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008041-3 05/01
For in vitro diagnostic use 
Identification system for streptococci
 API 20 STREP is a standardized method combining 20 biochemical tests that offer widespread capabilities. It enables group or speciesidentification of most streptococci encountered in medical bacteriology. The complete list of those organisms that it is possible toidentify with this system is given in the Identification Table at the end of this package insert.
PRINCIPLE
The API 20 STREP strip consists of 20 microtubes containingdehydrated substrates for the demonstration of enzymatic activityor the fermentation of sugars.The enzymatic tests are inoculated with a dense suspension of organisms, made from a pure culture, which is used to rehydratethe enzymatic substrates. The metabolic end products producedduring the incubation period are either revealed throughspontaneous colored reactions or by the addition of reagents.The fermentation tests are inoculated with an enriched mediumwhich reconstitutes the sugar substrates. Fermentation of carbohydrates is detected by a shift in the pH indicator.The reactions are read according to the Interpretation of Reactions (Table 2) and the identification is obtained by referringto the Analytical Profile Index or using the identification software.
REAGENTS
Kit contents (25 tests) :
-25API 20 STREP strips-25incubation boxes-25ampules of GP Medium-25result sheets-25swabs-1package insert
Additional products (not included in the kit) :
-Suspension Medium, 2 ml (Prod. No. 70700)-Reagents : Ninhydrin (NIN)(Prod. No. 70490)Potassium hydroxide(Prod. No. V7053)VP2
or 
(Prod. No. 70430) _-naphthol(Prod. No. V7054)ZYME A(Prod. No. 70470)ZYME B(Prod. No. 70480)-Mineral oil (Prod. No. 70100)-Sterile Pasteur pipettes-McFarland Standard #4(Prod. No. 70900)-API 20 STREP Analytical Profile Index (Prod. No. 20690) or identification software (consult bioMérieux)-Ampule rack (Prod. No. 70200)-Columbia blood agar plates-Schaedler broth (optional)
Required laboratory equipment :
-35-37°C incubator -Refrigerator (2-8
°
C)-Bunsen burner -Marker pen-Anaerobic jar -Inoculating loop
COMPOSITION OF MEDIA AND REAGENTS
SuspensionMedium
2 mlDemineralized water 
GP Medium
2 mlCystine0.5 gTryptone20 gSodium chloride5 gSodium sulfite0.5 gPhenol red0.17 gDemineralized water to make 1000 mlpH : 7.8
NIN reagent
5 mlNinhydrin7 g2-methoxyethanol100 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 skinand if swallowed.S53 : Avoid exposure (avoid contact with skin andeyes, vapor inhalation and brutal superheating).S45 : In case of accident or if you feel unwell, seekmedical advice immediately (show the label wherepossible).
Voges-Proskauer reagent:Potassiumhydroxide
30 ml40% aqueous KOH solution89%in demineralized water11%
CORROSIVE
R35 : Causes severe burns.S24/25: Avoid contact with skin and eyes.S26 : In case of contact with eyes, rinse immediatelywith plenty of water and seek medical advice.S36/37/39 : Wear suitable protective clothing, glovesand eye/face protection.S45: Incase of accident or if you feel unwell, seekmedical advice immediately (show the label wherepossible).
VogesProskauer reagent: VP25 ml or alpha-naphthol30 ml
alpha-naphthol6 gEthyl alcohol100mlalpha-naphthol 2.05 gto be diluted with 95% ethyl alcohol 29ml
FLAMMABLE AFTER RECONSTITUTION
R21/22: Harmful in contact with skin and if swallowed.S24/25: Avoid contact with skin and eyes.
ZYME A reagent
8 mlTris-hydroxymethyl-aminomethane25 gHydrochloric acid (37 %)11 mlSodium lauryl sulfate10 gH
2
O 100 ml
ZYME B reagent
8 mlFast Blue BB0.35 g2-methoxyethanol100 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 skinand if swallowed.S53 : Avoid exposure - obtain special instructionsbefore use.S45 : In case of accident or if you feel unwell, seekmedical advice immediately (show the label wherepossible).
 
