REF 20 100 / 20 160 07584D - GB - 2002/10

®
20 E IVD

Identification system for Enterobacteriaceae and other non-fastidious Gram-negative rods

SUMMARY AND EXPLANATION Material :
API 20 E is a standardized identification system for - Pipettes or PSIpettes
Enterobacteriaceae and other non-fastidious, Gram- - Ampule protector
negative rods which uses 21 miniaturized biochemical - Ampule rack
tests and a database. The complete list of those - General microbiology laboratory equipment
organisms that it is possible to identify with this system is
given in the Identification Table at the end of this package POSSIBLE ADDITIONAL REAGENTS :
insert. - API OF Medium (Ref. 50 110) :
Test for the determination of fermentative or oxidative
PRINCIPLE metabolism.
The API 20 E strip consists of 20 microtubes containing - API M Medium (Ref. 50 120) :
dehydrated substrates. These tests are inoculated with Test for motility of facultative anaerobic bacteria.
a bacterial suspension that reconstitutes the media.
During incubation, metabolism produces color changes WARNINGS AND PRECAUTIONS
that are either spontaneous or revealed by the addition of • For in vitro diagnostic use and microbiological
reagents. control.
The reactions are read according to the Reading Table • For professional use only.
and the identification is obtained by referring to the • This kit contains products of animal origin. Certified
Analytical Profile Index or using the identification software. knowledge of the origin and/or sanitary state of the
animals does not totally guarantee the absence of
CONTENT OF THE KIT transmissible pathogenic agents. It is therefore
Kit for 25 tests (ref. 20 100) recommended that these products be treated as
- 25 API 20 E strips potentially infectious, and handled observing the usual
- 25 incubation boxes safety precautions (do not ingest or inhale).
- 25 result sheets • All specimens, microbial cultures and inoculated
- 1 clip seal products should be considered infectious and handled
- 1 package insert appropriately. Aseptic technique and usual precautions
for handling the bacterial group studied should be
Kit for 100 tests (ref. 20 160) observed throughout this procedure. Refer to "NCCLS
- 100 API 20 E strips (4x25 strips) M29-A, Protection of Laboratory Workers from
- 100 incubation boxes Instrument Biohazards and Infectious Disease
- 100 result sheets Transmitted by Blood, Body Fluids, and Tissue;
- 1 clip seal Approved Guideline - December 1997". For additional
- 1 package insert handling precautions, refer to "Biosafety in
Microbiological and Biomedical Laboratories, HHS
COMPOSITION OF THE STRIP Publication No. (CDC) 93-8395, 3rd Edition (May
1993)", or to the regulations currently in use in each
The composition of the API 20 E strip is given in the
country.
Reading Table of this package insert.
• Do not use reagents past the expiration date.
• Before use, check that the packaging of the various
REAGENTS AND MATERIAL REQUIRED BUT NOT components is intact.
PROVIDED • Do not use strips which have been damaged : cupules
Reagents : deformed, desiccant sachet open, ...
- API NaCl 0.85 % Medium, 5 ml (Ref. 20 230) or • The performance data presented were obtained using
API Suspension Medium, 5 ml (Ref. 20 150) the procedure indicated in this package insert. Any
- API 20 E reagent kit (Ref. 20 120) or change or modification in the procedure may affect the
individual reagents : TDA (Ref. 70 402) results.
JAMES (Ref. 70 542) • Interpretation of the test results should be made taking
VP 1 + VP 2 (Ref. 70 422) into consideration the patient history, the source of the
NIT 1 + NIT 2 (Ref. 70 442) specimen, colonial and microscopic morphology of the
- Zn reagent (Ref. 70 380) strain and, if necessary, the results of any other tests
- Oxidase (Ref. 55 635*) performed, particularly the antimicrobial susceptibility
* reference not sold in certain countries : use an patterns.
equivalent reagent.
- Mineral oil (Ref. 70 100)
- API 20 E Analytical Profile Index (Ref. 20 190) or
identification software (consult bioMérieux)

