Modified Oxidase and Benzidine Tests for Separation of Staphylococci from Micrococci

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1 JOURNAL OF CLNCAL MCROBOLOGY, June 1981, p /81/ $02.00/0 Vol. 13, No. 6 Modified Oxidase and Benzidine Tests for Separation of Staphylococci from Micrococci ANTON FALLER AND KARL-HENZ SCHLEFER* Lehrstuhl fûr Mikrobiologie, Technische Universitat Munchen, 8000 Munich 2, Federal Republic of Germany Received 3 February 1981/Accepted 24 February 1981 Two simple and rapid methods for the separation of staphylococci from micrococci are described. They are based on modified oxidase and benzidine tests. Micrococci and Staphylococcus sciuri yield a blue color with a 6% solution of tetramethylphenylenediamine in dimethyl sulfoxide, whereas all of the other staphylococci exhibit no coloration. Best results were obtained with overnight cultures on blood agar. The presence of c-type cytochromes in micrococci and S. sciuri could be detected with benzidine; all noncovalently linked heme groups are removed before the addition of the benzidine reagent. The oxidase test is the simplest and most rapid method for the separation of staphylococci (except S. sciuri) from micrococci, if the nutritional requirements and the time of incubation are strictly followed. This test is especially recommended for the examination of clinical material in which S. sciuri is usually not found. The oxidation-fermentation test is the classical method for the separation of staphylococci from micrococci (31). However, this test does not provide clear results. t has been applied in the system of Baird-Parker (1, 2) and has led to numerous misclassifications of staphylococci as micrococci and vice versa (9, 16, 22). The guanosine and cytosine content of deoxyribonucleic acid (4, 14, 27) and the chemical composition of cell wall components (12, 13, 17-20, 23) are more reliable characters for distinguishing staphylococci from micrococci. However, determination of these characters is rather laborious and time consuming, and they are, therefore, not suitable for routine laboratory studies. A rapid test system was consequently developed (21) depending on the lysostaphin sensitivity of staphylococci. During the last few years further methods for routine separation of staphylococci and micrococci have been published, e.g., a serological approach (26), a phage adsorption test (25), and selective media (6, 24). n this paper, two even more simple and rapid methods based on modified oxidase and benzidine tests are described. casein, 5 g of yeast extract, 5 g of NaC, 5 g of glucose, 12.5 g of agar, and 1,000 ml of tap water (ph 7.5); (ii) peptone-yeast extract agar (PY), which is the same medium as listed 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 were incubated under aerobic conditions. (ài) Reagents and test. The following reagents were used for the test: tetramethylphenylenediamine (TMPD), tetramethylphenylenediamine-hydrochloride (TMPD-hydrochloride), dimethyl sulfoxide (DMSO), and sodium ascorbate. As soon as colonies formed on blood agar plates (approximately 15 to 18 h after inoculation), one loop 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 within 2 min. n the case of the other three media the oxidase test cannot be performed before 3 days of growth. Positive reaction occurred within 5 to 10 min, depending on the medium. The following solutions were prepared for the oxidase reaction: 1% TMPD in DMSO, 1% TMPDhydrochloride in H20 (15), 1% TMPD-hydrochloride in H20 plus 0.1% sodium ascorbate (30), 6% TMPDhydrochloride in H20, and 6% TMPD in DMSO. Benzidine test. (i) Culture conditions. The strains were cultivated on PYG under the same conditions as for the oxidase test. MATERALS AND METHODS Oxidase test. (i) Culture conditions. Strains of (ii) Test and reagent. Two loops of a bacterial staphylococci and micrococci (see Table 1) were cultivated on blood agar with the following composition: mixture of 1 N trichloroacetic acid and 1 N perchloric colony were suspended in 1 ml of a 1:1 (vol/vol) standard 1 nutrient agar (E. Merck AG, Darmstadt, acid (HC104). The suspension was thoroughly mixed, Germany, article no. 7881) and 7% sheep blood. As a incubated for approximately 3 min, and centrifuged in comparison, three other media were tested, which are an Eppendorf centrifuge, and the supernatant fluid used in routine laboratories: (i) peptone-yeast extractglucose agar (PYG) consisting of 10 g of peptone from ml of distilled water and poured into 40 ml of an icewas discarded. The pellet was then homogenized in

