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 bacteria 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
PRINCIPLE reagent:
in demineralized water 11%
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 6g
Proskauer
REAGENTS reagent:
Ethyl alcohol 100 ml
Kit contents (25 tests) : VP2 5 ml
- 25 API STAPH strips
- 25 incubation boxes
FLAMMABLE AFTER RECONSTITUTION
- 25 ampules of API STAPH Medium
R21/22: Harmful in contact with skin and if
- 25 result sheets swallowed
- 1 package insert S24/25: Avoid contact with skin and eyes.
Nitrate reduction Sulfanilic acid 0.8%
Additional products (not included in the kit) : reagent: Acetic acid 5N 99.2%
- Mineral oil (Prod. No. 70100) NIT 1
- Reagents : VP1 (Prod. No. 70422) 5 ml
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 incubator 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 5g 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.

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ZYME B Fast Blue BB 0.35 g
reagent 2-methoxyethanol 100 ml
8 ml TOXIC
R60 : May impair fertility.
R61 : May cause harm to the unborn child.
R10 : Flammable.
R20/21/22 : Harmful by inhalation, in contact
with skin and if swallowed.
S53 : Avoid exposure (avoid contact with skin
and eyes, vapour inhalation and brutal
superheating).
S45 : In case of accident or if you feel unwell,
seek medical advice immediately (show the
label where possible).
STORAGE OF THE STRIPS AND MEDIA
The strips and media should be stored at 2-8°C until the
expiration date indicated on the packaging.
STORAGE OF THE REAGENTS
VP1, NIT 1 and NIT 2 reagents should be stored at 15-30°C until
the expiration date indicated on the packaging. VP2 should be
stored at 2-8°C until the expiration date if this comes first.
Record the date opened on the bottle label.
The ZYME B reagent may be kept for up to 1 month after the
ampule has been opened and the reagent transferred into the
dropper-bottle, (or until the expiration date if this comes first) :
record the date opened on the bottle label. ZYME B reagent
should be stored in the dark at 2-8°C. It is very sensitive to light.
Wrap the bottle in aluminum foil and only leave it out of the
refrigerator while being used. Do not leave it on the bench for
prolonged periods of time. The ZYME B reagent is normally
yellow in color. Dispose of the reagent if any tint of pink (sign of
deterioration) is observed.
The ZYME A reagent should be stored at 2-30°C, and may be
kept for up to 1 month after the ampule has been opened and
the reagent transferred into the dropper bottles, (or until the
expiration date if this comes first). Record the date opened on the
bottle label.
At 2-8°C, the ZYME A reagent may form a precipitate which does
not affect any of the properties of the reagent and which may be
redissolved by gently heating.
USE OF THE REAGENTS
• Allow reagents to come to room temperature (20-30°C) before
using.
• Open the ampule of VP1, VP2, ZYME A and ZYME B reagents
as indicated in the paragraph "Warnings and Precautions"
(ampule with dropper-cap).
• Dispense one drop of reagent.
• Carefully close the bottle after use and store it as indicated in
the paragraph "Storage of the reagents".
WARNINGS AND PRECAUTIONS
• For in vitro diagnostic use only.
• Qualified laboratory personnel should use aseptic technique
and established precautions for infectious agents.
• Do not pipette specimens or reagents by mouth.
• Do not use reagents past the expiration date.
• Do not allow reagents to come into contact with skin, eyes or
clothing.
• Do not interchange consumables between different lot
numbers.
• Upon removal from refrigerator, allow reagents to come to
room temperature (20-30°C) before using.
Open ampules carefully as follows :
* For ampule with no dropper-cap :
- Carefully remove the cap.
* For ampule with dropper-cap :
- Turn the ampule upside down and maintain it in
a vertical position.
- Hold the ampule in one hand in a vertical
position (white plastic cap uppermost).
- Press the cap down as far as possible.
- Cover the flattened part of the cap with the
upper part of the thumb.
- Apply thumb pressure in an outward motion
to the flattened part of the cap to snap off the
top of the ampule inside the cap.
- Squeeze on the cap to transfer all the reagent
into the dropper-bottle.
• All inoculated products should be considered infectious and
handled appropriately.
• All patient specimens and microbial cultures are potentially
infectious and should be treated with universal precautions
(NCCLS M29-A: Protection of Laboratory Workers from
Instrument Biohazards and Infectious Disease Transmitted by
Blood, Body Fluids, and Tissue: Approved Guideline. 1997).
• After completing test, reading and interpretation, all specimens,
spills and inoculated products must be autoclaved, incinerated
or immersed in a germicide prior to disposal.
• The performance data presented were obtained using the
procedure indicated. Any changes or modification in the
procedure may affect results.
• Interpretation of the test results should be made by a
competent microbiologist who should also take into
