negative V. (metschnikovii) cholerae. It shouldbe noted that, whereas the corrected TMPD oxi-dation Q(02) value for the oxidase-positive V.parahaemolyticus strain SAK3 was not muchgreater than the value shown for the oxidase-negative P. maltophilia (27 versus 25), therewas a major difference in their respectiveTMPD/endogenous ratios. This difference is at-tributed to the extremely low endogenous respi-ratory Q(02) values that appeared to be charac-teristic for all ofthe Vibrio spp. analyzed in thisstudy.Table 2 presents the TMPD oxidase Q(02)

values for all other Kovacs oxidase-positivebacteria examined in this study. This physio-logically diverse group of bacteria possessescorrected TMPD oxidase Q(02) values rangingfrom 64 to 2,154 and the TMPD/endogenousratios ranging from 5 to 197. Azotomonas inso-lita had the highest TMPD oxidase Q(02) valueof any organism tested, a TMPD oxidase Q(02)of 2,154 and a TMPD/endogenous, ratio of 197.Included in Table 2 are the values obtainedfor two Micrococcus (Sarcina) luteus strains,which are considered oxidase variable (3, 4).Although the Kovacs oxidase test data showedthese two strains to be oxidase positive, theydid possess both the lowest corrected TMPDoxidase Q(02) values (64 and 105) and lowestTMPD/endogenous ratios (5 and 13) of all theoxidase-positive bacteria examined. Sporosar-cina (Sarcina) ureae exhibited a corrected

APPL. ENVIRON. MICROBIOL.

TMPD oxidase Q(02) value of 650 and a ratio of94, both values being significantly higher thanthose obtained for M. (Sarcina) luteus, the ge-nus with which it formerly was classified. Thisrelationship is similar to the situation alreadynoted for the Branhamella versus Neisseriaspp., which showed that the TMPD oxidaseassay is sensitive enough for recognition ofquantitative differences within genera.Table 3 presents data for the quantitative

TMPD oxidase reaction for organisms of thefamily Enterobacteriaceae that have beenknown to be oxidase-negative bacteria. The en-terobacteria exhibited low but still measurableTMPD oxidase Q(02) values; the correctedTMPD oxidation Q(02) values r4nged from 0 to33, and TMPD/endogenous ratios ranged from 1to 5. The TMPD oxidase Q(02) values for anumber ofEscherichia coli strains also are pre-sented. The data specifically show the extent towhich this quantitative reaction can varywithin a species that oxidizes TMPD at lowrates. The corrected TMPD oxidase Q(02) val-ues for the E. coli strains ranged from 5 to 33,and the TMPD/endogenous ratios ranged from2 to 5.Table 4 presents data for the quantitative

TMPD oxidase study using oxidase-negativebacteria, other than the enterobacteria. Thecorrected TMPD oxidase Q(02) values for theseorganisms ranged from 0 to 24, and the TMPD/endogenous ratios ranged from 1 to 5. Amongthese oxidase-negative bacteria, one finds theKovacs oxidase-variable species, M. (lysodeik-ticus) luteus, which possesses a TMPD oxidaseQ(02) value considerably lower than the twooxidase-positive M. luteus strains discussedpreviously (see Table 2). Also shown are thedata obtained for Bacillus spp., which are gen-erally considered to be oxidase variable (38).All Bacillus spp. examined were found to beoxidase negative, having low corrected TMPDoxidase Q(02) values ranging from 0 to 10 andratios ranging from 1 to 5. Surprisingly, Bacil-lus cereus did exhibit a relatively high TMPDoxidase value [Q(02) = 66], but when this valuewas corrected for the endogenous respiration[Q(02) = 56] no difference was noted in theanalyses of TMPD oxidase patterns.From the data presented in Tables 1 through

