.%5cdocuments%5cmicro M403%5capi%5capi20e Insert 173zd
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20100/20160/20109/201790
008040-6 02/02
For in vitro diagnostic use Identification system for Enterobacteriaceae and other Gram-negative rods API 20 E is an identification system for Enterobacteriaceae and other non-fastidious Gram-negative rods which uses 23 standardized and miniaturized biochemical tests and a database. The complete list of those organisms that it is possible to identify with this system is given in the Identification Table at the end of this package insert.
PRINCIPLE The API 20 E strip consists of 20 microtubes containing dehydrated substrates. These tests are inoculated with a bacterial suspension which reconstitutes the media. During incubation, metabolism produces color changes that are either spontaneous or revealed by the addition of reagents. The reactions are read according to the Interpretation of Reactions (Table 2) and the identification is obtained by referring t the Analytical Profile Index or using the identification software. REAGENTS Kit contents ( Prod. no. 20100 OR 20109 - 25 tests) : - 25 API 20 E strips - 25 incubation boxes - 25 result sheets - 1 clip seal - 1 package insert
COMPOSITION OF MEDIA AND REAGENTS NaCl 0.85 % Medium 5 ml or Suspension Medium 5 ml
Required laboratory equipment : -
8.5 g 1000 ml
Demineralized water
Ferric chloride 30 ml Kovacs’ reagent 30 ml
10% aqueous ferric chloride solution in demineralized water Avoid skin Paradimethylaminobenzaldehyde Concentrated hydrochloric acid N-amyl alcohol
5% 24% 71%
HARMFUL R10 : Flammable. R20 : Harmful by inhalation. R34: Causes burns. R37: Irritating to respiratory system. S24/25 : Avoid with skin and eyes. S16: Keep away from sources of ignition. No smoking. S26: In case of with eyes, rinse immediately with plenty of water and seek medical advice. S36/37/39: Wear suitable protective clothing, gloves and eye/face protection. S45: In case of accident or if you feel unwell, seek medical advice immediately (show the label where possible).
Kit contents (Prod. No. 20160 OR 20179 - 100 tests) : - 100 API 20 E strips - 100 incubation boxes 100 result sheets 1 clip seal 1 package insert Additional products (not included in the kit) : - NaCl 0.85 % Medium, 5 ml (Prod. No. 20230) or Suspension Medium, 5ml (Prod. No. 20150) - Ferric chloride, 30ml (Prod. No. V7052) - VP1, 5ml (Prod. No. 70422) - VP2, 5ml (Prod. No. 70422) - NIT 1, 5ml (Prod. No. 70442) - NIT 2, 5ml (Prod. No. 70442) - Kovacs’ Reagent (Prod. No. V7050) - Hydrogen peroxide, 1.5% - Oxidase Test Kit (Prod. No. V7062) - Zinc Dust, 2x10g (Prod. No. 70380) - Mineral oil (Prod. No. 70100) - MacConkey Medium - Pasteur pipettes - API 20 E Analytical Profile Index (Prod. No. 20190) or identification software (consult bioMérieux) - Ampule rack (Prod. No. 70200) And possibly : - API OF Medium (Prod. No. 50110) : Test for the determination of fermentative or oxidative metabolism. - API M Medium (Prod No. 50120) Test for motility of facultative anaerobic bacteria.
Sodium chloride Demineralized water
VP1 5 ml
40% aqueous KOH solution in demineralized water
89% 11%
CORROSIVE R35 : Causes severe burns. S24/25: Avoid with skin and eyes. S26 : In case of with eyes, rinse immediately with plenty of water and seek medical advice. S36/37/39: Wear suitable protective clothing, gloves and eye/face protection. S45: In case of accident or if you feel unwell, seek medical advice immediately (show the label where possible).
VogesProskauer reagent : VP2 5 ml
NIT 1 5 ml
Alpha-naphthol Ethyl alcohol
6g 100 ml
FLAMMABLE AFTER RECONSTITUTION R21/22: Harmful in with skin and if swallowed. S24/25: Avoid with skin and eyes.
Sulfanilic acid Acetic acid 5N
0.8 % 99.2%
CORROSIVE R34 : Causes burns. S2 : Keep out of reach of children. S23 : Do not breathe vapours. S26 : In case of with eyes, rinse immediately with plenty of water and seek medical advice.
35-37°C incubator Refrigerator Bunsen burner Marker pen
NIT 2
5ml N,N-dimethyl-1-naphthylamine Acetic acid 5N
0.5% 99.5%
CORROSIVE R34 : Causes burns. S2 : Keep out of reach of children. S23 : Do not breathe vapours. S26 : In case of with eyes, rinse immediately with plenty of water and seek medical advice.
Oxidase Test Reagent
Oxidase Reagent: see Oxidase Test Kit Package Insert.
1
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api 20 E
Zinc dust 10 g
0008040-6 02/02
Zinc dust FLAMMABLE R15 : with water liberates highly flammable gases. R17 : Spontaneously flammable in air. S7/8 : Keep container tightly closed and dry. S43 : In case of fire caused by metals, use powder extinguishers. 'Never use water'.
STORAGE OF THE STRIPS The strips are supplied in an aluminum pouch with desiccant sachets. Once opened, the pouch should be re-sealed using the clip seal (included in the kit) to preserve the remaining strips with the desiccant sachets : place the open end of the pouch along the seal and carefully clamp between the two parts. The strips may then be kept for up to 10 months after the pouch has been opened, at 2-8°C (or until the expiration date indicated on the packaging, if this comes before).
STORAGE OF THE REAGENTS Kovacs’ reagent, VP1, NIT 1 and NIT 2 should be stored at 15-30°C until the expiration date indicated on the packaging. Ferric chloride should be stored at 2-30°C until the expiration date indicated on the packaging. Zinc dust should be stored at 8-30°C until the expiration date indicated on the packaging. VP2 should be stored in the dark at 2-8°C until the expiration date if this comes first. Record the date opened on the bottle label. See Oxidase Test Kit Package Insert for storage instructions.
USE OF THE REAGENTS Allow reagents to come to room temperature (20-30°C) before using.
1. Ferric chloride, VP1, NIT 1, NIT 2 and Kovacs’:
• Dispense one drop of reagent. • Carefully close the bottle after use and store it as indicated in the paragraph "Storage of the Reagents".
2. VP2 :
• Open the ampule of reagent as indicated in the paragaraph "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".
3. Zinc dust :
• Open the bottle • Take up an aliquot of powder (approximately 2-3 mg) using the spatula fixed to the cap and deposit this quantity in the reaction cupule. • Carefully close the bottle after use and store it as indicated in the paragraph "Storage of the reagents".
4. Oxidase Test Kit:
• Please note that the oxidase test must be performed as it is an integral part of the st final profile (21 identification test .) See the Oxidase Test Kit Package Insert for the use of the oxidase reagent.
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 allow reagents to come into with skin, eyes, or clothing. • Do not use reagents past the expiration date. • Upon removal from refrigerator, allow reagents to come to room temperature (2030°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 to the flattened part of the cap to snap off the top of the ampoule 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 in a vertical position. - Squeeze on the cap to transfer all the reagent into the dropperbottle. • 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. • 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. • The performance data presented were obtained using the procedure indicated. Any changes or modifications in the procedure may affect results.
INSTRUCTIONS FOR USE
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 Diseases 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.
Preparation of the strip • 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, CO2, 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. • Place the strip in the tray. • Perform the oxidase test on a colony identical to the colony which will be tested. Refer to the Oxidase Test Kit Package Insert. This reaction should be recorded on the result sheet as it constitutes the 21st test. NOTE : API 20 E should only be used with non-fastidious Gram-negative rods. Fastidious organisms having demanding nutritional requirements and requiring appropriate handling precautions (i.e., Brucella and Francisella) are not included in the API 20 E database. Alternative procedures must be used to exclude or confirm their presence.
Preparation of the inoculum • Open an ampule of NaCl 0.85 % Medium (5 ml) or an ampule of Suspension Medium (5ml) as indicated in the paragraph "Warnings and Precautions" or use any tube containing 5 ml of sterile NaCl 0.85% Medium, pH 5.5 - 7.0, or sterile distilled water, without additives. • With the aid of a pipette, remove a single well-isolated colony from an isolation plate. • Carefully emulsify to achieve a homogeneous bacterial suspension. NOTE : Most Vibrio species are halophilic. If Vibrio is suspected, suspend the bacteria in NaCl 0.85 % Medium.
Inoculation of the strip 2
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• With the same pipette, fill both tube and cupule of test CIT , VP and GEL with the bacterial suspension. • Fill only the tubes (and not the cupules) of the other tests. • Create anaerobiosis in the tests ____ ADH, ____ LDC , ____ ODC, ____ H2S and URE ____ by overlaying with mineral oil. • Close the incubation box. • Incubate at 35-37°C for 18-24 hours. • Weekend incubation : The biochemical reactions of the API 20 E strip should be read after 18-24 hours of incubation. If the strips cannot be read after 24 hours of incubation at 35-37°C, the strips should be removed from the incubator and stored at 2-8°C (refrigerator) until the reactions can be read.
Reading the strip • After 18-24 hours at 35-37°C, read the strip by referring to Interpretation of Reactions (Table 2.) • Record all spontaneous reactions on the result sheet. • If the glucose is positive or 3 or more tests are positive, reveal the tests which require the addition of reagents : - TDA Test : add 1 drop of Ferric chloride. A dark brown color indicates a positive reaction to be recorded on the result sheet. - IND Test : add 1 drop of Kovacs’ reagent. Wait 2 minutes. A red ring indicates a positive reaction to be recorded on the result sheet. - VP Test : add 1 drop of each of VP1 and VP2 reagents. Wait at least 10 minutes. A pink or red color indicates a positive reaction to be recorded on the result sheet. If a slightly pink color appears within 10 to 12 minutes, the reaction should be considered negative. NO2 Test : add 2 drops of each of NIT 1 and NIT 2 reagents to the GLU tube. Wait 2 to 3 minutes. A red color indicates a positive reaction. A negative reaction (yellow) may be due to the reduction to nitrogen (as sometimes evidenced by gas bubbles) : add 2 to 3 mg of Zinc dust to the GLU tube. After 5 minutes, if the tube remains yellow this indicates that (N2) is positive and is to be recorded on the result sheet. If the test turns pink-red, this is a negative reaction : the nitrates still present in the tube have been reduced by the Zinc. This reaction is useful when testing Gram-negative, oxidase positive rods. NOTE : The nitrate reduction and indole production tests must be performed last since these reactions release gaseous products which interfere with the interpretation of other tests on the strip. The plastic incubation lid should not be replaced after the addition of these reagents. • If the glucose is negative and the number of positive tests is less than or equal to 2, do not add reagents : - Inoculate 2 ampules of API OF Medium to confirm the metabolism of glucose. - Streak a MacConkey agar plate. - Check for motility : inoculate 1 ampule of API M Medium or observe between slide and cover slip under a microscope. - Reincubate the strip for 24 hours. - Add the reagents as described above. - Record the strip and supplementary test results on the result sheet by referring to Interpretation of Reactions (Table 2).
