20500/20509 008038-4 02/02

For in vitro diagnostic use

Identification system for staphylococci and micrococci API STAPH is an identification system for the genera Staphylococcus Kocuria and Micrococcus, using standardized and miniaturized biochemical tests with a specially adapted database. The complete list of those that it is possible to identify with this system can be found in the Identification Table at the end of this package insert. Voges- 40%aqueous KOH solution 89% Proskauer in demineralized water 11% PRINCIPLE reagent: API STAPH consists of a strip containing dehydrated test VP1 CORROSIVE substrates in individual microtubes. The tests are reconstituted by 5 ml R35 : Causes severe burns. adding to each tube an aliquot of API STAPH Medium that has S24/25: Avoid contact with skin and eyes. S26 : In case of contact with eyes, rinse been inoculated with the strain to be studied. immediately with plenty of water and seek The strip is then incubated for 18-24 hours at 35-37°C after which medical advice. the results are read and interpreted with reference to the S36/37/39 : Wear suitable protective clothing, information contained in this package insert. The identification is gloves and eye/face protection. facilitated by the use of the API STAPH Analytical Profile Index or S45: In case of accident or if you feel unwell, the identification software. The identification obtained is based on seek medical advice immediately (show the the classification of Kloos and Schleifer. label when possible). Voges- Alpha-naphthol 6 g REAGENTS Proskauer Ethyl alcohol 100 ml reagent: Kit contents (25 tests) : VP2 5 ml -25API STAPH strips -25incubation boxes FLAMMABLE AFTER RECONSTITUTION -25ampules of API STAPH Medium R21/22: Harmful in contact with skin and if -25result sheets swallowed -1package insert S24/25: Avoid contact with skin and eyes. Nitrate reduction Additional products (not included in the kit) : Sulfanilic acid 0.8% reagent: Acetic acid 5N 99.2% - Mineral oil (Prod. No. 70100) NIT 1 5 ml - Reagents : VP1 (Prod. No. 70422) CORROSIVE VP2 (Prod. No. 70422) R34 : Causes burns. NIT 1 (Prod. No. 70442) S2 : Keep out of reach of children. NIT 2 (Prod. No. 70442) S23 : Do not breathe vapours. ZYME A (Prod. No. 70472) S26 : In case of contact with eyes, rinse ZYME B (Prod. No. 70472) immediately with plenty of water and seek - McFarland Standard (Prod. No. 70900) medical advice. - API STAPH Analytical Profile Index (Prod. No. 20590) or Nitrate reduction N,N-dimethyl-1-naphthylamine 0.5% Identification software (consult bioMérieux) reagent: Acetic acid 5N 99.5% - Sterile Pasteur pipettes NIT 2 - Ampule stand (Prod. No. 70200) 5 ml CORROSIVE R34 : Causes burns. S2 : Keep out of reach of children. Required laboratory equipment : S23 : Do not breathe vapours. - 35-37°C Bunsen burner S26 : In case of contact with eyes, rinse - Refrigerator Marker pen immediately with plenty of water and seek medical advice. COMPOSITION OF MEDIA AND REAGENTS ZYME A Tris-hydroxymethyl-aminomethane 25 g reagent Hydrochloric acid (37 %) 11 ml API STAPH Yeast extract 0.5 g 8 ml Sodium lauryl sulfate 10 g Medium Bacto peptone 10 g H2O 100 ml 6 ml NaCl 5 g IRRITANT Trace elements 10 ml Demineralized water to make 1000 ml R36/38 : Irritating to eyes and skin. pH : 7.0 - 7.4 S26 : In case of contact with eyes, rinse immediately with plenty of water and seek medical advice. S36 : Wear suitable protective clothing.