4, it is possible to resolve quantitatively thatpoint which separates the bacteria oxidase posi-tive by the Kovacs test from the oxidase-nega-tive ones. Oxidase-negative bacteria wouldhave to have both a corrected TMPD oxidaseQ(02) value no greater than 33 and a TMPD/endogenous ratio no greater than 5. These twoparameters have to be considered to evaluate


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on a quantitative basis whether or not an orga-nism is either oxidase positive or negative. Anyorganism having TMPD oxidation values justslightly higher (or lower) than these parame-ters would be most difficult to classify andwould be considered as a truly oxidase-variablespecies. Of all the organisms analyzed in thisstudy, none approached this point. The closestwere two V. parahaemolyticus strains, FC1011and SAK3, which exhibited low correctedTMPD oxidase Q(02) values of 34 and 27, re-spectively (see Table 1). However, the TMPD/endogenous ratios for these two strains were sohigh (35 and 28, respectively) that there was noquestion as to the extent that the TMPD oxi-dase reaction occurred. The exact opposite situ-ation exists for an oxidase-positive strain ofM.luteus (see Table 2). The TMPD/endogenousratio was low [Q(02) = 51, but the correctedTMPD oxidase Q(02) value was sufficientlyhigh [Q(02) = 641 so as to leave no doubt that ameaningful TMPD oxidation reaction had oc-curred.Figure 1 shows the relationship between the

corrected TMPD oxidase Q(02) values and theH-L 0/F test groups for each organism used inthis study. The abbreviations represent thebacterial generic names that are identified inTables 1 through 4. The 0/F groups are definedin the legend of Fig. 1 and reflect physiologicaldifferences in bacteria that are based on oxygenrequirements for growth on a complex mediumcontaining carbohydrate. Basically, the H-L 0/F test groups allow separation of bacteria thatexhibit an oxidative-type metabolic patternfrom those that are basically fermentative. Thecorrelation of the TMPD oxidase Q(02) value tothe H-L 0/F groups showed that high TMPDoxidase activity was found primarily in oxida-tive-type organisms, although some interestingexceptions were noted. The dashed line in Fig.1 separates the Kovacs oxidase-positive bacte-ria (all points above the line) from the oxidase-negative bacteria (all points below the line).The dashed line is discontinuous, to separatethe two oxidase-positive V. parahaemolyticusstrains from the oxidase-negative bacteria inH-L group Illa. From Fig. 1, it appears that thetwo V. parahaemolyticus strains have the samecorrected TMPD oxidase Q(02) values as threeoxidase-negative E. coli strains in group IIIband just slightly higher than those shown for P.maltophilia and Streptomyces griseus in groupI. This figure, however, does not take into ac-count the high TMPD/endogenous ratios thathave been discussed previously about these twoV. parahaemolyticus strains (see Table 1). Thedata shown in Fig. 1 are organized in summary

form in Table 5 with the genus and speciesname listed alphabetically for each of the H-Lgroups. The table also shows the ranges for thecorrected TMPD oxidase Q(02) values (footnotea) for each of the H-L groupings for both theoxidase-positive and -negative organisms.The bacteria in H-L group I produce alkaline