STORAGE OF THE STRIPS AND MEDIA
The strips and media should be stored at 2-8°C until theexpiration date indicated on the packaging.
STORAGE OF THE REAGENTS
The reagents should be stored in the dark at 2-8°C (exceptPotassium hydroxide and ZYME A which can be stored at 2-30°C) until the expiration date indicated on the packaging. VP2may be kept up to 1 month after the ampules have been openedand the reagent transferred to the dropper-bottle. After reconstitution, alpha naphthol should be stored at 2-8°C and maybe kept for up to 90 days, (or until the expiration date if thiscomes first) : record the reconstitution date on the bottle label.The ZYME A, ZYME B and NIN reagents may be kept for up to 1month after the ampules have been opened and the reagentstransferred into the dropper-bottles, (or until the expiration date if this comes first) : record the date opened on the bottle label.The NIN and ZYME B reagents are very sensitive tolight : wrap the bottles in aluminum foil and only leave them out of the refrigerator while being used. Do not leave them on the benchfor prolonged periods of time.The NIN reagent is very sensitive to traces of water and air :transfer the reagent into the dropper-bottle using a dry pipetteand keep the bottle tightly closed.The ZYME B reagent is normally yellow in color. Dispose of thereagent if any tint of pink (sign of deterioration) is observed. At 2-8°C, the ZYME A reagent may form a precipitate which doesnot affect any of the properties of the reagent and which may beredissolved by gently heating.
USE OF THE REAGENTS
 Allow reagents to come to room temperature (20-30°C) beforeusing.
1. ZYME A and ZYME B reagents :
•
Open the ampule of reagent 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 inthe paragraph "Storage of the reagents".
2. NIN reagent :
•
Open the ampule of reagent as indicated in the paragraph"Warnings and Precautions" (ampule with no dropper-cap).
•
Take up the contents of the ampule using a completely drypipette and transfer this liquid into the dropper-bottle.
•
Fit the dropper to the bottle.
•
Dispense one drop of reagent.
•
Carefully close the bottle after use and store it as indicated inthe paragraph "Storage of the reagents".
3. VP2:
•
Open the ampule of reagent as indicated in the paragraphWarnings and Precautions" (ampule with dropper cap).
•
Dispense one drop of reagent.
•
Carefully close the bottle after use an store it as indicated in theparagraph "Storage of the reagents".
4. Alpha-naphthol :
•
Reconstitute with 29 ml of 95% ethanol.
•
Carefully close the bottle.
•
Shake.
•
Reagent can be used after active ingredient is completelydisolved.
•
Dispense one drop of reagent.
•
Carefully close the bottle after use an store it as indicated in theparagraph "Storage of the reagents".
 
WARNINGS AND PRECAUTIONS
•
For 
in vitro
diagnostic use only.
•
Qualified laboratory personnel should use aseptic techniqueand 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 reagents or consumables between differentlot numbers.
•
Upon removal from refrigerator, allow reagents to come toroom temperature (20-30°C) before using.
•
Open ampules carefully as follows :
 
-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 tothe flattened part of the cap to snap off the top of the ampule inside the cap.
 
*For ampule with no dropper-cap :
 
- Carefully remove the cap.
 
*For ampule with dropper-cap :
 
- Turn the ampule upside down and maintain it ina vertical position.
 
-Squeeze on the cap to transfer all the reagentinto the dropper-bottle.
•
All inoculated products should be considered infectious andhandled appropriately.
•
All patient specimens and microbial cultures are potentiallyinfectious and should be treated with universal precautions(NCCLS M29-A:
Protection of Laboratory Workeres fromInstrument 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, incineratedor immersed in a germicide prior to disposal.
•
Interpretation of the test results should be made by acompetent microbiologist who should also take intoconsideration the patient history, the source of the specimen,colonial and microscopic morphology and, if necessary, theresults of any other tests performed, particularly theantimicrobial susceptibility patterns.
•
Any changes or modifications in the procedure may affect theresults.
 
 
 
INSTRUCTIONS FOR USE
 
Specimens and bacterial cultures should be considered infectiousand handled appropriately by trained and competent technicians.
 
 Aseptic technique and usual handling precautions for thebacterial group studied should be observed throughout thisprocedure ; refer to Universal Precautions (NCCLS M29-A:
Protection of Laboratory Workers from Instrument Biohazardsand Infectious Diseases Transmitted by Blood, Body Fluids, and Tissue
: Approved Guideline. 1997).
 
For additional handling precautions, refer to Biosafety inMicrobiological and Biomedical Laboratories, HHS PublicationNo. (CDC) 93-8395, 3rd Edition (May 1993), or to the regulationsof each country.
 