1 api Staph 0 008038-4 02/02 ZYME B Fast Blue BB 0.35 g • Upon removal from refrigerator, allow reagents to come to reagent 2-methoxyethanol 100 ml room temperature (20-30°C) before using. 8 ml TOXIC Open ampules carefully as follows : R60 : May impair fertility. * For ampule with no dropper-cap : R61 : May cause harm to the unborn child. - Carefully remove the cap. R10 : Flammable. * For ampule with dropper-cap : R20/21/22 : Harmful by inhalation, in contact - Turn the ampule upside down and maintain it in with skin and if swallowed. a vertical position. S53 : Avoid exposure (avoid contact with skin and eyes, vapour inhalation and brutal - Hold the ampule in one hand in a vertical superheating). S45 : In case of accident or if you feel unwell, position (white plastic cap uppermost). seek medical advice immediately (show the - Press the cap down as far as possible. label where possible). - Cover the flattened part of the cap with the upper part of the thumb. - Apply thumb pressure in an outward motion STORAGE OF THE STRIPS AND MEDIA to the flattened part of the cap to snap off the The strips and media should be stored at 2-8°C until the top of the ampule inside the cap. expiration date indicated on the packaging.

STORAGE OF THE REAGENTS - Squeeze on the cap to transfer all the reagent into the dropper-bottle. VP1, NIT 1 and NIT 2 reagents should be stored at 15-30°C until • All inoculated products should be considered infectious and the expiration date indicated on the packaging. VP2 should be handled appropriately. stored at 2-8°C until the expiration date if this comes first. • Record the date opened on the bottle label. All patient specimens and microbial cultures are potentially The ZYME B reagent may be kept for up to 1 month after the infectious and should be treated with universal precautions ampule has been opened and the reagent transferred into the (NCCLS M29-A: Protection of Laboratory Workers from dropper-bottle, (or until the expiration date if this comes first) : Instrument Biohazards and Infectious Disease Transmitted by record the date opened on the bottle label. ZYME B reagent Blood, Body Fluids, and Tissue: Approved Guideline. 1997). should be stored in the dark at 2-8°C. It is very sensitive to light. • After completing test, reading and interpretation, all specimens, Wrap the bottle in aluminum foil and only leave it out of the spills and inoculated products must be autoclaved, incinerated refrigerator while being used. Do not leave it on the bench for or immersed in a germicide prior to disposal. prolonged periods of time. The ZYME B reagent is normally • The performance data presented were obtained using the yellow in color. Dispose of the reagent if any tint of pink (sign of procedure indicated. Any changes or modification in the deterioration) is observed. procedure may affect results. The ZYME A reagent should be stored at 2-30°C, and may be • Interpretation of the test results should be made by a kept for up to 1 month after the ampule has been opened and competent microbiologist who should also take into the reagent transferred into the dropper bottles, (or until the consideration the patient history, the source of the specimen, expiration date if this comes first). Record the date opened on the colonial and microscopic morphology and, if necessary, the bottle label. results of any other tests performed, particularly the At 2-8°C, the ZYME A reagent may form a precipitate which does antimicrobial susceptibility patterns. not affect any of the properties of the reagent and which may be redissolved by gently heating. SPECIMEN COLLECTION USE OF THE REAGENTS API STAPH is not for use directly with clinical or other specimens. The microorganisms to be identified must first be isolated as • Allow reagents to come to room temperature (20-30°C) before separate colonies by the specimen onto appropriate using. culture media according to standard microbiological practices. • Open the ampule of VP1, VP2, ZYME A and ZYME B reagents NOTE: Specimens and bacterial cultures should be considered as indicated in the paragraph "Warnings and Precautions" infectious and handled appropriately by trained and competent (ampule with dropper-cap). technicians. • Dispense one drop of reagent. Aseptic technique and usual handling precautions for the • bacterial group studied should be observed throughout this Carefully close the bottle after use and store it as indicated in procedure ; refer to Universal Precautions (NCCLS M29-A, the paragraph "Storage of the reagents". Protection of Laboratory Workers from Instrument Biohazards and Infectious Disease Transmitted by Blood, Body Fluids, and WARNINGS AND PRECAUTIONS Tissue: Approved Guideline. 1997). • For in vitro diagnostic use only. For additional handling precautions, refer to Biosafety in • Qualified laboratory personnel should use aseptic technique Microbiological and Biomedical Laboratories, HHS Publication and established precautions for infectious agents. No. (CDC) 93-8395, 3rd Edition (May 1993), or to the regulations • Do not pipette specimens or reagents by mouth. of each country. • Do not use reagents past the expiration date. • Do not allow reagents to come into contact with skin, eyes or RECOMMENDED PROCEDURE clothing. • Do not interchange consumables between different lot Preparation of the strip numbers.