bioMérieux® sa English - 1

remove a single well. developed in the whole cupule indicates a positive reaction to be recorded on the result sheet. • Identification : without additives. The plastic incubation lid should not be replaced about 5 ml of distilled water or demineralized water after the addition of the reagent. tray. in order to avoid damaging the desiccant • After the incubation period. at 2-8°C • Incubate at 36°C ± 2°C for 18-24 hours. (Do not record the reference on the lid as it may be . before adding the reagents is less than 3 : combed wells of the tray to create a humid atmosphere. final profile (21st identification test). A reddish The microorganisms to be identified must first be iso- brown color indicates a positive reaction to be lated on a culture medium adapted to the culture of recorded on the result sheet. It is .Enter the 7-digit numerical profile manually via the suspension. Enterobacteriaceae and/or non-fastidious Gram-negative . the pouch should be re-sealed using the suspension. the recorded on the result sheet as it is an integral part of the reaction should be considered negative. By Preparation of the inoculum adding together the values corresponding to positive reactions within each group. CO2. [or any water without additives or chemicals which may • If the number of positive tests (including the GLU test) release gases (e. the tests are separated into groups must be used to exclude or confirm their presence. READING AND INTERPRETATION (*) Recommended method for opening the pouches : cut Reading the strip open the pouch just below the seal while holding the pouch upright. etc. This suspension must be used immediately after preparation. clip seal (included in the kit) to preserve the remaining • Fill only the tube (and not the cupule) of the other tests. 10 months after the pouch has been opened.GB . A pink color rods. keyboard. ODC. the Reading Table.g. Enterobacteriaceae and/or non-fastidious Gram-negative rods. it may be necessary to • Place the strip in the incubation box. The strips may then be kept for up to • Close the incubation box. bioMérieux® sa English . INSTRUCTIONS FOR USE . strips with the desiccant sachets : place the open end of • Create anaerobiosis in the tests ADH..) (see previous paragraph). A pink or red color indicates The oxidase test must be performed according to the a positive reaction to be recorded on the result sheet. according to standard microbiological techniques. the two parts. • If 3 or more tests (GLU test + or –) are positive. • Remove the strip from its packaging. The result should be If a slightly pink color appears after 10 minutes.2002/10 STORAGE CONDITIONS Inoculation of the strip The strips are supplied in an aluminum pouch with • Using the same pipette. 2 or 4 is indicated for each. VP and GEL with the bacterial Once opened (*).85 % Medium (5 ml) or obtained for the 20 tests of the API 20 E strip. read the strip by referring to sachets. fill both tube and cupule of desiccant sachets. suspend the bacteria in API NaCl 0. . of 3 and a value 1. NOTE : most Vibrio species are halophilous.0) • Using a pipette or PSIpette..e. .To complete the identification. precautions (i. LDC. record SPECIMENS (COLLECTION AND PREPARATION) all the spontaneous reactions on the result sheet and then reveal the tests which require the addition of API 20 E is not for use directly with clinical or other reagents : specimens. The an ampule of API Suspension Medium (5 ml) as oxidase reaction constitutes the 21st test and has a indicated in the paragraph "Warnings and Precautions" value of 4 if it is positive.api® 20 E 07584D . recommended to use young cultures (18-24 hours old). Oxidase test Wait at least 10 minutes.Look up the numerical profile in the list of profiles. (or until the expiration date indicated on the packaging. the tests CIT .TDA Test : add 1 drop of TDA reagent. NOTE : The indole production test must be performed last since this reaction releases gaseous products which Preparation of the strip interfere with the interpretation of other tests on the • Prepare an incubation box (tray and lid) and distribute strip.2 . a 7-digit profile number is • Open an ampule of API NaCl 0.85 % Medium.)] into the honey. This is performed using the database (V4. If a Vibrio is suspected. H2S the pouch along the seal and carefully clamp between and URE by overlaying with mineral oil. Alternative procedures On the result sheet. * with the identification software : • Carefully emulsify to achieve a homogeneous bacterial . Brucella and Francisella) are not • Determination of the numerical profile : included in the API 20 E database. manufacturer's instructions for use. . of the package insert for these products.IND Test : add 1 drop of JAMES reagent.Reincubate the strip for a further 24 hours (± 2 hours) • Record the strain reference on the elongated flap of the without adding any reagents. Fastidious organisms having demanding nutritional Interpretation requirements and requiring appropriate handling Identification is obtained with the numerical profile. Cl2. if this comes before). perform supplementary tests (refer to Identification NOTE : API 20 E should only be used with paragraph).VP Test : add 1 drop each of VP 1 and VP 2 reagents.Reveal the tests requiring the addition of reagents misplaced during the procedure. * with the Analytical Profile Index : isolated colony from an isolation plate. or use any tube containing 5 ml of sterile saline or sterile distilled water.