Selection of colonies
 
Once the microorganism to be identified has been isolated andverified to be a member of the genus
Streptococcus
or relatedgenera shown in Table 3 (Gram, catalase test) :
•
 Note the type of hemolysis on the result sheet(21st test).
•
Pick a well-isolated colony (Note 1) and suspend it in 0.3 ml of sterile water. Homogenize well.
•
Flood a Columbia sheep blood agar plate (Note 2) with thissuspension (or aseptically swab the entire surface of the agar).
•
Incubate anaerobically for 18-24 hours at 35-37°C.
 
NOTE 1 :
Alpha-hemolytic streptococci and enterococci producesufficiently large colonies after 24 hours of incubation. For other streptococci, it is preferable to select a colony after 48 hours of incubation. For fastidious strains (producing minute colonies after 48 hours), the following procedure is recommended :
 
-Culture the colony in 1 ml of Schaedler broth at 35-37°C for 5hours.
 
-Flood a Columbia sheep blood agar plate with the entireculture. Remove any excess liquid.
 
-Incubate anaerobically for 18 hours at 35-37°C.
 
NOTE 2 :
In the case of suspected pneumococci, it is advisableto prepare 2 plates in order to obtain sufficient growth.
 
Preparation of the strip
•
Prepare an incubation box (tray and lid) and distribute about 5ml of distilled water or demineralized water [or any water without additives or chemicals which may release gases (e.g.,Cl
2
, CO
2
, etc.)] into the honeycombed wells of the tray to createa humid atmosphere.
•
Record the strain reference on the elongated flap of the tray.
•
Remove the strip from its packaging and place it in the tray.
 
Preparation of the inoculum
•
Open an ampule of Suspension Medium (2 ml) as indicated inthe paragraph "Warnings and Precautions" (ampule with nodropper cap) or use any tube containing 2 ml of distilled water without additives.
 
•
Using a swab, harvest all the culture from the previouslyprepared subculture plate. Make a dense suspension with a
turbidity greater than 4 McFarland.
 
Inoculation of the strip
•
In the first half of the strip (tests VP to ADH
 ____ 
) distribute thesuspension with a sterile pipette, avoiding the formation of bubbles (tilt the strip slightly forwards) :
 
- For the tests VP to LAP : distribute approximately 100 µl intoeach cupule (3 drops with a Pasteur pipette ).
 
-For the ADH
 ____ 
test : fill the tube only.
•
In the second half of the strip (tests RIB
 ___ 
to GLYG
 ______ 
) :
 
-Open an ampule of GP Medium as indicated in theparagraph "Warnings and Precautions" (ampule with nodropper-cap) and transfer the rest of the suspension into it(appr. 0.5 ml). Mix well.
 
-Distribute this new suspension into the tubes only.
•
Fill the cupule of the underlined tests (ADH
 ____ 
to GLYG
 ______ 
) withmineral oil to form a convex meniscus.
•
Place the lid on the tray.
•
Incubate at 35-37°C for 4 hours to obtain a first reading and for 24 hours to obtain a second reading if this is required.
 
Reading of the strip
 
 After 4 hours of incubation :
•
Add the reagents :
 
-VP Test : 1 drop of each of Potassium hydroxide and VP2
or 
alpha-naphthol
.
 
-HIP Test : 2 drops of NIN reagent.
 
-PYRA, _GAL, _GUR, _GAL, PAL and LAP Tests : 1 drop of each of ZYME A and ZYME B reagents.
•
Wait 10 minutes, then read the reactions by referring to theInterpretation of Reactions Table (Table 2). If necessary,expose the strip to a strong light (10 seconds with a 1000 Wlamp) to decolorize any excess reagents in the tubes PYRA toLAP.
 
Reincubation is necessary :
 
-if the profile cannot be found in the API 20 STREP AnalyticalProfile Index
 
-if the profile is given with the following note :
 
IDENTIFICATION NOT VALIDBEFORE 24 HOURS OF INCUBATION
 
 After 24 hours, reread the reactions ESC, ADH
 ____ 
, and RIB
 ___ 
toGLYG
 ______ 
. Do not reread the enzymatic reactions (HIP, PYRA, _GAL, _GUR, _GAL, PAL, LAP) and VP. Record the reactions onthe result sheet.
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