Identification Identification can be obtained : • 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 for the 20 tests of the API 20 E strip. The oxidase reaction constitutes the 21st test and has a value of 4 if it is positive. • using the identification software to obtain information on any profile not listed in the Analytical Profile Index, call the Voice Response System at 800-645-7056. In some cases, the 7-digit profile is not discriminatory enough and the following supplementary tests need to be carried out : -
Reduction of nitrates to nitrites (NO2) Reduction of nitrates to N2 gas (N2) Motility (MOB) Growth on MacConkey agar medium (McC) Oxidation of glucose (OF-O) Fermentation of glucose (OF-F)
These supplementary tests, indicated in the Analytical Profile Index, may be used to form a 9-digit profile. Identification is then obtained using the identification software.
5 315 173 (57) Enterobacter gergoviae
DISPOSAL OF USED MATERIAL After use, all ampules, pipettes, strips and incubation boxes should be autoclaved, incinerated, or immersed in a disinfectant for decontamination prior to disposal.
RANGE OF EXPECTED VALUES Consult the Identification Table at the end of this package insert for the range of expected values for the various biochemical reactions.
LIMITATIONS • The API 20 E system is intended uniquely for the identification of those nonfastidious, Gram-negative rods 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. • Discrepancies with respect to conventional methods may be observed due to the different principles of the reactions used in the API technique. • If Salmonella spp., Shigella spp., are identified, serological testing must be performed to confirm the bacterial identification.
QUALITY CONTROL The media, strips, and reagents are systematically quality controlled at various stages of their manufacture. For those who wish to perform their own quality control tests with the strip, it is recommended that the following stock cultures be used, to obtain the results below : Table 1 ONPG ____ ADH ____ LDC ODC ____ 1. 2. 3. 4. 5.
+ + – + +
– + – – –
V – – + +
– + + – +
CIT V + V + –
1. Stenotrophomonas maltophilia 2. Enterobacter cloacae 3. Proteus mirabilis
H2S URE ____ TDA IND VP – – + – –
– – + V –
– – + – –
– – – – +
– + – V –
ATCC 51331 ATCC 13047 ATCC 35659
GEL GLU MAN INO SOR RHA SAC MEL AMY ARA NO2 N2 + – V – –
– + + + +
– + – + +
– V – + –
– + – + +
– + – + +
– + V + –
– + – + +
– + – + –
– + – + +
– + + + +
– – – – –
4. Klebsiella pneumoniae ssp pneumoniae ATCC 35657 5. Escherichia coli ATCC 25922
ATCC : American Type Culture Collection, 10801 University Boulevard, Manassas, VA 20110-2209, USA. • Profile obtained after 24-48 hours of incubation for the strain ATCC 51331, using cultures grown on Trypticase Soy agar + blood • Profiles obtained after 18-24 hours of incubation for the other strains, using cultures grown on Trypticase Soy agar + blood • Bacterial suspension prepared in NaCl 0.85 % Medium
PERFORMANCE CHARACTERISTICS The Profile Recognition System has been evaluated by the National Institutes of Health, Bethesda, Maryland. The API 20E System has been evaluated extensively worldwide by institutions such as the Centers for Disease Control, Atlanta, Georgia; the Mayo Clinic, Rochester, Minnesota; the Pasteur 3
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0008040-6 02/02
Institute, Lyon, ; Sherbrooke University, Sherbrooke, Quebec, Canada. In fact, the API 20E System was one of the original miniaturized biochemical detection systems and has evolved over the years as the standard against which other identification systems have been compared. Recently, the Centers for Disease Control reevaluated the identification capabilities of the API 20 E System and reported that at 24 hours, 20 E correctly identified to genus and species level 87.7% of a clinically weighted assortment of isolates. At 48 hours, 96.3% of the isolates were identified correctly (O’Hara, C.M., D.L. Rhoden, and J.M. Miller, 1992. J. Clin. Microbiol. 30: 123-125). These percentages reflect the product’s identification capabilities for taxa that are identified routinely on the 20 E System. In addition, the API 20E System permits recognition of biotypes within each species. Delineation of biotypes may be of value in determining the effectiveness of treatment and the existence of nosocomial infections as related to of the family Enterobacteriaceae, as well as for control of the latter.
bioMérieux sa
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INTERPRETATION OF REACTIONS Table 2 TESTS
SUBSTRATES
QTY
ENZYMATIC ACTIVITY OR REACTION TESTED
RESULTS NEGATIVE
POSITIVE
beta-galactosidase
colorless
yellow (1)
red / orange (2)
ortho-nitrophenyl-‚ -Dgalactopyranoside(ONPG)
0.2 mg
isopropylthiogalactopyranoside (IPTG)
8.0 µg
ADH ____
arginine
2.0 mg
arginine dihydrolase
yellow
LDC ____
lysine
2.0 mg
lysine decarboxylase
yellow
red/orange (2)
ODC ____
ornithine
2.0 mg
ornithine decarboxylase
yellow
red / orange (2)
sodium citrate
0.8 mg
citrate utilization
pale green / yellow
blue-green / blue (3)
H2S ___
sodium thiosulfate
80.0 µg
H2S production
colorless / greyish
black deposit / thin line
URE ____
urea
0.8 mg
urease
yellow
red / orange (2)
tryptophane deaminase
Ferric chloride (1 drop) / immediate yellow brown-red
ONPG
CIT
TDA
tryptophane
0.4 mg
Kovacs’ reagent (1 drop) / 2 min IND
VP GEL
tryptophane
0.2 mg
creatine
0.9 mg
sodium pyruvate
2.0 mg
Kohn's charcoal gelatin
0.6 mg
indole production
IND yellow
IND red ring
VP1 (1 drop) + VP2 (1 drop) / acetoin production gelatinase
10 min
colorless
pink / red (5)
no diffusion of black pigment
diffusion of black pigment
GLU
glucose
2.0 mg
fermentation / oxidation (4)
blue / blue-green
yellow / greyish yellow
MAN
mannitol
2.0 mg
fermentation / oxidation (4)
blue / blue-green
yellow
INO
inositol
2.0 mg
fermentation / oxidation (4)
blue / blue-green
yellow
SOR
sorbitol
2.0 mg
fermentation / oxidation (4)
blue / blue-green
yellow
RHA
rhamnose
2.0 mg
fermentation / oxidation (4)
blue / blue-green
yellow
SAC
sucrose
2.0 mg
fermentation / oxidation (4)
blue / blue-green
yellow
MEL
melibiose
2.0 mg
fermentation / oxidation (4)
blue / blue-green
yellow
AMY
amygdalin
2.0 mg
fermentation / oxidation (4)
blue / blue-green
yellow
ARA
arabinose
2.0 mg
fermentation / oxidation (4)
blue / blue-green
yellow
OX
on filter paper
Oxidase reagent / 1-2 min colorless violet
cytochrome-oxidase
NIT 1 (2 drops) + NIT 2 (2 drops) / 2-3 min GLU tube Nitrate Reduction
MOB
yellow
NO2 production Potassium nitrate
API M Medium or microscope
80.0 µl
red Zinc Dust / 5 min
reduction to N2 gas
orange-red
yellow
motility
non-motile
motile
McC
MacConkey medium
growth
absence
presence
OF-F OF-O
glucose (API OF Medium)
fermentation : under mineral oil oxidation : exposed to the air
green green
yellow yellow
(1) A very pale yellow should also be considered positive. (2) An orange color after 36-48 hours incubation must be considered negative. (3) Reading made in the cupule (aerobic).
(4) Fermentation begins in the lower portion of the tubes, oxidation begins in the cupules. (5) A slightly pink color after 10 minutes should be considered negative.
5
api 20 E
0
008040-6 02/02 RECOMMENDED METHODOLOGY
1 colony
NaCl 0.85 % Medium 5 ml / Suspension Medium 5 ml
- | CIT | - | VP | - | GEL | api 20 E ADH ODC H2S - URE
18:00-24:00 / 48:00
37°C
Tests ⊕ ≤ 2 and GLU (-) api 20 E
TDA IND VP GLU (NO2) OX
: Ferric chloride : Kovacs’ reagent : VP1 + VP2 : NIT 1 + NIT 2 : Oxidase test reagent
api 20 E
+ - + - + -
6
20100/20160/20109/201790
008040-6 02/02
For in vitro diagnostic use Identification system for Enterobacteriaceae and other Gram-negative rods API 20 E is an identification system for Enterobacteriaceae and other non-fastidious Gram-negative rods which uses 23 standardized and miniaturized biochemical tests and a database. The complete list of those organisms that it is possible to identify with this system is given in the Identification Table at the end of this package insert.