2 api Staph 0 008038-4 02/02 • Prepare an incubation box (tray and lid) and distribute about 5 ml of distilled water or demineralized water [or any water Identification without additives or chemicals which may release gases (e.g., Identification can be made : Cl2, CO2, etc.)] into the honeycombed wells of the tray to create • using the Analytical Profile Index : the pattern of the reactions a humid atmosphere. obtained must be coded into a numerical profile. • Record the strain reference on the elongated flap of the tray. On the result sheet, the tests are separated into groups of 3 • Remove the strip from its packaging and place it in the and a number 1, 2 or 4 is indicated for each. By adding the incubation box. numbers corresponding to positive reactions within each group, a 7-digit profile number is obtained. Resistance to lysostaphin Preparation of the inoculum (200 µg/ml), which is characteristic of micrococci, constitutes • Subculture the organism onto blood agar (or Agar P) and the 21st test and has a value of 4 if it is positive. incubate 18-24 hours at 35-37°C. • Check that the strain belongs to the Micrococcaceae family To obtain information on any profile not listed in the codebook, (morphology, , , etc.) and also check that call the Voice Response System at 800-645-7056. the culture is pure. • using the identification software. • Open an ampule of API STAPH Medium as indicated in the paragraph "Warnings and Precautions" (ampule with no dropper-cap). • Prepare a homogeneous bacterial suspension with a turbidity equal to 0.5 McFarland.

Inoculation of the strip 6 706 113 Staphylococcus epidermidis • Using a sterile pipette, fill the microtubes with the inoculated API STAPH Medium. Only fill the tube portion of the DISPOSAL OF USED MATERIAL microtubes, not the cupules (slightly underfill the microtubes). After use, all ampules, pipettes, incubation boxes and strips Avoid the formation of bubbles by holding the tip of the pipette should be incinerated, autoclaved or immersed in a disinfectant against the side of the cupule. for decontamination prior to disposal. • Ensure anaerobiosis in the ADH____ and URE____ tests by filling the cupules with mineral oil to form a convex meniscus. LIMITATIONS • Close the incubation box. The API STAPH system is designed uniquely for the identification • Incubate at 35-37°C for 18-24 hours. of the species included in the database (see Identification Table at the end of this package insert). It cannot be used to identify Reading the strip any other microorganisms or to exclude their presence. • Develop the reactions by adding 1 drop of each of the following reagents : RANGE OF EXPECTED VALUES - VP test : VP1 and VP2 reagents. Wait 10 minutes. A violet- The API STAPH tests produce a color reaction which indicates a pink color indicates a positive reaction. A pale pink or light positive or negative result (see Tables 2 and 3.) pink color obtained after 10 minutes should be considered negative. - NIT test : NIT 1 and NIT 2. PERFORMANCE CHARACTERISTICS Wait 10 minutes. A red color indicates a positive reaction. Evaluations of the API STAPH system were performed by - PAL test : ZYME A and ZYME B reagents. Gemmell and Dawson. These studies have shown that the API Wait 10 minutes. A violet color indicates a positive reaction. STAPH system identifies clinically isolated staphylococci as • rapidly and accurately as conventional methods. Read all the reactions by referring to the Interpretation of For specific performance characteristics, refer to Table 2. Reactions (Table 2). • Record the results on the result sheet.