consideration the patient history, the source of the specimen,
colonial and microscopic morphology and, if necessary, the
results of any other tests performed, particularly the
antimicrobial susceptibility patterns.
SPECIMEN COLLECTION
API STAPH is not for use directly with clinical or other specimens.
The microorganisms to be identified must first be isolated as
separate colonies by streaking the specimen onto appropriate
culture media according to standard microbiological practices.
NOTE: Specimens and bacterial cultures should be considered
infectious and handled appropriately by trained and competent
technicians.
Aseptic technique and usual handling precautions for the
bacterial group studied should be observed throughout this
procedure ; refer to Universal Precautions (NCCLS M29-A,
Protection of Laboratory Workers from Instrument Biohazards
and Infectious Disease Transmitted by Blood, Body Fluids, and
Tissue: Approved Guideline. 1997).
For additional handling precautions, refer to Biosafety in
Microbiological and Biomedical Laboratories, HHS Publication
No. (CDC) 93-8395, 3rd Edition (May 1993), or to the regulations
of each country.
RECOMMENDED PROCEDURE
Preparation of the strip
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• Prepare an incubation box (tray and lid) and distribute about 5
ml of distilled water or demineralized water [or any water
without additives or chemicals which may release gases (e.g.,
Cl2, CO 2, etc.)] into the honeycombed wells of the tray to create
a humid atmosphere.
• Record the strain reference on the elongated flap of the tray.
• Remove the strip from its packaging and place it in the
incubation box.
Preparation of the inoculum
• Subculture the organism onto blood agar (or Agar P) and
incubate 18-24 hours at 35-37°C.
• Check that the strain belongs to the Micrococcaceae family
(morphology, Gram stain, catalase, etc.) and also check that
the culture is pure.
• 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
• Using a sterile pipette, fill the microtubes with the inoculated
API STAPH Medium. Only fill the tube portion of the
microtubes, not the cupules (slightly underfill the microtubes).
Avoid the formation of bubbles by holding the tip of the pipette
against the side of the cupule.
• Ensure anaerobiosis in the ADH ____ and URE
____ tests by filling the
cupules with mineral oil to form a convex meniscus.
• Close the incubation box.
• Incubate at 35-37°C for 18-24 hours.
Reading the strip
• Develop the reactions by adding 1 drop of each of the
following reagents :
- VP test : VP1 and VP2 reagents. Wait 10 minutes. A violet-
pink color indicates a positive reaction. A pale pink or light
pink color obtained after 10 minutes should be considered
negative.
- NIT test : NIT 1 and NIT 2.
Wait 10 minutes. A red color indicates a positive reaction.
- PAL test : ZYME A and ZYME B reagents.
Wait 10 minutes. A violet color indicates a positive reaction.
• Read all the reactions by referring to the Interpretation of
Reactions (Table 2).
• Record the results on the result sheet.
Identification
Identification can be made :
• using the Analytical Profile Index : the pattern of the reactions
obtained must be coded into a numerical profile.
On the result sheet, the tests are separated into groups of 3
and a number 1, 2 or 4 is indicated for each. By adding the
numbers corresponding to positive reactions within each group,
a 7-digit profile number is obtained. Resistance to lysostaphin
(200 µg/ml), which is characteristic of micrococci, constitutes
the 21st test and has a value of 4 if it is positive.
To obtain information on any profile not listed in the codebook,
call the Voice Response System at 800-645-7056.
• using the identification software.
6 706 113 Staphylococcus epidermidis
DISPOSAL OF USED MATERIAL
After use, all ampules, pipettes, incubation boxes and strips
should be incinerated, autoclaved or immersed in a disinfectant
for decontamination prior to disposal.
LIMITATIONS
The API STAPH system is designed uniquely for the identification
of the species included in the database (see Identification Table
at the end of this package insert). It cannot be used to identify
any other microorganisms or to exclude their presence.
RANGE OF EXPECTED VALUES
The API STAPH tests produce a color reaction which indicates a
positive or negative result (see Tables 2 and 3.)
PERFORMANCE CHARACTERISTICS
Evaluations of the API STAPH system were performed by
Gemmell and Dawson. These studies have shown that the API
STAPH system identifies clinically isolated staphylococci as
rapidly and accurately as conventional methods.
For specific performance characteristics, refer to Table 2.
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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 + + V V + + + + + – –
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
0 No substrate Negative control red —
GLU D-Glucose (Positive control)
FRU D-Fructose
MNE D-Mannose
MAL Maltose Acidification due to
LAC Lactose carbohydrate utilization yellow
red
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 Urease 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