end products when grown on the H-L 0/F me-dia both under aerobic and anaerobic condi-tions. Classifying organisms in this group as"nonoxidizers" is misleading in that all orga-nisms in group I exhibit basically an oxidative(respiratory) metabolic pattern. Apparently,the inability ofmany organisms in this group tometabolize the test carbohydrates oxidativelycreates problems in characterizing them ac-cording to the H-L scheme. For this reason, thebacteria listed in H-L group I showed the leastcorrelation with TMPD oxidase activity, and itis quite apparent that listed among these bacte-ria are the asaccharolytic types, which areknown to be strongly oxidase positive andshown in this study to have high TMPD oxidaseQ(02) values. Included in this catagory wouldbe the three strains of B. catarrhalis, Alcali-genes faecalis, and Moraxella osloensis. H-Lgroup I also includes the oxidase-variable orga-nisms previously discussed that were reclassi-fied recently as M. luteus, M. (lysodeikticus)luteus, M. (Sarcina flava) luteus, and M. (Sar-cina) luteus. These bacteria exhibited quantita-tive TMPD oxidase Q(02) values coming closestto being oxidase variable. Among the orga-nisms in H-L group I are many bacteria thathave been known to possess an "oxidative" me-tabolism yet are oxidase negative and exhibitlow TMPD oxidase Q(02) values. The lowTMPD oxidase activity may result from (i) thelack of necessary electron transport compo-nents, i.e., type c cytochrome, as in the case ofP. maltophilia (37); (ii) the organism possessesa terminal or cytochrome oxidase componentthat cannot be assayed using TMPD as an elec-tron donor; and (iii) TMPD may be imper-meable to whole-cell oxidations in some bac-teria. Preliminary experiments in thislaboratory suggest that the latter possibilitymay be the case for most organisms of the orderActinomycetales (S. griseus, Mycobacteriumphlei, M. smegmatis, and M. fortuitum).The organisms listed in H-L group II have

the capability of oxidizing carbohydrates to acidend products only under aerobic conditions. Alloxidase-positive organisms in H-L group II areunquestionably"oxidative" bacteria, exhibitinghigh TMPD oxidase Q(02) values. Included arethe Pseudomonas spp. (P. aeruginosa, P. fluo-rescens, P. stutzeri, and P. acidovorans), Neis-

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TMPD OXIDASE - HL OXFERM CORRELATION STUDY

11HUmH - LEIFSON

111a Nib(OXFERM) GROWS

FIG. 1. TMPD oxidase -H-L 0IF test correlation study for heterotrophically grown bacteria. The bacteriasurveyed are grouped according to the results obtain for the quantitation ofthe TMPD oxidase and the H-L 0/

F test; the abbreviations are identified in Tables 1 to 4. The ordinate presents on a logarithmic scale the Q(02)value (microliters of 02 consumed per hour per milligram [dry weight] at 30 C) for TMPD oxidation, whichwas corrected by substraction of the endogenous respiration Q(02) value. The abscissa presents the H-L 0IFtest groups, which are based on the utilization ofoxygen during carbohydrate metabolism. The groups as de-fined by Hugh and Leifson (23) are: group I, nonoxidizers, nonfermenters; group II, oxidizers, nonfermenters;group IIIa, fermenters (anaerogenic); group IIIb, fermenters (aerogenic); and group IIIc, fermenters andoxidizers. None of the bacteria surveyed for TMPD oxidase was found to be in group IIIc. The dashed linerepresents that value established by the manometric TMPD oxidase reaction, which separates Kovacs oxidase-positive bacteria (all points above the line) from oxidase-negative bacteria (all points below the line).

seria spp., Rhizobium meliloti, and Achromo-bacter xerosis. Surprisingly, H-L group IIcontained oxidase-negative organisms (Table5), which, upon analysis, exhibited the ex-

pected low TMPD oxidase Q(02) values. For V.stercoraria, this low activity could be attrib-

uted to the lack of cytochrome c, although theterminal oxidase, cytochrome o, is present (8,30, 42). Nocardia asteroides whole cells willprobably be impermeable to TMPD, and no rea-

son, as yet, can be given for the lack ofTMPD oxidation by the other oxidase-negative

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TABLE 5. Summary ofTMPD oxidation rates ofbacteria correlated with H-L OIF test for both Kovacsoxidase-positive and -negative organisms

H-L group

Group I Group II Group lIla Group IIIb

Q(02) = 1,736 to 64a Q(02) = 2,154 to 280a Q(02) = 511 to 27a Q(02) = 139a

Oxidase positiveAlcaligenes faecalis Achromobacter xerosis Aeromonas (liquefaciens) Aeromonas hydrophilaBranhamella catarrhalis Agrobacterium tumefaciens hydrophila