PRINCIPLE The API 20 E strip consists of 20 microtubes containing dehydrated substrates. These tests are inoculated with a bacterial suspension which reconstitutes the media. During incubation, metabolism produces color changes that are either spontaneous or revealed by the addition of reagents. The reactions are read according to the Interpretation of Reactions (Table 2) and the identification is obtained by referring t the Analytical Profile Index or using the identification software. REAGENTS Kit contents ( Prod. no. 20100 OR 20109 - 25 tests) : - 25 API 20 E strips - 25 incubation boxes - 25 result sheets - 1 clip seal - 1 package insert
COMPOSITION OF MEDIA AND REAGENTS NaCl 0.85 % Medium 5 ml or Suspension Medium 5 ml
Required laboratory equipment : -
8.5 g 1000 ml
Demineralized water
Ferric chloride 30 ml Kovacs’ reagent 30 ml
10% aqueous ferric chloride solution in demineralized water Avoid skin Paradimethylaminobenzaldehyde Concentrated hydrochloric acid N-amyl alcohol
5% 24% 71%
HARMFUL R10 : Flammable. R20 : Harmful by inhalation. R34: Causes burns. R37: Irritating to respiratory system. S24/25 : Avoid with skin and eyes. S16: Keep away from sources of ignition. No smoking. S26: In case of with eyes, rinse immediately with plenty of water and seek medical advice. S36/37/39: Wear suitable protective clothing, gloves and eye/face protection. S45: In case of accident or if you feel unwell, seek medical advice immediately (show the label where possible).
Kit contents (Prod. No. 20160 OR 20179 - 100 tests) : - 100 API 20 E strips - 100 incubation boxes 100 result sheets 1 clip seal 1 package insert Additional products (not included in the kit) : - NaCl 0.85 % Medium, 5 ml (Prod. No. 20230) or Suspension Medium, 5ml (Prod. No. 20150) - Ferric chloride, 30ml (Prod. No. V7052) - VP1, 5ml (Prod. No. 70422) - VP2, 5ml (Prod. No. 70422) - NIT 1, 5ml (Prod. No. 70442) - NIT 2, 5ml (Prod. No. 70442) - Kovacs’ Reagent (Prod. No. V7050) - Hydrogen peroxide, 1.5% - Oxidase Test Kit (Prod. No. V7062) - Zinc Dust, 2x10g (Prod. No. 70380) - Mineral oil (Prod. No. 70100) - MacConkey Medium - Pasteur pipettes - API 20 E Analytical Profile Index (Prod. No. 20190) or identification software (consult bioMérieux) - Ampule rack (Prod. No. 70200) And possibly : - API OF Medium (Prod. No. 50110) : Test for the determination of fermentative or oxidative metabolism. - API M Medium (Prod No. 50120) Test for motility of facultative anaerobic bacteria.
Sodium chloride Demineralized water
VP1 5 ml
40% aqueous KOH solution in demineralized water
89% 11%
CORROSIVE R35 : Causes severe burns. S24/25: Avoid with skin and eyes. S26 : In case of with eyes, rinse immediately with plenty of water and seek medical advice. S36/37/39: Wear suitable protective clothing, gloves and eye/face protection. S45: In case of accident or if you feel unwell, seek medical advice immediately (show the label where possible).
VogesProskauer reagent : VP2 5 ml
NIT 1 5 ml
Alpha-naphthol Ethyl alcohol
6g 100 ml
FLAMMABLE AFTER RECONSTITUTION R21/22: Harmful in with skin and if swallowed. S24/25: Avoid with skin and eyes.
Sulfanilic acid Acetic acid 5N
0.8 % 99.2%
CORROSIVE R34 : Causes burns. S2 : Keep out of reach of children. S23 : Do not breathe vapours. S26 : In case of with eyes, rinse immediately with plenty of water and seek medical advice.
35-37°C incubator Refrigerator Bunsen burner Marker pen
NIT 2
5ml N,N-dimethyl-1-naphthylamine Acetic acid 5N
0.5% 99.5%
CORROSIVE R34 : Causes burns. S2 : Keep out of reach of children. S23 : Do not breathe vapours. S26 : In case of with eyes, rinse immediately with plenty of water and seek medical advice.
Oxidase Test Reagent
Oxidase Reagent: see Oxidase Test Kit Package Insert.
1
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api 20 E
Zinc dust 10 g
0008040-6 02/02
Zinc dust FLAMMABLE R15 : with water liberates highly flammable gases. R17 : Spontaneously flammable in air. S7/8 : Keep container tightly closed and dry. S43 : In case of fire caused by metals, use powder extinguishers. 'Never use water'.
STORAGE OF THE STRIPS The strips are supplied in an aluminum pouch with desiccant sachets. Once opened, the pouch should be re-sealed using the clip seal (included in the kit) to preserve the remaining strips with the desiccant sachets : place the open end of the pouch along the seal and carefully clamp between the two parts. The strips may then be kept for up to 10 months after the pouch has been opened, at 2-8°C (or until the expiration date indicated on the packaging, if this comes before).
STORAGE OF THE REAGENTS Kovacs’ reagent, VP1, NIT 1 and NIT 2 should be stored at 15-30°C until the expiration date indicated on the packaging. Ferric chloride should be stored at 2-30°C until the expiration date indicated on the packaging. Zinc dust should be stored at 8-30°C until the expiration date indicated on the packaging. VP2 should be stored in the dark at 2-8°C until the expiration date if this comes first. Record the date opened on the bottle label. See Oxidase Test Kit Package Insert for storage instructions.
USE OF THE REAGENTS Allow reagents to come to room temperature (20-30°C) before using.
1. Ferric chloride, VP1, NIT 1, NIT 2 and Kovacs’:
• Dispense one drop of reagent. • Carefully close the bottle after use and store it as indicated in the paragraph "Storage of the Reagents".
2. VP2 :
• Open the ampule of reagent as indicated in the paragaraph "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".
3. Zinc dust :
• Open the bottle • Take up an aliquot of powder (approximately 2-3 mg) using the spatula fixed to the cap and deposit this quantity in the reaction cupule. • Carefully close the bottle after use and store it as indicated in the paragraph "Storage of the reagents".
4. Oxidase Test Kit:
• Please note that the oxidase test must be performed as it is an integral part of the st final profile (21 identification test .) See the Oxidase Test Kit Package Insert for the use of the oxidase reagent.
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 allow reagents to come into with skin, eyes, or clothing. • Do not use reagents past the expiration date. • Upon removal from refrigerator, allow reagents to come to room temperature (2030°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 to the flattened part of the cap to snap off the top of the ampoule 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 in a vertical position. - Squeeze on the cap to transfer all the reagent into the dropperbottle. • 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. • 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. • The performance data presented were obtained using the procedure indicated. Any changes or modifications in the procedure may affect results.
INSTRUCTIONS FOR USE
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 Diseases 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.
Preparation of the strip • 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, CO2, 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. • Place the strip in the tray. • Perform the oxidase test on a colony identical to the colony which will be tested. Refer to the Oxidase Test Kit Package Insert. This reaction should be recorded on the result sheet as it constitutes the 21st test. NOTE : API 20 E should only be used with non-fastidious Gram-negative rods. Fastidious organisms having demanding nutritional requirements and requiring appropriate handling precautions (i.e., Brucella and Francisella) are not included in the API 20 E database. Alternative procedures must be used to exclude or confirm their presence.
Preparation of the inoculum • Open an ampule of NaCl 0.85 % Medium (5 ml) or an ampule of Suspension Medium (5ml) as indicated in the paragraph "Warnings and Precautions" or use any tube containing 5 ml of sterile NaCl 0.85% Medium, pH 5.5 - 7.0, or sterile distilled water, without additives. • With the aid of a pipette, remove a single well-isolated colony from an isolation plate. • Carefully emulsify to achieve a homogeneous bacterial suspension. NOTE : Most Vibrio species are halophilic. If Vibrio is suspected, suspend the bacteria in NaCl 0.85 % Medium.
Inoculation of the strip 2
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0008040-6 02/02
• With the same pipette, fill both tube and cupule of test CIT , VP and GEL with the bacterial suspension. • Fill only the tubes (and not the cupules) of the other tests. • Create anaerobiosis in the tests ____ ADH, ____ LDC , ____ ODC, ____ H2S and URE ____ by overlaying with mineral oil. • Close the incubation box. • Incubate at 35-37°C for 18-24 hours. • Weekend incubation : The biochemical reactions of the API 20 E strip should be read after 18-24 hours of incubation. If the strips cannot be read after 24 hours of incubation at 35-37°C, the strips should be removed from the incubator and stored at 2-8°C (refrigerator) until the reactions can be read.
Reading the strip • After 18-24 hours at 35-37°C, read the strip by referring to Interpretation of Reactions (Table 2.) • Record all spontaneous reactions on the result sheet. • If the glucose is positive or 3 or more tests are positive, reveal the tests which require the addition of reagents : - TDA Test : add 1 drop of Ferric chloride. A dark brown color indicates a positive reaction to be recorded on the result sheet. - IND Test : add 1 drop of Kovacs’ reagent. Wait 2 minutes. A red ring indicates a positive reaction to be recorded on the result sheet. - VP Test : add 1 drop of each of VP1 and VP2 reagents. Wait at least 10 minutes. A pink or red color indicates a positive reaction to be recorded on the result sheet. If a slightly pink color appears within 10 to 12 minutes, the reaction should be considered negative. NO2 Test : add 2 drops of each of NIT 1 and NIT 2 reagents to the GLU tube. Wait 2 to 3 minutes. A red color indicates a positive reaction. A negative reaction (yellow) may be due to the reduction to nitrogen (as sometimes evidenced by gas bubbles) : add 2 to 3 mg of Zinc dust to the GLU tube. After 5 minutes, if the tube remains yellow this indicates that (N2) is positive and is to be recorded on the result sheet. If the test turns pink-red, this is a negative reaction : the nitrates still present in the tube have been reduced by the Zinc. This reaction is useful when testing Gram-negative, oxidase positive rods. NOTE : The nitrate reduction and indole production tests must be performed last since these reactions release gaseous products which interfere with the interpretation of other tests on the strip. The plastic incubation lid should not be replaced after the addition of these reagents. • If the glucose is negative and the number of positive tests is less than or equal to 2, do not add reagents : - Inoculate 2 ampules of API OF Medium to confirm the metabolism of glucose. - Streak a MacConkey agar plate. - Check for motility : inoculate 1 ampule of API M Medium or observe between slide and cover slip under a microscope. - Reincubate the strip for 24 hours. - Add the reagents as described above. - Record the strip and supplementary test results on the result sheet by referring to Interpretation of Reactions (Table 2).