Identification is then be recorded on the result sheet.85 % Medium. It cannot be used to identify any other PERFORMANCE microorganisms or to exclude their presence. to negative. ONPG ADH LDC ODC CIT H2S URE TDA IND VP GEL GLU MAN INO SOR RHA SAC MEL AMY ARA NO2 N2* 1. carried out : . • Profile obtained after 24-48 hours of incubation for the strain ATCC 51331.61 % of the strains were not identified. Klebsiella pneumoniae ssp pneumoniae ATCC 35657 ATCC : American Type Culture Collection. QUALITY CONTROL The media. For those users who wish to perform their own quality control tests with the strip. 10801 University Boulevard. In tested: addition. insert for the range of expected results for the various fastidious. Manassas. Further tests may be proposed in case of low discrimination. . If the test turns orange. A red color indicates a API OF Medium (see package insert). positive reaction (NO2). belonging to species included in the database were • If Salmonella or Shigella are identified. Stenotrophomonas maltophilia ATCC 51331 4.GB . Refer to the identification software or Analytical Profile Index. such as Klebsiella or Proteus may revert from positive . (see Identification Table at the end of this package insert). Enterobacter cloacae ATCC 13047 5.api® 20 E 07584D . indicated in the introduction reagent to the GLU tube. NOTE : For the same reason as the indole test (see the 5 315 173 (57) Enterobacter gergoviae note in the paragraph "Reading the strip"). oxidase positive rods. Escherichia coli ATCC 25922 or else one of the following strains : 2. A negative reaction (yellow) . evidenced by gas bubbles) : add 2 to 3 mg of Zn These supplementary tests. This reaction is useful when testing Gram-negative.52 % of the strains were misidentified.3 . red.2. • On rare occasions.3.Oxidation of glucose (OF-O) : Inoculate an ampule of tube. This reaction will be recorded as a negative • Other non-fastidious Gram-negative rods : reaction. the glucose reactions for organisms .Growth on MacConkey agar medium (McC) : Streak a . Wait 2 to 5 minutes. + – + + – – – – + – – + + – + + – + – + + – 2. the 7-digit profile is not discriminatory . • Only pure cultures of a single organism should be used. serological tested : identification must be performed to confirm the bacterial . LIMITATIONS OF THE METHOD RANGE OF EXPECTED RESULTS • The API 20 E system is intended uniquely for the Consult the Identification Table at the end of this package identification of Enterobacteriaceae and those non. + + – + + – – – – + – + + – + + + + + + + – 4. and reagents are systematically quality controlled at various stages of their manufacture. this is a negative reaction : the nitrates still present in the tube have been reduced by the Zinc.32 % of the strains were correctly identified identification. • Bacterial suspensions prepared in API NaCl 0. using colonies grown on Trypticase Soy agar + blood. After 5 minutes. strips.Reduction of nitrates to nitrites (NO2) and N2 gas (N2) : MacConkey agar plate (see package insert).6. • Enterobacteriaceae : • Discrepancies with respect to conventional methods 5514 collection strains and strains of various origins may be observed. USA. • Profiles obtained after 18-24 hours of incubation for the other strains. it is preferable to use the strain 1. + – V – V – – – – – + – – – – – – – – – – – 3.92. bioMérieux® sa English . VA 20110-2209. the nitrate reduction test must be performed last. Such occurrences are reflected in the 2386 collection strains and strains of various origins percentages indicated in the Identification Table. It is the responsibility of the user to perform Quality Control in accordance with any local applicable regulations. add 1 drop each of NIT 1 and NIT 2 reagents to the GLU . (with or without supplementary tests). + – + – + – V – – V – + + + + + + + + + + – * The N2 (+) state may be observed for the strain ATCC 13047 and the strain ATCC 25922.4. Proteus mirabilis ATCC 35659 3.Fermentation of glucose (OF-F) : Inoculate an ampule of may be due to the reduction to nitrogen (as sometimes API OF Medium (see package insert).16 % of the strains were not identified. – – – + V + + + – – V + – – – – V – – – + – 5. obtained using the identification software. using colonies grown on Trypticase Soy agar + blood.90. substrate variations exist that also account for . .59 % of the strains were misidentified. Gram-negative rods included in the database biochemical reactions.Motility (MOB) : Inoculate an ampule of API M Medium enough and the following supplementary tests need to be (see package insert). (with or without supplementary tests).80 % of the strains were correctly identified percentage differences. if the tube section (Profile coding) of the Analytical Profile Index. They are due to the different belonging to species included in the database were principles of the reactions used in the API technique. may remains yellow this indicates a positive reaction (N2) to be used to form a 9-digit profile.2002/10 In some cases. in which instance a bluish-green color is seen.