(3)b Azotobacter vinelandii Vibrio parahaemolyticusMicrococcus (S.) luteus Azotomonas insolita (2)M. (S. flava) luteus Flavobacterium capsulatum V. parahaemolyticus bio-Moraxella osloensis Neisseria mucosa type alginolyticusNeisseria elongata N. siccaN. flava Rhizobium meliloti (2)Pseudomonas bathycetes Pseudomonas acidovoransSpirillum itersonii P. aeruginosaSporosarcina (S.) ureae P. fluorescens

P. stutzeri

Q(02) = 25 to 3a Q(02) = 17 to la Q(02) = 14 to 1l Q(02) = 33 to la

Oxidase negative

Acinetobacter calcoaceticus Acinetobacter calcoaceticus Bacillus cereus Enterobacter aerogenesCorynebacterium 7E1C Arthrobacter globiformis B. megaterium Escherichia coli (8)Micrococcus (lysodeikticus) Bacillus firmus B. pumilus Klebsiella pneumoniae

luteus Nocardia asteroides B. subtilis Proteus morganiiMycobacterium fortuitum Vitreoscilla stercoraria B. (vulgatus) subtilis P. vulgarisM. phlei Xanthomonas phaseoli Corynebacterium diphthe- Serratia marcescensM. smegmatis riae Salmonella typhimuriumPseudomonas maltophilia Escherichia coli (2)Streptomyces griseus Gaffkya tetragena (2)

Lactobacillus caseiStaphylococcus (albus)aureus

S. aureus (2)Pediococcus cerevisiaeStreptococcus faecalis (2)S. pneumoniaeS. pyogenesVibrio (metschnikovii)cholerae biotypeproteus

a Range of Q(02) values for the TMPD oxidation after correcting for the endogenous by subtraction.b Numbers in parentheses indicate the total number of strains that were analyzed for that organism.

bacteria falling into H-L group II. It would be ofinterest to examine these organisms to estab-lish which oxidation-reduction (or cytochrome)components are present that allow such bac-teria to be oxidative, yet lack the capabilityto oxidize TMPD.The bacteria of H-L group IIIa are recognized

by acid production without gas during growthat the expense of carbohydrates under bothaerobic and anaerobic conditions. Except forthree organisms, all bacteria in this group (i)possess a fermentative metabolic pattern, (ii)possess an oxidase-negative reaction, and (iii)show low TMPD oxidase activity. Although theTMPD oxidase activities were low for the fer-mentative organisms of this group, the assaystill was sensitive enough to allow further sepa-ration of the oxidase-negative organisms intotwo separate groups (see Fig. 1). Low, but stillmeasurable, TMPD oxidase activity was noted

for the organisms Corynebacterium diphthe-riae, Bacillus spp. (B. cereus, B. megaterium,and B. subtilis), and strains of Staphylococcusaureus, as well as others. However, there wereorganisms in this group that completely lackTMPD oxidation capabilities, among whichwere the Streptococcus spp. (Streptococcus pneu-moniae, S. pyogenes), P. cerevisiae, and Lacto-bacillus casei. The inability of these organismsto oxidize TMPD is undoubtedly due to the factthat they do not contain cytochrome compo-nents and thereby lack the conventional mem-brane-bound electron transport system(s)(7, 9).The unusual exceptions were three "fermen-

tative" bacteria that were oxidase positive andpossessed moderate to high TMPD oxidaseQ(02) values. Aeromonas liquefaciens had avery high TMPD oxidase Q(02) value, whereasthe three strains of V. parahaemolyticus