Identification Identification can be obtained : • 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 for the 20 tests of the API 20 E strip. The oxidase reaction constitutes the 21st test and has a value of 4 if it is positive. • using the identification software to obtain information on any profile not listed in the Analytical Profile Index, call the Voice Response System at 800-645-7056. In some cases, the 7-digit profile is not discriminatory enough and the following supplementary tests need to be carried out : -
Reduction of nitrates to nitrites (NO2) Reduction of nitrates to N2 gas (N2) Motility (MOB) Growth on MacConkey agar medium (McC) Oxidation of glucose (OF-O) Fermentation of glucose (OF-F)
These supplementary tests, indicated in the Analytical Profile Index, may be used to form a 9-digit profile. Identification is then obtained using the identification software.
5 315 173 (57) Enterobacter gergoviae
DISPOSAL OF USED MATERIAL After use, all ampules, pipettes, strips and incubation boxes should be autoclaved, incinerated, or immersed in a disinfectant for decontamination prior to disposal.
RANGE OF EXPECTED VALUES Consult the Identification Table at the end of this package insert for the range of expected values for the various biochemical reactions.
LIMITATIONS • The API 20 E system is intended uniquely for the identification of those nonfastidious, Gram-negative rods 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. • Discrepancies with respect to conventional methods may be observed due to the different principles of the reactions used in the API technique. • If Salmonella spp., Shigella spp., are identified, serological testing must be performed to confirm the bacterial identification.
QUALITY CONTROL The media, strips, and reagents are systematically quality controlled at various stages of their manufacture. For those who wish to perform their own quality control tests with the strip, it is recommended that the following stock cultures be used, to obtain the results below : Table 1 ONPG ____ ADH ____ LDC ODC ____ 1. 2. 3. 4. 5.
+ + – + +
– + – – –
V – – + +
– + + – +
CIT V + V + –
1. Stenotrophomonas maltophilia 2. Enterobacter cloacae 3. Proteus mirabilis
H2S URE ____ TDA IND VP – – + – –
– – + V –
– – + – –
– – – – +
– + – V –
ATCC 51331 ATCC 13047 ATCC 35659
GEL GLU MAN INO SOR RHA SAC MEL AMY ARA NO2 N2 + – V – –
– + + + +
– + – + +
– V – + –
– + – + +
– + – + +
– + V + –
– + – + +
– + – + –
– + – + +
– + + + +
– – – – –
4. Klebsiella pneumoniae ssp pneumoniae ATCC 35657 5. Escherichia coli ATCC 25922
ATCC : American Type Culture Collection, 10801 University Boulevard, Manassas, VA 20110-2209, USA. • Profile obtained after 24-48 hours of incubation for the strain ATCC 51331, using cultures grown on Trypticase Soy agar + blood • Profiles obtained after 18-24 hours of incubation for the other strains, using cultures grown on Trypticase Soy agar + blood • Bacterial suspension prepared in NaCl 0.85 % Medium
PERFORMANCE CHARACTERISTICS The Profile Recognition System has been evaluated by the National Institutes of Health, Bethesda, Maryland. The API 20E System has been evaluated extensively worldwide by institutions such as the Centers for Disease Control, Atlanta, Georgia; the Mayo Clinic, Rochester, Minnesota; the Pasteur 3
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0008040-6 02/02
Institute, Lyon, ; Sherbrooke University, Sherbrooke, Quebec, Canada. In fact, the API 20E System was one of the original miniaturized biochemical detection systems and has evolved over the years as the standard against which other identification systems have been compared. Recently, the Centers for Disease Control reevaluated the identification capabilities of the API 20 E System and reported that at 24 hours, 20 E correctly identified to genus and species level 87.7% of a clinically weighted assortment of isolates. At 48 hours, 96.3% of the isolates were identified correctly (O’Hara, C.M., D.L. Rhoden, and J.M. Miller, 1992. J. Clin. Microbiol. 30: 123-125). These percentages reflect the product’s identification capabilities for taxa that are identified routinely on the 20 E System. In addition, the API 20E System permits recognition of biotypes within each species. Delineation of biotypes may be of value in determining the effectiveness of treatment and the existence of nosocomial infections as related to of the family Enterobacteriaceae, as well as for control of the latter.
bioMérieux sa
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INTERPRETATION OF REACTIONS Table 2 TESTS
SUBSTRATES
QTY
ENZYMATIC ACTIVITY OR REACTION TESTED
RESULTS NEGATIVE
POSITIVE
beta-galactosidase
colorless
yellow (1)
red / orange (2)
ortho-nitrophenyl-‚ -Dgalactopyranoside(ONPG)
0.2 mg
isopropylthiogalactopyranoside (IPTG)
8.0 µg
ADH ____
arginine
2.0 mg
arginine dihydrolase
yellow
LDC ____
lysine
2.0 mg
lysine decarboxylase
yellow
red/orange (2)
ODC ____
ornithine
2.0 mg
ornithine decarboxylase
yellow
red / orange (2)
sodium citrate
0.8 mg
citrate utilization
pale green / yellow
blue-green / blue (3)
H2S ___
sodium thiosulfate
80.0 µg
H2S production
colorless / greyish
black deposit / thin line
URE ____
urea
0.8 mg
urease
yellow
red / orange (2)
tryptophane deaminase
Ferric chloride (1 drop) / immediate yellow brown-red
ONPG
CIT
TDA
tryptophane
0.4 mg
Kovacs’ reagent (1 drop) / 2 min IND
VP GEL
tryptophane
0.2 mg
creatine
0.9 mg
sodium pyruvate
2.0 mg
Kohn's charcoal gelatin
0.6 mg
indole production
IND yellow
IND red ring
VP1 (1 drop) + VP2 (1 drop) / acetoin production gelatinase
10 min
colorless
pink / red (5)
no diffusion of black pigment
diffusion of black pigment
GLU
glucose
2.0 mg
fermentation / oxidation (4)
blue / blue-green
yellow / greyish yellow
MAN
mannitol
2.0 mg
fermentation / oxidation (4)
blue / blue-green
yellow
INO
inositol
2.0 mg
fermentation / oxidation (4)
blue / blue-green
yellow
SOR
sorbitol
2.0 mg
fermentation / oxidation (4)
blue / blue-green
yellow
RHA
rhamnose
2.0 mg
fermentation / oxidation (4)
blue / blue-green
yellow
SAC
sucrose
2.0 mg
fermentation / oxidation (4)
blue / blue-green
yellow
MEL
melibiose
2.0 mg
fermentation / oxidation (4)
blue / blue-green
yellow
AMY
amygdalin
2.0 mg
fermentation / oxidation (4)
blue / blue-green
yellow
ARA
arabinose
2.0 mg
fermentation / oxidation (4)
blue / blue-green
yellow
OX
on filter paper
Oxidase reagent / 1-2 min colorless violet
cytochrome-oxidase
NIT 1 (2 drops) + NIT 2 (2 drops) / 2-3 min GLU tube Nitrate Reduction
MOB
yellow
NO2 production Potassium nitrate
API M Medium or microscope
80.0 µl
red Zinc Dust / 5 min
reduction to N2 gas
orange-red
yellow
motility
non-motile
motile
McC
MacConkey medium
growth
absence
presence
OF-F OF-O
glucose (API OF Medium)
fermentation : under mineral oil oxidation : exposed to the air
green green
yellow yellow
(1) A very pale yellow should also be considered positive. (2) An orange color after 36-48 hours incubation must be considered negative. (3) Reading made in the cupule (aerobic).
(4) Fermentation begins in the lower portion of the tubes, oxidation begins in the cupules. (5) A slightly pink color after 10 minutes should be considered negative.