colorless yellow (1) galactopyranoside Galactopyranosidase) ADH L-arginine 1.76 UREase yellow red / orange (2) TDA / immediate TDA L-tryptophane 0. and effluents produced according to their type and degree including any implied warranties of MERCHANTABILITY of hazardousness and to treat and dispose of them (or AND FITNESS FOR A PARTICULAR USE.2002/10 WASTE DISPOSAL WARRANTY It is the responsibility of each laboratory to handle waste bioMérieux disclaims all warranties.6 GELatinase no diffusion diffusion of black pigment (bovine origin) fermentation / oxidation GLU D-glucose 1.9 colorless pink / red (5) (Voges Proskauer) GEL Gelatin 0.9 blue / blue-green yellow (MANnitol) (4) fermentation / oxidation INO inositol 1.9 Lysine DeCarboxylase yellow red / orange (2) ODC L-ornithine 1.38 Tryptophane DeAminase yellow reddish brown JAMES / immediate colorless IND L-tryptophane 0. (4) Fermentation begins in the lower portion of the tubes.9 blue / blue-green yellow (SACcharose) (4) fermentation / oxidation MEL D-melibiose 1. oxidation begins in the cupule.19 INDole production pink pale green / yellow VP 1 + VP 2 / 10 min acetoin production VP sodium pyruvate 1.9 blue / blue-green yellow (MELibiose) (4) fermentation / oxidation AMY amygdalin 0. bioMérieux® sa English .756 CITrate utilization pale green / yellow blue-green / blue (3) H2S sodium thiosulfate 0. bioMérieux have them treated and disposed of) in accordance with shall not be liable for any incidental or consequential any applicable regulations. • The quantities indicated may be adjusted depending on the titer of the raw materials used. (2) An orange color after 36-48 hours incubation must be considered negative.9 blue / blue-green yellow (RHAmnose) (4) fermentation / oxidation SAC D-sucrose 1. • Certain cupules contain products of animal origin.GB .api® 20 E 07584D . express or implied.9 Ornithine DeCarboxylase yellow red / orange (2) CIT trisodium citrate 0.) NEGATIVE POSITIVE ß-galactosidase 2-nitrophenyl-ßD- ONPG 0. (5) A slightly pink color after 10 minutes should be considered negative.9 blue / blue-green yellow (ARAbinose) (4) OX (see oxidase test package insert) cytochrome-OXidase (see oxidase test package insert) (1) A very pale yellow should also be considered positive. (3) Reading made in the cupule (aerobic). IN NO EVENT SHALL BIOMERIEUX’S LIABLITY 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.9 blue / blue-green yellow (SORbitol) (4) fermentation / oxidation RHA L-rhamnose 1.223 (Ortho NitroPhenyl-ßD.57 blue / blue-green yellow (AMYgdalin) (4) fermentation / oxidation ARA L-arabinose 1.9 Arginine DiHydrolase yellow red / orange (2) LDC L-lysine 1.075 H2S production colorless / greyish black deposit / thin line URE urea 0. damages.9 blue / blue-green yellow / greyish yellow (GLUcose) (4) fermentation / oxidation MAN D-mannitol 1. notably peptones. READING TABLE QTY RESULTS TESTS ACTIVE INGREDIENTS REACTIONS/ENZYMES (mg/cup.4 .9 blue / blue-green yellow (INOsitol) (4) fermentation / oxidation SOR D-sorbitol 1.

oxidation : exposed to the green yellow air PROCEDURE p. 673 620 399 RCS LYON Durham. IV INDEX OF SYMBOLS p.076 GLU tube Zn / 5 min reduction to N2 gas orange-red yellow API M Medium or MOB .) NEGATIVE POSITIVE NIT 1 + NIT 2 / 2-5 min Nitrate reduction NO2 production yellow red potassium nitrate 0. growth absence presence OF-F .api® 20 E 07584D . (1) 919 620 20 00 Tel. I IDENTIFICATION TABLE p.GB . 33 (0)4 78 87 20 00 Fax (1) 919 620 22 11 Fax 33 (0)4 78 87 20 90 http://www.com Printed in France The logo is a registered and protected trademark of bioMérieux sa or one of its subsidiaries.biomerieux. motility non-motile motile microscope McC MacConkey medium . II LITERATURE REFERENCES p.2002/10 SUPPLEMENTARY TESTS RESULTS TESTS ACTIVE INGREDIENTS QTY REACTIONS/ENZYMES (mg/cup. V ® bioMérieux sa bioMérieux. fermentation : under green yellow mineral oil glucose (API OF Medium) OF-O . Inc au capital de 11 879 045 € Box 15969. NC 27704-0969 / USA 69280 Marcy-l'Etoile / France Tel. .