Modified oxidase and benzidine tests for separation of staphylococci from micrococci

JOURNAL OF CLINICAL MICROBIOLOGY, June1981, p. 1031-1035 0095-1137/81/061031-05$02.00/0

Vol.13, No. 6

Modified Oxidase and

Benzidine Tests for Separation

of

Staphylococci

from

Micrococci

ANTON FALLER ANDKARL-HEINZ SCHLEIFER*

LehrstuhlfûrMikrobiologie, Technische Universitat Munchen,8000Munich2,FederalRepublic of Germany

Received 3February1981/Accepted24February1981

Two simple and rapid methods for the separation of staphylococci from micrococciaredescribed. Theyarebasedonmodified oxidaseandbenzidinetests.

Micrococci and Staphylococcus sciuri yield a blue colorwith a 6% solution of tetramethylphenylenediamine in dimethyl sulfoxide, whereas all of the other staphylococci exhibit no coloration. Best results were obtained with overnight culturesonbloodagar. Thepresenceofc-typecytochromesinmicrococciandS. sciuri could bedetected with benzidine; all noncovalently linked hemegroups are removed before the addition of the benzidine reagent. The oxidase test is the simplest andmost rapid method for the separation of staphylococci (except S.

sciuri)frommicrococci, if thenutritional requirements and the time of incubation

arestrictlyfollowed. Thistestis especially recommended for the examination of clinicalmaterial inwhich S. sciuri is usuallynotfound.

The oxidation-fermentation testisthe classi-cal method for theseparation ofstaphylococci from micrococci (31). However, this test does notprovide clearresults.It has beenappliedin

thesystemof Baird-Parker (1,2) and has ledto numerousmisclassifications ofstaphylococcias

micrococci and vice versa (9, 16, 22). The

gua-nosine andcytosinecontentofdeoxyribonucleic acid (4, 14,27) and thechemicalcomposition of cell wallcomponents(12,13, 17-20,23) are more

reliable characters for distinguishing staphylo-cocci frommicrococci. However,determination ofthese characters is ratherlaborious andtime

consuming,andtheyare,therefore,notsuitable forroutine

laboratory

studies.Arapidtest sys-tem wasconsequentlydeveloped (21) depending

onthe lysostaphin sensitivity of

staphylococci.

During the last few years further methods for routine

separation

of

staphylococci

and

micro-cocci have been

published,

e.g., a

serological

approach

(26),a

phage adsorption

test

(25),

and

selective media

(6, 24).

Inthis paper, twoeven more

simple

andrapid methods basedon

modi-fied oxidase and

benzidine

tests aredescribed.

MATERIALS AND METHODS

Oxidasetest. (i) Culture conditions. Strainsof

staphylococciand micrococci (seeTable 1) were

cul-tivatedonblood agar withthefollowing composition:

standard 1 nutrient agar (E. Merck AG,Darmstadt,

Germany, articleno. 7881)and 7%sheepblood.Asa

comparison,three other mediaweretested,whichare

usedin routinelaboratories:(i)peptone-yeast

extract-glucose agar(PYG) consistingof10g of peptone from

casein, 5 g of yeast extract, 5 g ofNaCI, 5 g of glucose,

12.5g of agar, and 1,000 ml of tap water (pH 7.5);(ii)

peptone-yeast extract agar (PY), which is the same

mediumaslisted above, but without glucose; and (iii)

plate-count agar(PC)(E. Merck AG, article no. 5463).

The growth temperature was 30°C, and the strains

wereincubated underaerobicconditions.

(ài) Reagents and test. The following reagents

were used for thetest:tetramethylphenylenediamine

(TMPD),

tetramethylphenylenediamine-hydro-chloride (TMPD-hydrotetramethylphenylenediamine-hydro-chloride), dimethyl sulfoxide (DMSO), and sodium ascorbate.

As soon as colonies formed on blood agar plates

(approximately15to 18hafter inoculation), oneloop

of bacteria was smeared onto ordinary filter paper.

One drop of 6% TMPD in DMSO was added onto the bacterial material. Oxidase-positive bacteria turn dark

blue within2min.