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IDENTIFICATION TABLE % of positive reactions after 24-48 hrs at 35-37° ONPG 100
ADH 0
LDC 0
ODC 85
CIT 25
H2 S 0
URE 0
TDA 0
IND 0
VP 0
GEL 0
GLU 100
MAN 100
INO 0
SOR 1
RHA 99
SAC 0
MEL 92
AMY 99
ARA 100
OX 0
NO 2 100
N2 0
MOB 100
McC 100
OF/O 100
OF/F 100
Cedecea davisae 100
99
89
0
99
75
0
0
0
0
89
0
100
100
10
0
0
100
0
100
1
0
99
0
87
100
100
100
Cedecea lapagei
99
99
0
0
75
0
0
0
0
90
0
100
99
0
0
0
0
1
100
1
0
99
0
87
100
100
100
Citrobacter braakii
50
45
0
99
75
81
1
0
4
0
0
100
100
1
100
100
1
91
99
99
0
100
0
95
100
100
100
Citrobacter freundii
90
24
0
0
75
75
1
0
1
0
0
100
99
25
99
99
99
82
40
99
0
98
0
95
100
100
100
Citrobacter koseri/amalonaticus
99
75
0
100
97
0
1
0
99
0
0
100
100
25
99
99
1
1
98
99
0
100
0
95
100
100
100
Citrobacter koseri/farmeri
99
2
0
100
25
0
1
0
99
0
0
100
100
1
99
99
99
80
99
99
0
100
0
95
100
100
100
Citrobacter youngae
100
50
0
1
80
80
0
0
1
0
0
100
100
0
95
100
1
0
25
100
0
85
0
95
100
100
100
Edwardsiella hoshinae
0
0
100
99
50
94
0
0
99
0
0
100
100
0
0
1
100
0
0
1
0
100
0
100
100
100
100
Edwardsiella tarda
0
0
100
99
1
75
0
0
99
0
0
100
0
0
0
0
0
0
0
0
0
100
0
98
100
100
100
Enterobacter aerogenes
99
0
99
98
82
0
1
0
0
85
0
99
99
99
99
99
99
99
99
99
0
100
0
97
100
100
100
Enterobacter amnigenus 1
99
25
0
99
40
0
0
0
0
75
0
100
100
0
1
100
99
99
99
99
0
100
0
92
100
100
100
Enterobacter amnigenus 2
99
80
0
99
80
0
0
0
0
75
0
100
100
0
99
100
1
99
99
99
0
100
0
100
100
100
100
Enterobacter asburiae
100
25
0
99
80
0
0
0
0
10
0
100
99
25
100
0
99
0
100
100
0
100
0
95
100
100
100
Enterobacter cancerogenus
100
75
0
99
99
0
0
0
0
89
0
100
100
0
1
100
1
1
100
100
0
100
0
99
100
100
100
Enterobacter cloacae
98
82
1
92
90
0
1
0
0
85
0
99
99
12
90
85
96
90
99
99
0
100
0
95
100
100
100
Enterobacter gergoviae
99
0
32
100
75
0
99
0
0
90
0
100
99
23
1
100
99
100
99
100
0
100
0
90
100
100
100
Enterobacter intermedius
99
0
0
99
1
0
0
0
0
2
0
100
97
0
88
99
40
100
99
99
0
100
0
92
100
100
100
Enterobacter sakazakii
100
96
0
91
94
0
1
0
25
91
10
100
100
75
8
99
99
99
99
99
0
100
0
96
100
100
100
Escherichia coli 1
90
1
74
70
0
1
3
0
89
0
0
99
98
1
91
82
36
75
3
99
0
100
0
95
100
100
100
Escherichia coli 2
26
1
45
20
0
1
1
0
50
0
0
99
90
1
42
30
3
3
1
70
0
98
0
5
100
100
100
Escherichia fergusonii
96
1
99
100
1
0
0
0
99
0
0
100
99
1
0
87
0
1
99
99
0
100
0
93
100
100
100
Escherichia hermannii
100
0
1
100
1
0
0
0
99
0
0
100
100
0
0
99
25
0
99
99
0
100
0
99
100
100
100
Escherichia vulneris
100
30
50
0
0
0
0
0
0
0
0
100
100
0
1
95
7
95
95
99
0
100
0
100
100
100
100
Ewingella americana
98
0
0
0
75
0
0
0
0
95
1
99
99
0
0
1
0
1
50
1
0
100
0
60
100
100
100
Hafnia alvei 1
75
0
99
98
50
0
10
0
0
50
0
99
99
0
1
99
0
0
25
99
0
100
0
85
100
100
100
Hafnia alvei 2
50
0
99
99
1
0
1
0
0
10
0
99
98
0
1
1
1
0
0
1
0
100
0
0
100
100
100
Klebsiella ornithinolytica
100
0
99
99
99
0
85
0
100
65
0
100
100
99
100
100
100
100
100
100
0
100
0
0
100
100
100
Klebsiella oxytoca
99
0
80
0
89
0
78
0
99
80
0
100
100
99
100
99
99
100
100
100
0
100
0
0
100
100
100
Klebsiella pneumoniae ssp ozaenae
94
18
25
1
18
0
1
0
0
1
0
99
96
57
66
58
20
80
97
85
0
92
0
0
100
100
100
Klebsiella pneumoniae ssp pneumoniae
99
0
73
0
86
0
75
0
0
90
0
100
99
99
99
99
99
99
99
99
0
100
0
0
100
100
100
1
0
0
0
0
0
0
0
0
0
0
99
100
90
90
75
75
1
99
10
0
100
0
0
100
100
100
Klebsiella terrigena
100
0
99
6
52
0
0
0
0
75
0
99
99
99
99
99
100
100
100
99
0
100
0
0
100
100
100
Kluyvera spp
95
0
25
99
60
0
0
0
80
0
0
100
99
0
25
93
89
99
99
99
0
95
0
94
100
100
100
Leclercia adecarboxylata
99
0
0
0
0
0
1
0
99
0
1
100
99
0
2
100
66
99
99
100
0
100
0
100
100
100
100
Moellerella wisconsensis
97
0
0
0
40
0
0
0
15
1
0
100
1
0
0
0
100
99
0
0
0
90
0
0
100
100
100
1
0
10
98
1
1
99
93
99
0
0
99
0
0
0
0
1
0
0
0
0
88
0
95
100
100
100
Pantoea spp 1
85
1
0
0
13
0
1
0
1
9
1
100
99
1
26
1
98
26
59
61
0
85
0
85
100
100
100
Pantoea spp 2
99
1
0
0
99
0
1
0
53
62
4
100
99
36
82
90
98
91
99
99
0
85
0
85
100
100
100
Pantoea spp 3
99
1
0
0
21
0
1
0
1
86
15
100
99
34
1
97
93
23
65
97
0
85
0
85
100
100
100
Pantoea spp 4
86
1
0
0
29
0
1
0
59
1
1
99
100
10
32
99
72
89
99
99
0
85
0
85
100
100
100
Proteus mirabilis
1
0
0
99
50
75
99
98
1
1
82
98
0
0
0
0
1
0
0
0
0
93
0
95
100
100
100
Proteus penneri
1
0
0
0
1
20
100
99
0
0
50
99
0
0
0
0
100
0
1
0
0
99
0
85
100
100
100
Proteus vulgaris
1
0
0
0
12
83
99
99
92
0
74
99
1
1
0
1
89
0
66
1
0
100
0
94
100
100
100
Providencia alcalifaciens/rustigianii
0
0
0
0
80
0
0
100
99
0
0
99
1
1
0
0
1
0
0
1
0
100
0
96
100
100
100
API 20 E
V4.0
Buttiauxella agrestis
Klebsiella pneumoniae ssp rhinoscleromatis
Morganella morganii
7
api 20 E 0 008040-6 02/02 ______________________________________________________________________________________________________________________________________________________________________ __ Providencia rettgeri
1
1
0
0
74
0
99
99
90
0
0
98
82
78
1
50
25
0
40
1
0
98
0
94
100
100
100
Providencia stuartii
1
0
0
0
85
0
30
98
95
0
0
98
3
80
0
0
15
0
0
0
0
100
0
85
100
100
100
Rahnella aquatilis
100
0
0
0
50
0
0
1
0
99
0
100
100
0
98
99
100
97
100
98
0
100
0
6
100
100
100
Salmonella arizonae
98
75
97
98
75
99
0
0
1
0
0
100
99
0
99
99
1
78
0
99
0
100
0
99
100
100
100
Salmonella choleraesuis
0
15
99
99
6
64
0
0
0
0
0
100
99
0
98
99
0
20
0
0
0
100
0
95
100
100
100
Salmonella gallinarum
0
1
100
1
0
25
0
0
0
0
0
100
100
0
0
1
0
0
0
100
0
100
0
0
100
100
100
8
api 20 E 0 008040-6 02/02 ______________________________________________________________________________________________________________________________________________________________________ __ ONPG 0
ADH 5
LDC 0
ODC 99
CIT 0
H2 S 1
URE 0
TDA 0
IND 0
VP 0
GEL 0
GLU 100
MAN 99
INO 0
SOR 99
RHA 98
SAC 0
MEL 96
AMY 0
ARA 99
OX 0
NO 2 100
N2 0
MOB 95
McC 100
OF/O 100
OF/F 100
Salmonella pullorum
0
1
75
100
0
85
0
0
0
0
0
100
100
Salmonella typhi
0
1
99
0
0
8
0
0
0
0
0
100
99
0
0
100
0
99
0
0
0
0
75
0
100
0
99
0
0
0
100
0
0
100
100
100
0
97
100
100
Salmonella spp
1
56
82
93
65
83
0
0
1
0
1
99
100
40
99
86
1
90
1
99
1
100
100
0
95
100
100
Serratia ficaria
99
0
0
0
100
0
0
0
0
40
90
100
100
50
99
74
99
99
100
99
100
0
92
0
100
100
100
Serratia fonticola
99
0
73
99
75
0
0
0
0
0
0
100
100
97
100
99
30
99
99
100
99
0
99
0
91
100
100
Serratia liquefaciens
95
1
78
98
80
0
2
0
0
59
65
100
99
80
98
2
99
72
100
97
97
0
100
0
95
100
100
Serratia marcescens
94
0
95
95
96
0
25
0
1
70
87
100
99
85
98
1
99
100
68
97
25
0
95
0
97
100
100
Serratia odorifera 1
95
0
95
99
95
0
0
0
99
50
99
100
99
99
99
99
100
99
99
99
99
0
99
0
100
100
100
Serratia odorifera 2
95
0
96
1
95
0
0
0
99
50
99
100
99
99
99
100
99
1
99
99
95
0
99
0
100
100
100
Serratia plymuthica
99
0
0
0
65
0
0
0
0
65
50
100
90
70
100
70
1
99
85
98
98
0
99
0
50
100
100
Serratia rubidaea
99
0
30
0
92
0
1
0
0
71
82
99
99
100
75
1
3
99
95
99
99
0
100
0
85
100
100
1
0
0
1
0
0
0
0
29
0
0
99
100
63
0
7
7
1
20
0
50
0
100
0
0
100
100
Shigella sonnei
96
0
0
93
0
0
0
0
0
0
0
100
99
99
0
1
75
1
1
0
99
0
100
0
0
100
100
Yersinia enterocolitica
80
0
0
90
0
0
98
0
50
5
100
0
99
99
25
98
1
99
4
75
75
0
98
0
2
100
100
Yersinia frederiksenii/intermedia
99
0
0
75
1
0
99
0
99
100
1
0
100
99
25
99
99
99
1
99
99
0
98
0
5
100
100
Yersinia kristensenii
80
0
0
80
0
0
99
0
100
97
0
0
100
99
10
99
0
0
0
99
99
0
98
0
5
100
100
Yersinia pestis
68
0
0
0
0
0
0
100
0
0
1
0
99
99
0
70
0
0
0
30
30
0
47
0
0
99
100
Yersinia pseudotuberculosis
98
0
0
0
1
0
100
99
0
0
0
0
99
97
0
0
75
0
50
25
50
0
95
0
0
100
100
Aeromonas hydrophila gr. 1
98
90
25
1
25
100
0
0
0
85
25
90
99
99
1
3
5
97
1
75
75
100
97
0
95
99
99
Aeromonas hydrophila gr. 2
99
97
80
1
99
80
0
0
0
85
80
97
97
99
9
9
1
80
1
75
5
100
97
0
95
99
99
Aeromonas salmonicida ssp salmonicida
1
60
1
99
0
0
0
0
0
1
0
75
50
54
0
0
0
0
0
1
0
100
98
0
1
99
99
Photobacterium damsela
1
99
99
75
0
1
0
98
0
0
10
1
50
0
0
0
0
1
0