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(FC1011 and SAK3; V. parahaemolyticus bio-type alginolyticus) had moderately highTMPD oxidase activity (Tables 1 and 2). Noreason can be given at this time for the highTMPD oxidative activity for the fermentativegroup IIIa organisms A. (liquefaciens) hydro-phila.The bacteria found in H-L group IIIb produce

acid and gas from carbohydrate decompositionunder both aerobic and anaerobic conditions.Except A. hydrophila, H-L group IIlb con-tained essentially all bacteria of the family En-terobacteriaceae, which are unquestionably ox-idase negative, and possess low TMPD oxidaseQ(02) values. The fermentative enterobacteria,however, do have low, but still measurable,TMPD oxidase activity, with E. coli oxidizingTMPD at the upper limit observed for any oxi-dase-negative bacterium. The numerous E. colistrains analyzed show the actual extent of vari-ation that can occur, using the quantitativeTMPD oxidase assay described (Table 3).A. hydrophila was the only oxidase-positive

fermenter found in H-L group IIIb. Thisorganism was unquestionably oxidase posi-tive and possessed relatively high TMPD oxi-dase activity; a rate that was at least four timesgreater than that exhibited by the most activeE. coli strains. This finding is analogous to thatpreviously reported for A. (liquefaciens) hydro-phila, in H-L group IIIa, and is undoubtedly ameaningful one.None of the bacteria surveyed in this quanti-

tative TMPD oxidase assay fell into H-L groupIIlc, which originally included the "paracolon"bacteria (17).

DISCUSSIONThe principle underlying the microbiological

oxidase test is the visual analysis of a colorchange that results from the oxidation of a PPDderivative (with or without a-naphthol), causedby a cytochrome-dependent bacterial terminaloxidase reaction. A quantitative manometricprocedure is described herein that measuresthis same oxidase reaction by monitoring oxy-gen consumption as TMPD is oxidized by rest-ing-cell suspensions. Specifically, this TMPDoxidase reaction was utilized to measure termi-nal oxidase activity in a variety of physiologi-cally diverse bacteria. All resting-cell suspen-sions were prepared from organisms grown het-erotrophically, under identical nutritional con-ditions, and harvested at the late-logarithmicgrowth phase. Such conditions were kept con-stant to ensure that bacterial intracellular cy-tochrome type and content were characteristicof those commonly found in the logarithmic


growth phase. Bacterial heme content is knownto vary depending upon the environmental (orphysiological) growth conditions as well as thephase ofgrowth during which the bacterial pop-ulation is harvested (16, 24). For example, log-phase A. vinelandii strain 0 cells, grown onsucrose under nitrogen-fixing conditions, ex-hibited 10-fold lower TMPD oxidase Q(02) val-ues than did identically treated cells that weregrown on acetate as the sole source of carbon(25). Another report of this nature states thatthe oxidase-negative M. leprae becomes cyto-chrome oxidase positive when freshly testedisolates are analyzed from tissues (4).Another factor than can affect the TMPD

oxidation by bacterial whole cells is the perme-ability of the electron donor (TMPD) itself.Since TMPD oxidation is carried out by mem-brane-bound terminal oxidase(s) (2, 18), it isnecessary that TMPD penetrate the outer cellwall envelope. For both A. vinelandii and Neis-seria spp., reduced TMPD readily oxidized bythe intact whole cells at rates which indicatethat no surface permeability restrictions exist(23, 25). Conover (6) also has demonstrated thatreduced TMPD readily penetrates the nuclearmembrane barrier and is oxidized by prepara-tions of intact calf thymus nuclei. However,ferrocytochrome c oxidation could not be car-ried out in such preparations. The intact nucleipreparation remained impermeable to the nat-ural electron donor, cytochrome c. Although inthis study it was assumed that TMPD is perme-able for all oxidase-positive bacteria, the degreeof permeability could undoubtedly affect thequantitative Q(02) values obtained. The differ-ences in activity observed between some oxi-dase-positive and -negative bacteria must, inpart, reflect a measure ofpermeability kinetics.This possibility, however, does not detract fromthe usefulness of this quantitative TMPD oxi-dation reaction. Its use here is analogous to thequalitative oxidase test, except that it allowsone to establish the degree to which this reac-tion can occur in bacteria and use this value fortaxonomic purposes. High TMPD oxidase Q(02)values were found for Pseudomonas, Neisseria,and Vibrio spp., and this finding could havebeen predicted since these bacteria have longbeen suspected of possessing potent terminaloxidase (4, 15, 38). Among these organismswere two exceptions, P. maltophilia and V.(metschnikovii) cholerae biotype proteus. Bothbacteria previously have been reported as oxi-dase-negative species (4, 37) and as such wouldbe expected to have a low TMPD oxidase activ-ity, which they did (see Table 1). Without ex-ception, all oxidase-negative bacteria had low