3 api Staph 0 008038-4 02/02 QUALITY CONTROL The media, strips and reagents are systematically controlled at various stages of their manufacture. For those who wish to perform their own quality control tests with the strips, it is recommended to use the following stock cultures : Table 1 0 GLU FRU MNE MAL LAC TRE MAN XLT MEL NIT PAL VP RAF XYL SAC MDG NAG ADH URE 1. ––– ––––––– –V–––––––+ 2. – ++++++++– ++V–++–+ –+ 3. – +++++++V+ +VV+++++ –– 4. – +++–––V–– +–+––V––+– Profiles obtained after culture on sheep blood agar

1. Micrococcus spp ATCC 700405 3. Staphylococcus lentus ATCC 700403 2. Staphylococcus xylosus ATCC 700404 4. Staphylococcus capitis ATCC 35661 ATCC : American Type Culture Collection, 10801 University Boulevard, Manassas, VA 20110-2209, USA.

INTERPRETATION OF REACTIONS

Table 2

TESTS SUBSTRATE REACTIONS / ENZYMES RESULT NEGATIVE POSITIVE 0No substrate Negative control red — GLU D-Glucose (Positive control) FRU D-Fructose MNE D-Mannose MAL Maltose Acidification due to carbohydrate utilization LAC Lactose red yellow TRE D-Trehalose MAN D-Mannitol XLT Xylitol MEL D-Melibiose NIT 1 (1 drop) + NIT 2 (1 drop) / 10 min. NIT Potassium nitrate Reduction of nitrate to nitrite colorless-light pink red ZYME A (1 drop) + ZYME B (1 drop) / 10 min PAL _-naphthyl-acid phosphate Alkaline phosphatase yellow violet VP1 (1 drop) + VP2 (1 drop) / 10 min VP Sodium pyruvate Acetyl-methyl-carbinol production colorless-light pink violet-pink RAF Raffinose XYL Xylose SAC Sucrose Acidification due to red yellow MDG _-methyl-D-glucoside carbohydrate utilization NAG N-acetyl-glucosamine ____ADH Arginine Arginine dihydrolase yellow orange-red ____URE Urea yellow red-violet The acidification tests should be compared to the negative (0) and positive (GLU) controls.

When MNE and XLT are preceded or followed by positive tests, then an orange test should be considered negative.