0
0
100
100
0
25
99
99
Plesiomonas shigelloides
95
99
99
100
100
0
0
0
0
100
0
0
99
0
99
0
0
0
0
0
0
100
99
0
95
99
99
99
0
0
98
75
60
0
1
0
100
10
75
99
100
0
1
0
100
0
10
1
100
47
0
100
99
94
94
Vibrio cholerae
98
1
94
97
75
0
0
0
99
58
92
98
98
0
0
0
94
0
5
0
100
96
0
100
96
99
99
Vibrio fluvialis
95
99
0
0
1
0
0
0
80
0
75
75
80
0
1
0
75
0
36
75
100
100
0
100
99
99
99
Vibrio hollisae
1
0
0
0
0
0
0
0
94
0
0
10
0
0
0
0
0
0
0
0
100
100
0
0
99
99
99
Vibrio mimicus
99
0
99
99
50
0
0
0
99
1
99
99
99
0
0
0
0
0
0
0
100
95
0
100
95
99
99
0
0
100
99
50
0
1
0
100
1
75
100
99
0
0
1
1
0
12
50
100
63
0
100
98
99
99
Vibrio vulnificus
99
0
91
90
25
0
0
0
99
1
99
99
75
0
0
0
1
0
90
0
99
54
0
100
99
99
99
Pasteurella aerogenes
99
0
0
80
0
0
99
0
0
0
0
99
0
97
0
1
99
0
0
75
75
100
0
0
100
100
100
Pasteurella multocida 1
4
0
0
25
0
0
0
0
99
0
0
29
1
0
1
0
75
0
0
0
99
90
0
0
2
23
23
Pasteurella multocida 2
7
0
0
45
0
0
0
0
99
0
0
44
99
0
99
0
99
0
0
0
89
90
0
0
2
23
23
Pasteurella pneumotropica/haemolytica
60
0
1
10
0
0
25
0
15
7
3
35
12
12
12
1
35
1
2
1
80
99
0
0
9
33
33
Acinetobacter baumannii/calcoaceticus
0
0
0
0
51
0
1
0
0
5
5
99
0
0
0
0
0
99
1
99
0
3
0
0
90
98
0
Bordetella/Alcaligenes/Moraxella spp *
0
0
0
0
52
0
14
1
0
25
1
0
0
0
0
0
0
0
0
0
95
62
1
75
75
0
0
50
0
25
16
78
0
0
0
0
1
43
60
1
0
0
0
13
0
7
20
90
40
0
99
88
97
0
0
99
0
0
75
0
0
0
14
0
99
99
0
0
0
0
10
0
0
0
99
75
0
99
99
99
99
86
75
0
0
94
0
0
0
0
25
13
84
0
1
0
1
1
15
1
85
0
30
0
100
91
94
0
5
0
0
0
12
0
90
0
75
0
80
0
0
0
0
0
0
0
0
0
99
20
0
0
57
90
10
API 20 E
V4.0
Salmonella paratyphi A
Shigella spp
Vibrio alginolyticus
Vibrio parahaemolyticus
Burkholderia cepacia Chromobacterium violaceum Chryseomonas luteola Chryseobacterium indologenes Chryseobacterium meningosepticum
77
0
0
0
20
0
1
0
85
0
90
0
0
0
0
0
0
0
0
0
99
6
0
0
48
93
6
Eikenella corrodens
0
0
75
99
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
100
95
0
1
1
49
49
Flavimonas oryzihabitans
0
0
0
0
89
0
0
0
0
25
1
10
0
1
0
1
0
10
0
45
0
7
0
100
99
99
0
Myroides /Chryseobacterium indologenes
0
0
0
0
50
0
75
0
0
1
75
0
0
0
0
0
0
0
0
0
99
0
0
0
84
2
2
15
0
0
0
30
0
25
1
0
15
0
1
0
0
0
0
0
0
0
10
90
42
60
99
99
47
0
Ochrobactrum anthropi
8
api 20 E 0 008040-6 02/02 ______________________________________________________________________________________________________________________________________________________________________ __ Pseudomonas aeruginosa
0
89
0
0
92
0
25
0
0
1
75
50
0
0
0
0
1
10
1
25
97
12
56
97
100
98
0
Pseudomonas fluorescens/putida
0
75
0
0
75
0
0
0
0
10
27
25
0
0
0
0
0
25
1
20
99
26
0
100
96
93
0
Non-fermenter spp
1
1
0
0
37
0
1
0
0
15
9
9
0
0
0
1
1
1
1
1
93
48
35
99
85
49
0
Shewanella putrefaciens
0
0
0
80
75
75
1
0
0
0
75
1
0
0
0
0
1
0
0
2
99
96
0
100
96
9
0
70
0
75
1
75
1
0
0
0
0
90
1
0
0
0
0
0
0
0
0
1
26
1
100
91
49
0
Stenotrophomonas maltophilia
* Brucella spp possible / möglich / posible / possibile
8
api 20 E 0
008040-6 02/02
BIBLIOGRAPHY 1. APPELBAUM P.C., STAVITZ J., BENTZ M.S., VON KUSTER L.C. Four Methods for Identification of Gram-Negative Nonfermenting Rods : Organisms more Commonly Encountered in Clinical Specimens. (1980) J. Clin. Microbiol. 12, 271-278.
5. McLAUGHLIN J.K., ZUCKERMAN B.D., TENENBAUM S., WOLF B.A. Comparison of the API 20E, Flow, and Minitek systems for the identification of enteric and nonfermentative bacteria isolated from cosmetic raw materials. (1984) J. Soc. Cosmet. Chem. 35, 253-263.
2. BROOKS K.A., JENS M., SODEMAN T.M. A Clinical Evaluation of the API Microtube System for Identification of Enterobacteriaceae. (1974) Am. J. Med. Techn. 40, 55-61.
6. NORD C.E., LINDBERG A.A., DAHLBÄCK A. Evaluation of Five Test-Kits, API, AuxoTab, Enterotube, PathoTec and R/B, for Identification of Enterobacteriaceae. (1974) Med. Microbiol. Immunol. 159, 211-220.
3. CASTILLO C.B., BRUCKNER D.A. Comparative Evaluation of the Eiken and API 20E Systems and Conventional Methods for Identification of of the family Enterobacteriaceae. (1984) J. Clin. Microbiol. 20, 754-757.
7. SMITH P.B., TOMFOHRDE K.M., RHODEN D.L., BALOWS A. API System : A multitube Micromethod for Identification of Enterobacteriaceae. (1972) Applied Microbiol. 24, 449-452.
4. HAYEK L., WILLIS G.W. Identification of the Enterobacteriaceae : a Comparison of the Enterotube II with the API 20E. (1984) J. Clin. Pathol. 37, 344-347.
8. SWANSON E.C., COLLINS M.T. Use of the API 20E System to Identify Veterinary Enterobacteriaceae. (1980) J. Clin. Microbiol. 12, 10-14.
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.
9
api 20 E 0
008040-6 02/02
Fabricant / Manufacturer bioMérieux sa bioMérieux sa
bioMérieux, Inc.
au capital de 77 421 420 F 673 620 399 RCS LYON 69280 Marcy-l’Etoile / tel. (33) 4 78 87 20 00 / fax (33) 4 78 87 20 90 http://www.biomerieux.com
595 Anglum Road Hazelwood, MO 63042-2320 / USA tel. (1) 314-731-8500 / fax (1) 314-731-8800 Imprimé en / Printed in USA
008040-6 02/02
For in vitro diagnostic use Identification system for Enterobacteriaceae and other Gram-negative rods API 20 E is an identification system for Enterobacteriaceae and other non-fastidious Gram-negative rods which uses 23 standardized and miniaturized biochemical tests and a database. The complete list of those organisms that it is possible to identify with this system is given in the Identification Table at the end of this package insert.
PRINCIPLE The API 20 E strip consists of 20 microtubes containing dehydrated substrates. These tests are inoculated with a bacterial suspension which reconstitutes the media. During incubation, metabolism produces color changes that are either spontaneous or revealed by the addition of reagents. The reactions are read according to the Interpretation of Reactions (Table 2) and the identification is obtained by referring t the Analytical Profile Index or using the identification software. REAGENTS Kit contents ( Prod. no. 20100 OR 20109 - 25 tests) : - 25 API 20 E strips - 25 incubation boxes - 25 result sheets - 1 clip seal - 1 package insert
COMPOSITION OF MEDIA AND REAGENTS NaCl 0.85 % Medium 5 ml or Suspension Medium 5 ml
Required laboratory equipment : -
8.5 g 1000 ml
Demineralized water
Ferric chloride 30 ml Kovacs’ reagent 30 ml
10% aqueous ferric chloride solution in demineralized water Avoid skin Paradimethylaminobenzaldehyde Concentrated hydrochloric acid N-amyl alcohol
5% 24% 71%
HARMFUL R10 : Flammable. R20 : Harmful by inhalation. R34: Causes burns. R37: Irritating to respiratory system. S24/25 : Avoid with skin and eyes. S16: Keep away from sources of ignition. No smoking. S26: In case of with eyes, rinse immediately with plenty of water and seek medical advice. S36/37/39: Wear suitable protective clothing, gloves and eye/face protection. S45: In case of accident or if you feel unwell, seek medical advice immediately (show the label where possible).
Kit contents (Prod. No. 20160 OR 20179 - 100 tests) : - 100 API 20 E strips - 100 incubation boxes 100 result sheets 1 clip seal 1 package insert Additional products (not included in the kit) : - NaCl 0.85 % Medium, 5 ml (Prod. No. 20230) or Suspension Medium, 5ml (Prod. No. 20150) - Ferric chloride, 30ml (Prod. No. V7052) - VP1, 5ml (Prod. No. 70422) - VP2, 5ml (Prod. No. 70422) - NIT 1, 5ml (Prod. No. 70442) - NIT 2, 5ml (Prod. No. 70442) - Kovacs’ Reagent (Prod. No. V7050) - Hydrogen peroxide, 1.5% - Oxidase Test Kit (Prod. No. V7062) - Zinc Dust, 2x10g (Prod. No. 70380) - Mineral oil (Prod. No. 70100) - MacConkey Medium - Pasteur pipettes - API 20 E Analytical Profile Index (Prod. No. 20190) or identification software (consult bioMérieux) - Ampule rack (Prod. No. 70200) And possibly : - API OF Medium (Prod. No. 50110) : Test for the determination of fermentative or oxidative metabolism. - API M Medium (Prod No. 50120) Test for motility of facultative anaerobic bacteria.