+ - + - + -

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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 0 0 83 97 78 1 0 97 2 90 80 80 0
Staphylococcus auricularis 0 100 99 36 72 10 90 9 0 0 81 0 1 0 0 40 0 15 90 1 0
Staphylococcus capitis 0 100 99 80 43 22 2 36 0 0 86 23 90 0 0 50 0 1 85 35 0
Staphylococcus caprae 0 100 99 70 10 75 74 10 0 0 99 95 99 0 0 0 0 1 99 60 0
Staphylococcus carnosus 0 100 100 99 0 99 99 99 0 0 99 83 83 0 0 0 0 100 100 0 0
Staphylococcus chromogenes 0 100 100 99 79 100 100 13 0 0 96 96 1 0 1 100 0 31 89 95 0
Staphylococcus cohnii ssp cohnii 0 100 99 66 99 2 97 88 33 0 21 66 94 0 0 2 0 9 2 1 0
Staph.cohnii ssp urealyticum 0 100 100 99 98 98 100 94 64 0 1 94 87 0 0 0 0 98 0 99 0
Staphylococcus epidermidis 0 100 99 70 99 81 2 0 0 1 80 84 68 1 0 97 4 18 73 88 0
Staphylococcus haemolyticus 0 99 75 5 99 80 91 60 0 1 78 3 57 0 0 98 13 83 85 1 0
Staphylococcus hominis 0 98 94 41 97 50 86 28 0 1 82 27 70 1 0 97 4 50 43 84 0
Staphylococcus hyicus 0 100 99 99 0 87 99 0 0 0 90 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 0 1 0
Staphylococcus lugdunensis 0 100 89 88 99 66 99 0 0 0 99 16 99 0 0 100 0 90 1 50 0
Staphylococcus saprophyticus 0 100 99 2 97 90 99 88 22 0 35 14 79 1 0 96 1 70 30 65 0
Staphylococcus schleiferi 0 100 80 100 0 1 71 0 0 0 99 97 99 0 0 0 0 94 99 0 0
Staphylococcus sciuri 0 99 99 99 99 70 93 98 0 0 83 67 30 0 16 95 7 68 0 0 0
Staphylococcus simulans 0 100 100 57 11 95 92 73 4 0 83 27 38 0 4 97 2 90 97 84 0
Staphylococcus warneri 0 99 99 50 98 19 96 70 0 0 23 16 90 0 0 99 0 6 77 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 3 0 0 6 3 93 0 0 90 12 0 0 0 97
Kocuria varians/rosea 0 91 92 8 1 1 8 1 0 0 75 4 8 4 8 4 0 1 1 29 95
Micrococcus spp 0 2 4 0 1 0 1 0 0 0 8 15 1 0 0 1 0 1 11 11 91

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BIBLIOGRAPHY

1. BALDELLON CH., MEGRAUD F.

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

WARRANTY
bioMérieux, Inc. disclaims all warranties, express or implied, including any implied warranties of MERCHANTABILITY AND FITNESS
FOR A PARTICULAR USE. bioMérieux shall not be liable for any incidental or consequential damages. IN NO EVENT SHALL
BIOMERIEUX’S LIABILITY TO CUSTOMER UNDER ANY CLAIM EXCEED A REFUND OF THE AMOUNT PAID TO BIOMERIEUX
FOR THE PRODUCT OR SERVICE WHICH IS THE SUBJECT OF THE CLAIM.

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bioMérieux sa bioMérieux, Inc.

au capital de 77 421 420 F
673 620 399 RCS LYON
69280 Marcy-l’Etoile / France
tel. (33) 4 78 87 20 00 / fax (33) 4 78 87 20 90
http://www.biomerieux.com
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