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RECOMMENDED METHODOLOGY

- Cocci - Gram + - Catalase +

Blood agar

0.5 McF

api Staph Medium

0 URE Resistance to lysostaphin api Staph ADH URE

24:00 37°C

VP : VP1 + VP2 NIT : NIT 1 + NIT 2 api Staph PAL : ZYME A + ZYME B

+ - + - + -

5 api Staph0 008038-4 02/02 IDENTIFICATION TABLE Table 3 % of reactions positive after 24 hrs. at 35-37°C API STAPH V4.0 0 GLU FRU MNE MAL LAC TRE MAN XLT MEL NIT PAL VP RAF XYL SAC MDG NAG ADH URE LSTR Staphylococcus aureus 0 100 100 95 96 88 91 80 0083 97 78 1097 2 90 80 80 0 Staphylococcus auricularis 0 100 99 36 72 10 90 90081 01 0040 01590 10 Staphylococcus capitis 0 100 99 80 43 22 2 36 0086 23 90 0050 0185 35 0 Staphylococcus caprae 0 100 99 70 10 75 74 10 0 0 99 95 99 0 000 199 60 0 Staphylococcus carnosus 0 100 100 99 0 99 99 99 0099 83 83 0 000100 100 00 Staphylococcus chromogenes 0 100 100 99 79 100 100 13 0 0 96 96 101100 0 31 89 95 0 Staphylococcus cohnii ssp cohnii 0 100 99 66 99 2 97 88 33 02166 94 0 020 921 0 Staph.cohnii ssp urealyticum 0 100 100 99 98 98 100 94 64 0194 87 0 00098 0 99 0 Staphylococcus epidermidis 0 100 99 70 99 81 200180 84 68 1097 41873 88 0 Staphylococcus haemolyticus 0 99 75 5 99 80 91 60 0178 3 57 0098 13 83 85 10 Staphylococcus hominis 0 98 94 41 97 50 86 28 0182 27 70 1097 4 50 43 84 0 Staphylococcus hyicus 0 100 99 99 0 87 99 00090 90 15 0 0 99 2 93 100 68 0 Staphylococcus lentus 0 100 100 100 100 100 100 100 7 99 92 21 57 100 100 100 28 100 01 0 Staphylococcus lugdunensis 0 100 89 88 99 66 99 00099 16 99 00100 0 90 1 50 0 Staphylococcus saprophyticus 0 100 99 2 97 90 99 88 22 0 35 14 79 1096 1 70 30 65 0 Staphylococcus schleiferi 0 100 80 100 0171 00099 97 99 0 00094 99 00 Staphylococcus sciuri 0 99 99 99 99 70 93 98 0083 67 30 01695 7 68 00 0 Staphylococcus simulans 0 100 100 57 11 95 92 73 4083 27 38 0497 2 90 97 84 0 Staphylococcus warneri 0 99 99 50 98 19 96 70 002316 90 0099 0677 97 0 Staphylococcus xylosus 0 100 100 92 81 85 95 90 30 9 82 75 67 11 82 87 10 80 5 90 0 Kocuria kristinae 0 99 96 99 90 9 84 3006393 0090 12 0 0 0 97 Kocuria varians/rosea 0 91 92 8 11810075 48 4840 1129 95 Micrococcus spp 02 4 0 10100081510010 1111191

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BIBLIOGRAPHY

1. BALDELLON CH., MEGRAUD F. Characterization of Micrococcaceae Strains Isolated from the 5. LANGLOIS B.E., HARMON R.J., AKERS K. Human Urogenital Tract by the Conventional Scheme and a Identification of Staphylococcus Species of Bovine Origin Micromethod. with the DMS STAPH-TRAC System. (1985) J. Clin. Microbiol., 21, 474-477. (1984) J. Clin. Microbiol., 20, 227-230. 2. BRUN Y., FLEURETTE J., FOREY F. 6. MADDUX R.L., KOEHNE G. Micromethod for Biochemical Identification of Identification of Staphylococcus hyicus with the Negative Staphylococci. API STAPH Strip. (1978) J. Clin. Microbiol., 8, 503-508. (1982) J. Clin. Microbiol., 15, 984-986. 3. GEMMELL C.G., DAWSON J.E. 7. PASCOLI L., CHIARADIA V., MUCIGNAT G., SANTINI G. Identification of Coagulase-Negative Staphylococci with the Identification of Staphylococci by the API STAPH, Sceptor, API STAPH system. Rosco and Simplified Lyogroup Systems. (1982) J. Clin. Microbiol., 16, 874-877. (1986) Eur. J. Clin. Microbiol., 5, 669-671. 4. GIGER O., CHARILAOU C.C. and CUNDY K.R. 8. RADEBOLD K., ESSERS L. Comparison of the Staph-Ident and DMS Staph-Trac Evaluation of the API-STAPH Micro-System for Routine Systems with Conventional Methods used for the Identification of Staphylococci. Identification of Coagulase-Negative Staphylococci. (1980) Arzt. Lab 26, 236-238. (1984) J. Clin. Microbiol., 19, 68-72.

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Fabricant / Manufacturer bioMérieux sa

bioMérieux sa bioMérieux, Inc. au capital de 77 421 420 F 595 Anglum Road 673 620 399 RCS LYON Hazelwood, MO 63042-2320 / USA 69280 Marcy-l’Etoile / France tel. (1) 314-731-8500 / fax (1) 314-731-8800 tel. (33) 4 78 87 20 00 / fax (33) 4 78 87 20 90 Imprimé en / Printed in USA http://www.biomerieux.com