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TMPD oxidation values (see Tables 3 and 4).Organisms formerly reported to be oxidase var-iable also were examined by this quantitativeoxidase test. By examining the oxidase-varia-ble strains ofM. luteus (3, 4), one could differ-entiate the oxidase-positive strains, formerlySarcina spp., from the oxidase-negative strainM. (lysodeikticus) luteus (Tables 2 and 4).From the data obtained, it was possible for

the first time to resolve quantitatively thatpoint which separates the oxidase-positive bac-teria from the oxidase negative. Oxidase-nega-tive bacteria have endogenous corrected TMPDoxidase Q(02) values of s33 and uncorrectedTMPD/endogenous ratios of c5, whereas alloxidase-positive bacteria have TMPD oxidaseQ(02) values and ratios greater than both ofthese parameters.As realized from the earlier studies, the oxi-

dase test has been particularly useful for taxo-nomic studies with gram-negative bacteria (4,38). Most gram-positive bacteria are oxidasenegative, and only a few are oxidase variable(38). This point was confirmed readily by thisquantitative TMPD oxidase study. The onlygram-positive bacterium exhibiting a highTMPD oxidase value was Sporosarcina ureae;the next most active was M. (Sarcina) luteus,which was considered oxidase variable (4). Allother gram-positive organisms were oxidasenegative, having low TMPD oxidase Q(02) val-ues. Interestingly, among the gram-positive or-ganisms, one finds the obligately aerobic bacte-ria, which must possess a respiratory mecha-nism (H-L groups I and II); yet these are oxi-dase negative and have low TMPD oxidaseQ(02) values. One such organism is M. phlei,which possesses both type c cytochrome(s) aswell as the terminal oxidase components, cyto-chromes a + a3 and o (5, 32, 33). The isolatedelectron transport particle of the oxidase-nega-tive M. phlei oxidized TMPD at rates of approx-imately 0.02 to 0.20 uatoms of 02 consumed permin per mg of protein (33). This activity is lowwhen compared with a similar type particleisolated from the oxidase-positive A. vinelan-dii, which oxidized TMPD at a minimal rate of4.1 ,uatoms of 02 consumed per min per mg ofprotein (19, 21, 24). These results suggest thatthe low TMPD oxidase activity in M. phlei isprobably due to the chemical nature (or compo-sition) of the electron transport system, whichjust does not allow for maximal TMPD oxida-tion. TMPD simply may not serve equally wellas an electron donor for all bacterial terminaloxidases. Many bacteria of the mycobacterial-nocardial group have the capability of growingon hydrocarbons (4, 22, 28), which implies that


they must possess active "oxygenating mecha-nisms"; yet they apparently are all oxidase neg-ative and lack the capability of utilizing oxygenfor TMPD oxidation. The other possibility, pre-viously mentioned, is that the mycobacterial-nocardial-type bacterial cells are impermeableto TMPD. Our data suggest that this might bethe case for many organisms of the order Acti-nomycetales. Preliminary studies in our labora-tory indicate that by merely sonicating resting-cell suspensions of M. smegmatis one can in-crease the uncorrected TMPD oxidase Q(02)value fivefold, the new Q(02) value obtained forthe ruptured cells now falling in a range [Q(02)= 90] commonly found for many oxidase-posi-tive organisms.