Sodium chloride Demineralized water
VP1 5 ml
40% aqueous KOH solution in demineralized water
89% 11%
CORROSIVE R35 : Causes severe burns. S24/25: Avoid with skin and eyes. S26 : In case of with eyes, rinse immediately with plenty of water and seek medical advice. S36/37/39: Wear suitable protective clothing, gloves and eye/face protection. S45: In case of accident or if you feel unwell, seek medical advice immediately (show the label where possible).
VogesProskauer reagent : VP2 5 ml
NIT 1 5 ml
Alpha-naphthol Ethyl alcohol
6g 100 ml
FLAMMABLE AFTER RECONSTITUTION R21/22: Harmful in with skin and if swallowed. S24/25: Avoid with skin and eyes.
Sulfanilic acid Acetic acid 5N
0.8 % 99.2%
CORROSIVE R34 : Causes burns. S2 : Keep out of reach of children. S23 : Do not breathe vapours. S26 : In case of with eyes, rinse immediately with plenty of water and seek medical advice.
35-37°C incubator Refrigerator Bunsen burner Marker pen
NIT 2
5ml N,N-dimethyl-1-naphthylamine Acetic acid 5N
0.5% 99.5%
CORROSIVE R34 : Causes burns. S2 : Keep out of reach of children. S23 : Do not breathe vapours. S26 : In case of with eyes, rinse immediately with plenty of water and seek medical advice.
Oxidase Test Reagent
Oxidase Reagent: see Oxidase Test Kit Package Insert.
1
00
api 20 E
Zinc dust 10 g
0008040-6 02/02
Zinc dust FLAMMABLE R15 : with water liberates highly flammable gases. R17 : Spontaneously flammable in air. S7/8 : Keep container tightly closed and dry. S43 : In case of fire caused by metals, use powder extinguishers. 'Never use water'.
STORAGE OF THE STRIPS The strips are supplied in an aluminum pouch with desiccant sachets. Once opened, the pouch should be re-sealed using the clip seal (included in the kit) to preserve the remaining strips with the desiccant sachets : place the open end of the pouch along the seal and carefully clamp between the two parts. The strips may then be kept for up to 10 months after the pouch has been opened, at 2-8°C (or until the expiration date indicated on the packaging, if this comes before).
STORAGE OF THE REAGENTS Kovacs’ reagent, VP1, NIT 1 and NIT 2 should be stored at 15-30°C until the expiration date indicated on the packaging. Ferric chloride should be stored at 2-30°C until the expiration date indicated on the packaging. Zinc dust should be stored at 8-30°C until the expiration date indicated on the packaging. VP2 should be stored in the dark at 2-8°C until the expiration date if this comes first. Record the date opened on the bottle label. See Oxidase Test Kit Package Insert for storage instructions.
USE OF THE REAGENTS Allow reagents to come to room temperature (20-30°C) before using.
1. Ferric chloride, VP1, NIT 1, NIT 2 and Kovacs’:
• Dispense one drop of reagent. • Carefully close the bottle after use and store it as indicated in the paragraph "Storage of the Reagents".
2. VP2 :
• Open the ampule of reagent as indicated in the paragaraph "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".
3. Zinc dust :
• Open the bottle • Take up an aliquot of powder (approximately 2-3 mg) using the spatula fixed to the cap and deposit this quantity in the reaction cupule. • Carefully close the bottle after use and store it as indicated in the paragraph "Storage of the reagents".
4. Oxidase Test Kit:
• Please note that the oxidase test must be performed as it is an integral part of the st final profile (21 identification test .) See the Oxidase Test Kit Package Insert for the use of the oxidase reagent.
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 allow reagents to come into with skin, eyes, or clothing. • Do not use reagents past the expiration date. • Upon removal from refrigerator, allow reagents to come to room temperature (2030°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 to the flattened part of the cap to snap off the top of the ampoule 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 in a vertical position. - Squeeze on the cap to transfer all the reagent into the dropperbottle. • 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. • 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. • The performance data presented were obtained using the procedure indicated. Any changes or modifications in the procedure may affect results.
INSTRUCTIONS FOR USE
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 Diseases 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.
Preparation of the strip • 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, CO2, 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. • Place the strip in the tray. • Perform the oxidase test on a colony identical to the colony which will be tested. Refer to the Oxidase Test Kit Package Insert. This reaction should be recorded on the result sheet as it constitutes the 21st test. NOTE : API 20 E should only be used with non-fastidious Gram-negative rods. Fastidious organisms having demanding nutritional requirements and requiring appropriate handling precautions (i.e., Brucella and Francisella) are not included in the API 20 E database. Alternative procedures must be used to exclude or confirm their presence.
Preparation of the inoculum • Open an ampule of NaCl 0.85 % Medium (5 ml) or an ampule of Suspension Medium (5ml) as indicated in the paragraph "Warnings and Precautions" or use any tube containing 5 ml of sterile NaCl 0.85% Medium, pH 5.5 - 7.0, or sterile distilled water, without additives. • With the aid of a pipette, remove a single well-isolated colony from an isolation plate. • Carefully emulsify to achieve a homogeneous bacterial suspension. NOTE : Most Vibrio species are halophilic. If Vibrio is suspected, suspend the bacteria in NaCl 0.85 % Medium.
Inoculation of the strip 2
00
api 20 E
0008040-6 02/02
• With the same pipette, fill both tube and cupule of test CIT , VP and GEL with the bacterial suspension. • Fill only the tubes (and not the cupules) of the other tests. • Create anaerobiosis in the tests ____ ADH, ____ LDC , ____ ODC, ____ H2S and URE ____ by overlaying with mineral oil. • Close the incubation box. • Incubate at 35-37°C for 18-24 hours. • Weekend incubation : The biochemical reactions of the API 20 E strip should be read after 18-24 hours of incubation. If the strips cannot be read after 24 hours of incubation at 35-37°C, the strips should be removed from the incubator and stored at 2-8°C (refrigerator) until the reactions can be read.
Reading the strip • After 18-24 hours at 35-37°C, read the strip by referring to Interpretation of Reactions (Table 2.) • Record all spontaneous reactions on the result sheet. • If the glucose is positive or 3 or more tests are positive, reveal the tests which require the addition of reagents : - TDA Test : add 1 drop of Ferric chloride. A dark brown color indicates a positive reaction to be recorded on the result sheet. - IND Test : add 1 drop of Kovacs’ reagent. Wait 2 minutes. A red ring indicates a positive reaction to be recorded on the result sheet. - VP Test : add 1 drop of each of VP1 and VP2 reagents. Wait at least 10 minutes. A pink or red color indicates a positive reaction to be recorded on the result sheet. If a slightly pink color appears within 10 to 12 minutes, the reaction should be considered negative. NO2 Test : add 2 drops of each of NIT 1 and NIT 2 reagents to the GLU tube. Wait 2 to 3 minutes. A red color indicates a positive reaction. A negative reaction (yellow) may be due to the reduction to nitrogen (as sometimes evidenced by gas bubbles) : add 2 to 3 mg of Zinc dust to the GLU tube. After 5 minutes, if the tube remains yellow this indicates that (N2) is positive and is to be recorded on the result sheet. If the test turns pink-red, this is a negative reaction : the nitrates still present in the tube have been reduced by the Zinc. This reaction is useful when testing Gram-negative, oxidase positive rods. NOTE : The nitrate reduction and indole production tests must be performed last since these reactions release gaseous products which interfere with the interpretation of other tests on the strip. The plastic incubation lid should not be replaced after the addition of these reagents. • If the glucose is negative and the number of positive tests is less than or equal to 2, do not add reagents : - Inoculate 2 ampules of API OF Medium to confirm the metabolism of glucose. - Streak a MacConkey agar plate. - Check for motility : inoculate 1 ampule of API M Medium or observe between slide and cover slip under a microscope. - Reincubate the strip for 24 hours. - Add the reagents as described above. - Record the strip and supplementary test results on the result sheet by referring to Interpretation of Reactions (Table 2).
Identification Identification can be obtained : • 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 for the 20 tests of the API 20 E strip. The oxidase reaction constitutes the 21st test and has a value of 4 if it is positive. • using the identification software to obtain information on any profile not listed in the Analytical Profile Index, call the Voice Response System at 800-645-7056. In some cases, the 7-digit profile is not discriminatory enough and the following supplementary tests need to be carried out : -
Reduction of nitrates to nitrites (NO2) Reduction of nitrates to N2 gas (N2) Motility (MOB) Growth on MacConkey agar medium (McC) Oxidation of glucose (OF-O) Fermentation of glucose (OF-F)
These supplementary tests, indicated in the Analytical Profile Index, may be used to form a 9-digit profile. Identification is then obtained using the identification software.
5 315 173 (57) Enterobacter gergoviae
DISPOSAL OF USED MATERIAL After use, all ampules, pipettes, strips and incubation boxes should be autoclaved, incinerated, or immersed in a disinfectant for decontamination prior to disposal.
RANGE OF EXPECTED VALUES Consult the Identification Table at the end of this package insert for the range of expected values for the various biochemical reactions.
LIMITATIONS • The API 20 E system is intended uniquely for the identification of those nonfastidious, Gram-negative rods 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. • Discrepancies with respect to conventional methods may be observed due to the different principles of the reactions used in the API technique. • If Salmonella spp., Shigella spp., are identified, serological testing must be performed to confirm the bacterial identification.
QUALITY CONTROL The media, strips, and reagents are systematically quality controlled at various stages of their manufacture. For those who wish to perform their own quality control tests with the strip, it is recommended that the following stock cultures be used, to obtain the results below : Table 1 ONPG ____ ADH ____ LDC ODC ____ 1. 2. 3. 4. 5.