In thecaseofthe other three media the oxidasetest

cannotbeperformedbefore3days ofgrowth. Positive

reactionoccurred within5 to 10min,dependingonthe

medium. The following solutions were prepared for

the oxidase reaction: 1% TMPD inDMSO, 1%

TMPD-hydrochlorideinH20 (15), 1%TMPD-hydrochloride

inH20 plus 0.1% sodium ascorbate (30), 6%

TMPD-hydrochloride inH20, and 6% TMPD in DMSO.

Benzidine test. (i) Culture conditions. The

strainswerecultivatedonPYG underthesame

con-ditions as for the oxidase test.

(ii) Test and reagent. Two loops of abacterial

colony were suspended in 1 ml of a 1:1 (vol/vol)

mixtureof1Ntrichloroacetic acidand 1Nperchloric

acid(HC104). Thesuspensionwasthoroughlymixed,

incubatedforapproximately3min,andcentrifugedin

an Eppendorf centrifuge, and the supernatant fluid

wasdiscarded.Thepelletwasthenhomogenizedin 1

ml ofdistilledwaterandpouredinto 40 mlofan ice-1031

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cold acetone-24 NHCl mixture (49:1, vol/vol) under vigorous stirring.The extractionproceduretakes 10to 15 min, after which the bacterial material is sucked through a filter paper disk (diameter, 0.9 cm;

Schleicher & Schull, Dassel, Germany, no. 309015). The benzidinetestwasthenperformed, bythemethod of Deibeland Evans(7),usingbenzidine baseinstead of benzidine-HCl.A blue colorationonthe filterpaper

is designatedasbenzidinepositive. RESULTS

Oxidase test. Ail of the strains ofmicrococci

andstaphylococciweretested foroxidase activ-ity by the application of various TMPD

solu-tions. It was found that in the case of a 6%

solution of TMPD in DMSO all micrococci

turnedblue,whereasnocolorationwasobserved

onstaphylococci,with theexceptionofS. sciuri

strains. Thedisadvantagesofthe otherreagents

are shown in Table 1. Moreover, the TMPD

derivativessolubilized inDMSOwerestable at

room temperature for several weeks, when the solution was protected against light. Aqueous

solutions, onthe other hand, arerather

autoxi-dizable andmustbe freshly prepared for every

test. Because of this autoxidation, even most oxidase-negative bacteria turned blue after 5 min.The addition of0.1% sodium ascorbate (30)

to prevent autoxidation is inadvisable, since

nearlyall strains provided negative resultsdue

to the strong reducingpower of sodium

ascor-bate.

Different media also affected the oxidase

re-action (Table 2). Better growth could be ob-servedonagarscontaining glucose thanon

glu-cose-deficient media, and the oxidase reaction

was more pronounced. On PYG, PY, and PC

media, the oxidase reaction should be carried outafter 3daysattheearliest. The blue color-ationthen occurredwithin 5 minand,inthecase

of PC agar, within 10 min. Best results were

TABLE 1. Influenceofdifferent concentrationsandsolutionsofTMPD(andDMPD)ontheoxidase reaction'

Oxidase reaction 1%

6% 1% TMPD- 1%

Strain' 6% 1% TMPD- TMPD- hydro-

DMPD-TMPDin TMPDin hydro- hydro- chloride hydrochlo-DMSO DMSO chloride chloride inH20 ride in

inH20 in H20 plus H120(13) ascorbate

M.luteus CCM169 +C WC + + w +

M.lylae ATCC27566 + + + + w +

M. roseus W.BackH15 + + + -C - w

M.nishinomiyaensis W.KloosKL146 + + + w - +

M.sedentarius W. Kloos TW93 + w +

M.kristinae ATCC27570 + + + w - +

M.varians CCM884 + + + - w w

S.saprophyticus CCM883 - - w - - ND('

S.xylosus DSM20266 - - w - - ND

S. cohnii DSM20260 - - - ND

S.haemolyticus ATCC20263 - - w - - ND

S.hominis ATCC27844 - - w - - ND

S.epidermidis ATCC 14990 - - - ND

S.capitis ATCC27840 - - - ND

S.warneri ATCC27836 - - - ND

S.simulans ATCC27848 - - - ND

S.intermedius CCM5739 - - - ND

S. aureus ATCC 12600 - - w -- - ND

S.hyicus NCTC10350 - - - ND

S. sciuri ATCC 29062 + - + + - ND

S. sciuri ATCC 29070 + - + - - ND

All strains werecultivated on PYG agar. DMPD,N,N,-dimethyl-p-phenylenediaminemonohydrochloride.

