Use Quantitative Oxidase Test Characterizing Oxidative ... oxidase-positive and -negative bacteria

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

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

    Use of a Quantitative Oxidase Test for Characterizing Oxidative Metabolism in Bacteria PETER 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 bacterial resting-cell suspensions and demonstrate the usefulness of this reaction for taxonomic purposes. Resting-cell suspensions of physiologically diverse bacteria were examined for their capabilities of oxidizing N,N,N',N'-tetramethyl-p- phenylenediamine (TMPD) using a manometric assay. For organisms having this capability, it was possible to calculate the conventional TMPD oxidase Q(02) value (microliters of 02 consumed per hour per milligram [dry weight]). All cultures were grown heterotrophically at 30 C, under identical nutritional conditions, and were harvested at the late-logarithmic growth phase. The TMPD oxidase Q(02) values showed perfect correlation with the Kovacs oxidase test and, in addition, it was possible to define quantitatively that point which- separated oxidase-positive from oxidase-negative bacteria. Oxidase-negative bacteria 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. This quantitative study also demonstrated that organisms that (i) lack a type c cytochrome, or (ii) lack a cytochrome-containing electron transport system, like the 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 oxidative relationships 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), are basic dyes that can function as biological elec- tron donors. Both derivatives have been used extensively in microbiological studies as quali- tative indicators of oxidase activity (10, 14, 15, 29). Originally Gordon and McLeod (15) found the oxidase test to be particularly useful for the rapid identification ofNeisseria gonorrhea and Vibrio cholerae. Kovacs (29) standardized the oxidase test procedure by using the more sensi- tive TMPD derivative and defined the time in- terval (10 s) during which a bacterial colony must turn blue in color to be considered oxidase positive. Gaby and Hadley (14), using a test tube cytochrome oxidase test, monitored the oxidation of DMPD-oxalate, in the presence of a-naphthol, and obtained results comparable to the Kovacs oxidase test (13). The taxonomic importance of the qualitative

    oxidase test has become apparent from the studies 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 using the oxidase test. Steel (38) examined 1,660 orga- nisms of various genera and showed the Kovacs oxidase reaction to be a useful taxonomic tool. The oxidase test also has been used in studies with 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 the PPD a-naphthol oxidation reaction in tissues; in this instance, it is more commonly referred to as the indophenol oxidase reaction (27, 40, 41). The early attempts at quantitating the TMPD oxidase reaction in bacteria were hindered by the complexity of the reaction and its uncertain relationship to catalase and per- oxidase activity (12, 31). Yamagutchi (43) im- proved upon the quantitation procedure and measured manometrically both the PPD and DMPD oxidation reactions, which he related to


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  • VOL. 31, 1976

    the dry weight of the bacterial cells. Oxidase- positive bacteria exhibited higher activities for PPD 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 viable cell count. Most recently the quantitative man- ometric TMPD oxidase analysis has been ex- tended to measure the potent terminal oxidase activity in Azotobacter vinelandii (25), where it has shown that the TMPD oxidation kinetics by whole cells were remarkably similar to those obtained for the electron transport particle (19). Reduced TMPD readily penetrates the intact A. vinelandii cell, and its oxidation by the electron transport system is comparable to the terminal oxidase reaction measured by cytochrome c oxi- dation (20, 24). This same quantitative TMPD oxidation reaction was also used successfully for 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 TMPD oxidase reaction could be used as a quantitative index for establishing the extent that terminal oxidase activity occurred in a large variety of oxidase-positive and -negative bacteria. Sev-


    enty-nine bacterial strains, representing 36 genera, were examined quantitatively for their capabilities of oxidizing TMPD; the results were then correlated to the data obtained for both the Kovacs oxidase test and the Hugh- Leifson Oxferm (H-L O/F) test. For the latter test, an attempt was made to establish whether or not TMPD oxidation could be correlated to the different oxidative and/or fermentative metabolic patterns exhibited by bacteria (17).


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

    Resting-cell suspensions were prepared from bac- teria grown at 30 C in nutrient broth (Difco), which contained 1% (wt/vol) sucrose supplemented with 0.5% (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 stercoraria was grown on this same medium, which was supple- mented with 0.25% (wt/vol) K2HPO4, and glucose was substituted for sucrose. All seed cultures were allowed adequate time to adapt on the above me- dium, and batch cultures were prepared in 400-ml quantities, 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 commonly oxidase positive

    Q(02) valuer Abbrevi-

    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 1,736 110 B. catarrhalis Gp4 U Maryland Nc 14 1,612 1,598 115 B. catarrhalis NC31 U Maryland Nc 17 1,630 1,613 96

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

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

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

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


    a Names in parentheses indicate the nomenclature used in Bergey's Manual, 7th ed. All numbers in parentheses are the ATCC 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) valuec Abbrevi-

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

    Endog og Achromobacter xerosis (14780) ATCC Ax 14 407 393 29 Aeromonas hydrophila (9071) UTHGSBS Ah 12 151 139 13 A. liquefaciens (14715) UTHGSBS Al 23 534 511 23 Agrobacterium tumefaciens (15955) ATCC Agt 12 1,686 1,674 141 Alcaligenes faecalis (8750) ATCC Alf 16 1,083 1,067 68 Azotobacter vinelandii 0 (12518) U Houston Av 19 1,221 1,202 64 Azotomonas insolita (12412) ATCC Ai 11 2,165 2,154 197 Flavobacterium capsulatum (14666) ATCC Fc 38 318 280 8 Micrococcus (Sarcina) luteus U Houston Si 16 80 64 5 M. (S. flava) luteus UTHGSBS Sf 9 114 105 13 Moraxella osloensis U Maryland Mo 8 1,001 993 125 Rhizobium meliloti F-2