2 1032 FALLER AND SCHLEFER cold acetone-24 N HCl mixture (49:1, vol/vol) under vigorous stirring. The extraction procedure takes 10 to 15 min, after which the bacterial material is sucked through a filter paper disk (diameter, 0.9 cm; Schleicher & Schull, Dassel, Germany, no ). The benzidine test was then performed, by the method of Deibel and Evans (7), using benzidine base instead of benzidine-hcl. A blue coloration on the filter paper is designated as benzidine positive. RESULTS Oxidase test. Ail of the strains of micrococci and staphylococci were tested for oxidase activity by the application of various TMPD solutions. t was found that in the case of a 6% solution of TMPD in DMSO all micrococci turned blue, whereas no coloration was observed on staphylococci, with the exception of S. sciuri strains. The disadvantages of the other reagents are shown in Table 1. Moreover, the TMPD derivatives solubilized in DMSO were stable at TABLE 1. room temperature for several weeks, when the solution was protected against light. Aqueous solutions, on the other hand, are rather autoxidizable and must be freshly prepared for every test. Because of this autoxidation, even most oxidase-negative bacteria turned blue after 5 min. The addition of 0.1% sodium ascorbate (30) to prevent autoxidation is inadvisable, since nearly all strains provided negative results due to the strong reducing power of sodium ascorbate. Different media also affected the oxidase reaction (Table 2). Better growth could be observed on agars containing glucose than on glucose-deficient media, and the oxidase reaction was more pronounced. On PYG, PY, and PC media, the oxidase reaction should be carried out after 3 days at the earliest. The blue coloration then occurred within 5 min and, in the case of PC agar, within 10 min. Best results were nfluence of different concentrations and solutions of TMPD (and DMPD) on the oxidase reaction' Oxidase reaction J. CLN. MCROBOL. 1% 6% 1% TMPD- 1% Strain' 6% 1% TMPD- TMPD- hydro- DMPD- TMPD in TMPD in hydro- hydro- chloride hydrochlo- DMSO DMSO chloride chloride in H20 ride in in H20 in H20 plus H120 (13) ascorbate M. luteus CCM 169 +C WC + + w + M. lylae ATCC w + M. roseus W. Back H C - w M. nishinomiyaensis W. Kloos KL w - + M. sedentarius W. Kloos TW93 + w + M. kristinae ATCC w - + M. varians CCM w w S. saprophyticus CCM w - - ND(' S. xylosus DSM w - - ND S. cohnii DSM ND S. haemolyticus ATCC w - - ND S. hominis ATCC w - - ND S. epidermidis ATCC ND S. capitis ATCC ND S. warneri ATCC ND S. simulans ATCC ND S. intermedius CCM ND S. aureus ATCC w -- - ND S. hyicus NCTC ND S. sciuri ATCC ND S. sciuri ATCC ND All strains were cultivated on PYG agar. DMPD, N,N,-dimethyl-p-phenylenediamine monohydrochloride. Sources of strains are as follows: ATCC, American Type Culture Collection; W. Back, Technische Universitat München, Weihenstephan, Germany; CCM, Czechoslovak Collection of Microorganisms; DSM, Deutsche Sammlung für Mikroorganismen; W. Kloos, North Carolina State University, Raleigh, N.C.; NCTC, National Collection of Type Cultures. +, Positive oxidase reaction; -, negative oxidase reaction; w, weakly positive oxidase reaction. dnd, Not done.

3 VOL. 13, 1981 TABLE 2. nfluence of different media on the oxidase reaction Oxidase reaction 6% TMPD in DMSO 6% TMPD- Strain Strain PYG Blood ~~~~~~~hydro- chloride PY PY (3 PC (3 agar in H20 days) days) days) (18- (blood 18 h) agar, 15- ~~~18 h) 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. sciuri subsp sciuri S. sciuri subsp. + + _ + + lentus a ncubation times are given within parentheses. +, Positive oxidase reaction; -, negative oxidase reaction; w, weakly positive oxidase reaction. obtained with overnight cultures on blood agar. A growth of 15 to 18 h was sufficient to obtain precise results, and a positive oxidase reaction should appear within 2 min on filter paper. Moreover, one should not use cultures from blood agar plates that are more than 24 h old, since many staphylococci react positively after a prolonged incubation time. Besides the type strains of micrococci and staphylococci (Table 1), 310 gram-positive, catalase-positive cocci isolated from milk, cheese, and dried sausage were tested with the oxidase reagent. Based on the 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 test within the period of 15 to 18 h. Benzidine test. n 1960, Deibel and Evans distinguished cytochrome-containing bacteria _ SEPARATON OF STAPHYLOCOCC FROM MCROCOCC 1033 from those lacking cytochromes 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 method described here is not identical to that of Deibel and Evans (7). To remove all heme-containing proteins except c-type cytochromes, the ceils have to be treated beforehand with trichloroacetic acid-hcl04 and acetone-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 benzidine base, benzidine-hcl was also tested, but this substance was less sensitive and revealed unuseful results since many micrococci reacted negatively. The more sensitive benzidine base was therefore applied in our test 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. whole cells 0.5 ml of trichloroacetic acid (2 N) ml of HC104 (2 N) + homogenize incubation, 3 min centrifuge supernatant fluid pellet (discard) (homogenize in 1 ml of distilled water) filtrate (discard) 40 ml of acetone-hcl (24 N) (49:1 [vol/vol]) extraction, 10 to 15 min s suck through a filter pellet (on the filter) benzidine reagent blue color no color: micrococci and staphylococci S. sciuri FG. 1. Separation of micrococci and staphylococci by means of the benzidine reagent.