Sourcesof strains are asfollows: ATCC, American Type Culture Collection; W. Back, Technische

Universitat

München, Weihenstephan, Germany; CCM, Czechoslovak Collection of Microorganisms; DSM, Deutsche

SammlungfürMikroorganismen; W.Kloos, NorthCarolina State University, Raleigh, N.C.; NCTC,National

CollectionofTypeCultures.

+,Positiveoxidasereaction; -, negative oxidase reaction; w, weakly positiveoxidase

reaction.

dND,

Notdone.

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SEPARATION OF STAPHYLOCOCCI FROM MICROCOCCI 1033

TABLE

2. Influenceof

different

media on the oxidase reaction

Oxidasereaction 6% TMPD inDMSO 6%

TMPD-Strain

~~~~~~~hydro-Strain PYG Blood chloride

PY PY (3 PC (3 agar inH20

days) days) days)

(18-

(blood

18h) agar,

15-~~~18h)

M.luteus + w + + +

M.lylae + + + + +

M.roseus + + + + +

M.nishinomiyaen- + + + + +

sis

M.sedentarius + w + + +

M.kristinae + + + + +

M.varians + + + + +

S. saprophyticus - - -

-S.xylosus - - - - w

S. cohnii - - - -

-S.haemolyticus - - - - w

S.hominis - - -

-S.epidermidis - - - - w

S.capitis - - - - _

S. warneri - - -

-S. simulans - - - - +

S.intermedius - - - - w

S.aureus - - -

-S.hyicus - w - - w

S.sciurisubsp. + + + + +

sciuri

S.sciurisubsp. + + _ + +

lentus _

a Incubation timesaregivenwithin

parentheses.

+,

Positiveoxidasereaction; -, negative oxidase reaction;

w,

weakly positive

oxidase reaction.

obtained withovernight culturesonbloodagar. Agrowth of15to 18 h wassufficienttoobtain precise results, and a

positive

oxidase reaction should appear within 2 min on

filter

paper.

Moreover, one should not use cultures from blood agarplates that are morethan24 h

old,

since many

staphylococci

react

positively

after

a

prolonged

incubationtime.

Besides the type strains of micrococci and

staphylococci

(Table 1),

310

gram-positive,

cat-alase-positive cocci isolated from

milk, cheese,

and driedsausagewere tested with theoxidase reagent. Based onthe oxidase test,302 strains

could be

properly

identified,

whereas

only

eight

isolates

(2.3%)

were misclassified. Most of the

wrongly

identified strains had grown

poorly

on the blood agar, so that there was not

enough

material for the testwithin the

period

of15 to 18h.

Benzidine test. In

1960,

Deibel and Evans

distinguished

cytochrome-containing

bacteria

fromthose lackingcytochromes by means of the benzidine reagent. This substance reacts with the heme groups, producing a blue color. The reaction is rather sensitive, and even small

amounts of cytochromes can be detected. The methoddescribed here is notidenticaltothat of

Deibeland Evans (7). Toremove all heme-con-taining proteins except c-typecytochromes,the ceilshave to be treatedbeforehandwith trichlo-roaceticacid-HCl04andacetone-HCl.The exact procedure for carrying out the test is shown in Fig. 1. All micrococci and strains of S. sciuri exhibited a positive reaction, whereas the other

staphylococci reacted negatively. Besides ben-zidine base, benzidine-HCl was also tested, but this substance was less sensitive and revealed unuseful results since manymicrococcireacted negatively. The more sensitive benzidine base wasthereforeapplied in ourtest system.

There are no special nutritional requirements; every medium which is free of azide and cyanide and which is suitable for good growth can be used.

wholecells

0.5ml oftrichloroacetic acid (2 N)

+

0.5mlofHC104 (2 N)

+

homogenize

incubation,3 min

centrifuge

supernatant fluid pellet

(discard) (homogenize

in 1ml ofdistilled water)

I

40mlofacetone-HCl(24N)

(49:1[vol/vol])

I

extraction, 10 to 15min

s

suckthroughafilter

I

filtrate(discard) pellet

(onthefilter)

I

benzidinereagent

blue color nocolor:

micrococci and staphylococci

S. sciuri

FIG. 1. Separation of micrococci and

staphylo-coccibymeansofthe benzidinereagent.

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DISCUSSION