Finally, the quantitative TMPD oxidase as-say described is analogous to the cytochrome coxidase assay. There is evidence which indi-cates that type c cytochrome(s) is required forthe TMPD oxidation by bacterial electrontransport systems, as is the case for cytochromeoxidase activity of tissues and mitochondria(27). The organisms P. maltophilia and V. ster-coraria were examined because of reports thatboth lack type c cytochromes (8, 30, 37, 42).Both were found to be oxidase negative and hadlow TMPD oxidase Q(02) values (Tables 1 and4). A similar finding would be expected for thephytopathogen P. syringae, which also is anoxidase-negative pseudomonad that lacks typec cytochrome (35). The low TMPD oxidase val-ues noted for Xanthomonas spp. and V. (met-schnikovii) cholerae may eventually be ex-plained on the same basis, that both either lacktype c cytochrome(s) or are devoid of terminalcytochrome oxidase component(s). The exactopposite appears to be the case for Aeromonasspp. These organisms exhibit a fernentativemetabolism similar to those organisms classi-fied in the family Enterobacteriaceae; yet theyhave high TMPD oxidase values (Table 2), likePseudomonas spp. (4). Aeromonas spp. are alloxidase positive, as reported earlier in studiesusing the cytochrome oxidase test (14), and itwas this characteristic that allowed for its sepa-ration from other enterobacteria (11). The highTMPD oxidase values for Aeromonas spp. sug-gest that these organisms do possess type ccytochrome(8) when grown under aerobic condi-tions, unlike the type c cytochrome(s) noted forE. coli, which is induced only when the orga-nism is grown under anaerobic conditions, oraerobically in the presence of nitrate.There are reports which indicate that type c

cytochromes are actually found in aerobicallygrown E. coli (36), which might account for theTMPD oxidase activities noted for the various


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E. coli strains examined. E. coli was found tohave the highest TMPD oxidase values of anyof the oxidase-negative bacteria examined.This activity, although minimal, might reflectthe small concentration of cytochrome c that isactually present. One study (36) describes anoxidase-positive mutant that has been isolatedfrom E. coli Hfr strain H1060, which exhibitedsmall but distinct alpha-band absorption char-acteristics for cytochrome c in the 550- to 553-nm region. The quantitative TMPD oxidase re-action has yet to be performed for such an oxi-dase-positive E. coli mutant. Except for thisone report, there is no other evidence to suggestthat oxidase-positive mutants can be isolatedfrom a wild-type oxidase-negative strain. Bac-teria that completely lack cytochrome compo-nents (and therefore are deficient in terminaloxidases) would be unable to oxidize TMPD toany meaningful degree. In this study, such oxi-dase-negative bacteria are represented byStreptococcus spp., L. casei, P. cerevisiae, andGaffkya tetragena (Table 4).

ACKNOWLEDGMENTSWe wish to thank the following investigators for provid-

ing the bacterial cultures: B. Wesley Catlin, the MedicalCollege of Wisconsin (M C Wisconsin); Rita Colwell, Uni-versity of Maryland (U Maryland); the culture collection ofthe late J. W. Foster, University of Texas at Austin (UTAustin); Millicent C. Goldschmidt, University of TexasMedical School (UTHMS); S. Kester, North Texas StateUniversity (NTSU); T. S. Matney for the E. coli strains, andManley Mandel for all of the other cultures that were ob-tained from the University of Texas at Houston GraduateSchool of Biomedical Sciences (UTHGSBS); J. R. Postgate,University of Sussex (U Sussex); Reuben Wende, HoustonHealth Department (Houston H D); Robert C. Wood, Uni-versity of Texas Medical Branch at Galveston (UT Galves-ton); as well as the American Type Culture Collection(ATCC), Rockville, Md. We also acknowledge the valuabletechnical assistance of Olga Marcucci during the earlyphases of this survey study.

This investigation was supported by Public Health Ser-vice grant GM 17607-02 from the National Institute of Gen-eral Medical Sciences and Biochemical Sciences Supportgrant 5 S05 RR07147-03.

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