4 1034 FALLER AND SCHLEFER DSCUSSON The oxidase reaction is a valuable diagnostic method in separating oxidase-positive Pseudomonas species from oxidase-negative members of the family Enterobacteriaceae (15, 29). Nothing, however, is known about the oxidizing enzymes or enzyme systems. At first it was thought that this reaction depended on peroxidases (10), and it was thought to depend then on cytochrome oxidases (11, 29). Later, Stanier et al. (28) suggested that the oxidase test is an indirect proof for the presence of a c-type cytochrome. This seems to be in accordance with the cytochrome patterns of micrococci and staphylococci (8), since all micrococci contain cytochrome c, whereas all staphylococci with the exception of S. sciuri lack this type of cytochrome. n previous reports, in which oxidase activity of micrococci and staphylococci was tested by using 1% aqueous solutions of TMPD-hydrochloride, 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 reacted positively. t is possible that the cell envelope of the gram-positive bacteria inhibits the permeation of the aqueous solution. For these reasons, TMPD was dissolved in DMSO, a substance which can rapidly permeate cell envelopes, and, in addition, the concentration of TMPD was increased from 1 to 6%. Furthermore, the oxidase reaction of the organisms can be influenced by the growth medium. Very little is known about this topic (3, 30). t was found that growth in the presence of glucose tends to result in a stronger oxidase reaction (Table 2). PC medium is suitable for the test, but its disadvantages are the long cultivation time required (3 days) and the slow development (10 min) of a positive reaction. On blood agar, however, the test can be performed in only 15 to 18 h, and a positive reaction appears within 2 min. Therefore, no false results are obtained due to autoxidation. Since most strains exhibit good growth on blood agar, its application in routine clinical laboratories should be possible. Simultaneously, the occurrence of hemolysis can be observed. The second method described in this paper is a modification of the benzidine test. The benzidine test has been applied to detect the presence of cytochromes (7). Both staphylococci and micrococci contain cytochromes. However, as has previously been shown (8), only micrococci and S. sciuri exhibit c-type cytochromes. These are respiratory chain proteins in which the heme J. CLN. MCROBOL. groups are covalently linked to the protein moiety. For this reason, the only heme-containing proteins remaining in the cells after treatment with trichloroacetic acid-hcl04 and acetone- HCl are c-type cytochromes. They can be detected with benzidine. The modified oxidase and benzidine tests provide the simplest and most rapid methods for separating staphylococci from micrococci. They are ideal for routine clinical laboratories since they do not require expensive chemicals or equipment. Since S. sciuri is usually not found in clinical material, the sole application of one of the two tests is sufficient to provide a clear separation of staphylococci from micrococci. f the nutritional requirements and the time of incubation are strictly followed, the method of choice is the oxidase test. This is the simplest and most rapid method, in particular if one uses cultures grown overnight on blood agar. A combination of lysostaphin and modified oxidase or benzidine tests is recommended if S. sciuri is also encountered in the test sample, e.g., in samples from certain animals. S. sciuri can easily be identified by its sensitivity to lysostaphin and production of acid, aerobically, from D-(+)-cellobiose or D-(+)-fucose or both. ACKNOWLEDGMENTS This work was supported by a grant from the Deutsche Forschungsgemeinshaft. We are grateful to V. Fowler for reading the manuscript. LTERATURE CTED 1. Baird-Parker, A. C A classification of micrococci and staphylococci based on physiological and biochemical tests. J. Gen. Microbiol. 30: Baird-Parker, A. C The classification of staphylococci and micrococci from world-wide sources. J. Gen. Microbiol. 38: Billing, E The bacterial genera Pseudomonas and Achromobacter. Nature (London) 188: Bohaeek, J., M. Kocur, and T. Martinec DNA base composition and taxonomy of some micrococci. J. Gen. Microbiol. 46: Boswell, P. A., G. F. Batstone, and R. G. 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