The oxidase reaction isavaluable

diagnostic

methodin separating

oxidase-positive

Pseudo-monasspecies from oxidase-negative members of thefamily Enterobacteriaceae

(15, 29).

Noth-ing, however, isknown about the oxidizing en-zymes or enzyme systems.At first itwasthought thatthis reactiondependedonperoxidases (10), and it was thought to depend then on

cyto-chrome oxidases (11, 29). Later, Stanier et al.

(28)suggestedthat theoxidasetestisanindirect prooffor the presence ofa c-type cytochrome. This seems to be inaccordance with the

cyto-chromepatternsof micrococciand

staphylococci

(8), sinceall micrococci contain cytochrome c, whereasall staphylococciwith theexceptionof S. sciuri lack this typeofcytochrome.

Inpreviousreports, in whichoxidase

activity

of micrococci and staphylococci was tested by

using 1% aqueous solutions of TMPD-hydro-chloride, no distinct

separation

of micrococci and

staphylococci

could be achieved

(5,

13,

30).

The concentration of TMPD was too low to

yield a positive reaction with all micrococci.

Only extremely oxidase-active organisms

re-acted

positively.

It is

possible

that the cell

en-velope of thegram-positivebacteriainhibits the permeation ofthe aqueous solution. For these reasons, TMPDwasdissolvedinDMSO,a sub-stance which can

rapidly

permeate cell enve-lopes, and, in addition, the concentration of

TMPD was increased from 1 to 6%.

Further-more,theoxidasereaction oftheorganismscan beinfluencedbythegrowth medium. Very little

is known about this topic (3, 30). Itwas found

thatgrowthin the presence ofglucosetends to result in a stronger oxidase reaction (Table 2).

PC medium issuitablefor the test, but its

dis-advantages are the long cultivation time

re-quired (3 days) and the slow development (10

min)ofapositivereaction. On bloodagar, how-ever, the testcanbeperformedinonly15 to 18

h,and apositive reaction appears within 2 min.

Therefore, no falseresults are obtained due to autoxidation. Since most strains exhibit good

growth onbloodagar, its application in routine clinical laboratories should be possible.

Simul-taneously, the occurrence of hemolysis can be observed.

The secondmethod described in this paper is amodificationof the benzidine test. The benzi-dine testhas beenapplied to detect the presence ofcytochromes(7). Both staphylococci and mi-crococci containcytochromes. However, as has

previouslybeen shown (8), only micrococci and S.sciuri exhibit c-type cytochromes. These are

respiratory chain proteins in which the heme

groups arecovalently linked to the protein

moi-ety. For this reason, the only heme-containing proteins remaining in the cellsafter treatment with trichloroacetic acid-HCl04 and

acetone-HCl

are c-type cytochromes. They canbe de-tected withbenzidine.

The modified oxidase andbenzidinetests pro-vide the simplest and most rapid methods for separatingstaphylococci from micrococci. They are ideal for routine clinical laboratories since they do not require expensive chemicals or

equipment. Since S. sciuriis

usually

notfound

in clinical material, thesole application ofone of the two tests is sufficient toprovide a clear separation of staphylococci from micrococci. If

the nutritional requirements and the time of incubation are strictly followed, the methodof

choice is the oxidase test. This is the simplest andmostrapid method,inparticular ifone uses

culturesgrownovernightonbloodagar. A

com-bination oflysostaphin and modified oxidase or

benzidine tests is recommended ifS. sciuri is also encountered in the test sample, e.g., in samples from certain animals. S. sciurican

easily

be identifiedbyits sensitivitytolysostaphinand

production of acid, aerobically, from D-(+)-cel-lobiose orD-(+)-fucose or both.

ACKNOWLEDGMENTS

This workwassupported byagrantfrom the Deutsche Forschungsgemeinshaft.

WearegratefultoV.Fowler forreading the manuscript. LITERATURE CITED

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