+ + – + +
– + – – –
V – – + +
– + + – +
CIT V + V + –
1. Stenotrophomonas maltophilia 2. Enterobacter cloacae 3. Proteus mirabilis
H2S URE ____ TDA IND VP – – + – –
– – + V –
– – + – –
– – – – +
– + – V –
ATCC 51331 ATCC 13047 ATCC 35659
GEL GLU MAN INO SOR RHA SAC MEL AMY ARA NO2 N2 + – V – –
– + + + +
– + – + +
– V – + –
– + – + +
– + – + +
– + V + –
– + – + +
– + – + –
– + – + +
– + + + +
– – – – –
4. Klebsiella pneumoniae ssp pneumoniae ATCC 35657 5. Escherichia coli ATCC 25922
ATCC : American Type Culture Collection, 10801 University Boulevard, Manassas, VA 20110-2209, USA. • Profile obtained after 24-48 hours of incubation for the strain ATCC 51331, using cultures grown on Trypticase Soy agar + blood • Profiles obtained after 18-24 hours of incubation for the other strains, using cultures grown on Trypticase Soy agar + blood • Bacterial suspension prepared in NaCl 0.85 % Medium
PERFORMANCE CHARACTERISTICS The Profile Recognition System has been evaluated by the National Institutes of Health, Bethesda, Maryland. The API 20E System has been evaluated extensively worldwide by institutions such as the Centers for Disease Control, Atlanta, Georgia; the Mayo Clinic, Rochester, Minnesota; the Pasteur 3
00
api 20 E
0008040-6 02/02
Institute, Lyon, ; Sherbrooke University, Sherbrooke, Quebec, Canada. In fact, the API 20E System was one of the original miniaturized biochemical detection systems and has evolved over the years as the standard against which other identification systems have been compared. Recently, the Centers for Disease Control reevaluated the identification capabilities of the API 20 E System and reported that at 24 hours, 20 E correctly identified to genus and species level 87.7% of a clinically weighted assortment of isolates. At 48 hours, 96.3% of the isolates were identified correctly (O’Hara, C.M., D.L. Rhoden, and J.M. Miller, 1992. J. Clin. Microbiol. 30: 123-125). These percentages reflect the product’s identification capabilities for taxa that are identified routinely on the 20 E System. In addition, the API 20E System permits recognition of biotypes within each species. Delineation of biotypes may be of value in determining the effectiveness of treatment and the existence of nosocomial infections as related to of the family Enterobacteriaceae, as well as for control of the latter.
bioMérieux sa
4
api 20 E
0
008040-6 02/02
INTERPRETATION OF REACTIONS Table 2 TESTS
SUBSTRATES
QTY
ENZYMATIC ACTIVITY OR REACTION TESTED
RESULTS NEGATIVE
POSITIVE
beta-galactosidase
colorless
yellow (1)
red / orange (2)
ortho-nitrophenyl-‚ -Dgalactopyranoside(ONPG)
0.2 mg
isopropylthiogalactopyranoside (IPTG)
8.0 µg
ADH ____
arginine
2.0 mg
arginine dihydrolase
yellow
LDC ____
lysine
2.0 mg
lysine decarboxylase
yellow
red/orange (2)
ODC ____
ornithine
2.0 mg
ornithine decarboxylase
yellow
red / orange (2)
sodium citrate
0.8 mg
citrate utilization
pale green / yellow
blue-green / blue (3)
H2S ___
sodium thiosulfate
80.0 µg
H2S production
colorless / greyish
black deposit / thin line
URE ____
urea
0.8 mg
urease
yellow
red / orange (2)
tryptophane deaminase
Ferric chloride (1 drop) / immediate yellow brown-red
ONPG
CIT
TDA
tryptophane
0.4 mg
Kovacs’ reagent (1 drop) / 2 min IND
VP GEL
tryptophane
0.2 mg
creatine
0.9 mg
sodium pyruvate
2.0 mg
Kohn's charcoal gelatin
0.6 mg
indole production
IND yellow
IND red ring
VP1 (1 drop) + VP2 (1 drop) / acetoin production gelatinase
10 min
colorless
pink / red (5)
no diffusion of black pigment
diffusion of black pigment
GLU
glucose
2.0 mg
fermentation / oxidation (4)
blue / blue-green
yellow / greyish yellow
MAN
mannitol
2.0 mg
fermentation / oxidation (4)
blue / blue-green
yellow
INO
inositol
2.0 mg
fermentation / oxidation (4)
blue / blue-green
yellow
SOR
sorbitol
2.0 mg
fermentation / oxidation (4)
blue / blue-green
yellow
RHA
rhamnose
2.0 mg
fermentation / oxidation (4)
blue / blue-green
yellow
SAC
sucrose
2.0 mg
fermentation / oxidation (4)
blue / blue-green
yellow
MEL
melibiose
2.0 mg
fermentation / oxidation (4)
blue / blue-green
yellow
AMY
amygdalin
2.0 mg
fermentation / oxidation (4)
blue / blue-green
yellow
ARA
arabinose
2.0 mg
fermentation / oxidation (4)
blue / blue-green
yellow
OX
on filter paper
Oxidase reagent / 1-2 min colorless violet
cytochrome-oxidase
NIT 1 (2 drops) + NIT 2 (2 drops) / 2-3 min GLU tube Nitrate Reduction
MOB
yellow
NO2 production Potassium nitrate
API M Medium or microscope
80.0 µl
red Zinc Dust / 5 min
reduction to N2 gas
orange-red
yellow
motility
non-motile
motile
McC
MacConkey medium
growth
absence
presence
OF-F OF-O
glucose (API OF Medium)
fermentation : under mineral oil oxidation : exposed to the air
green green
yellow yellow
(1) A very pale yellow should also be considered positive. (2) An orange color after 36-48 hours incubation must be considered negative. (3) Reading made in the cupule (aerobic).
(4) Fermentation begins in the lower portion of the tubes, oxidation begins in the cupules. (5) A slightly pink color after 10 minutes should be considered negative.
5
api 20 E
0
008040-6 02/02 RECOMMENDED METHODOLOGY
1 colony
NaCl 0.85 % Medium 5 ml / Suspension Medium 5 ml
- | CIT | - | VP | - | GEL | api 20 E ADH ODC H2S - URE
18:00-24:00 / 48:00
37°C
Tests ⊕ ≤ 2 and GLU (-) api 20 E
TDA IND VP GLU (NO2) OX
: Ferric chloride : Kovacs’ reagent : VP1 + VP2 : NIT 1 + NIT 2 : Oxidase test reagent
api 20 E
+ - + - + -
6
20100/20160/20109/201790
008040-6 02/02
For in vitro diagnostic use Identification system for Enterobacteriaceae and other Gram-negative rods API 20 E is an identification system for Enterobacteriaceae and other non-fastidious Gram-negative rods which uses 23 standardized and miniaturized biochemical tests and a database. The complete list of those organisms that it is possible to identify with this system is given in the Identification Table at the end of this package insert.
PRINCIPLE The API 20 E strip consists of 20 microtubes containing dehydrated substrates. These tests are inoculated with a bacterial suspension which reconstitutes the media. During incubation, metabolism produces color changes that are either spontaneous or revealed by the addition of reagents. The reactions are read according to the Interpretation of Reactions (Table 2) and the identification is obtained by referring t the Analytical Profile Index or using the identification software. REAGENTS Kit contents ( Prod. no. 20100 OR 20109 - 25 tests) : - 25 API 20 E strips - 25 incubation boxes - 25 result sheets - 1 clip seal - 1 package insert
COMPOSITION OF MEDIA AND REAGENTS NaCl 0.85 % Medium 5 ml or Suspension Medium 5 ml
Required laboratory equipment : -
8.5 g 1000 ml
Demineralized water
Ferric chloride 30 ml Kovacs’ reagent 30 ml
10% aqueous ferric chloride solution in demineralized water Avoid skin Paradimethylaminobenzaldehyde Concentrated hydrochloric acid N-amyl alcohol
5% 24% 71%
HARMFUL R10 : Flammable. R20 : Harmful by inhalation. R34: Causes burns. R37: Irritating to respiratory system. S24/25 : Avoid with skin and eyes. S16: Keep away from sources of ignition. No smoking. S26: In case of with eyes, rinse immediately with plenty of water and seek medical advice. S36/37/39: Wear suitable protective clothing, gloves and eye/face protection. S45: In case of accident or if you feel unwell, seek medical advice immediately (show the label where possible).
Kit contents (Prod. No. 20160 OR 20179 - 100 tests) : - 100 API 20 E strips - 100 incubation boxes 100 result sheets 1 clip seal 1 package insert Additional products (not included in the kit) : - NaCl 0.85 % Medium, 5 ml (Prod. No. 20230) or Suspension Medium, 5ml (Prod. No. 20150) - Ferric chloride, 30ml (Prod. No. V7052) - VP1, 5ml (Prod. No. 70422) - VP2, 5ml (Prod. No. 70422) - NIT 1, 5ml (Prod. No. 70442) - NIT 2, 5ml (Prod. No. 70442) - Kovacs’ Reagent (Prod. No. V7050) - Hydrogen peroxide, 1.5% - Oxidase Test Kit (Prod. No. V7062) - Zinc Dust, 2x10g (Prod. No. 70380) - Mineral oil (Prod. No. 70100) - MacConkey Medium - Pasteur pipettes - API 20 E Analytical Profile Index (Prod. No. 20190) or identification software (consult bioMérieux) - Ampule rack (Prod. No. 70200) And possibly : - API OF Medium (Prod. No. 50110) : Test for the determination of fermentative or oxidative metabolism. - API M Medium (Prod No. 50120) Test for motility of facultative anaerobic bacteria.
Sodium chloride Demineralized water
VP1 5 ml
40% aqueous KOH solution in demineralized water
89% 11%
CORROSIVE R35 : Causes severe burns. S24/25: Avoid with skin and eyes. S26 : In case of with eyes, rinse immediately with plenty of water and seek medical advice. S36/37/39: Wear suitable protective clothing, gloves and eye/face protection. S45: In case of accident or if you feel unwell, seek medical advice immediately (show the label where possible).
VogesProskauer reagent : VP2 5 ml
NIT 1 5 ml
Alpha-naphthol Ethyl alcohol
6g 100 ml
FLAMMABLE AFTER RECONSTITUTION R21/22: Harmful in with skin and if swallowed. S24/25: Avoid with skin and eyes.
Sulfanilic acid Acetic acid 5N
0.8 % 99.2%
CORROSIVE R34 : Causes burns. S2 : Keep out of reach of children. S23 : Do not breathe vapours. S26 : In case of with eyes, rinse immediately with plenty of water and seek medical advice.
35-37°C incubator Refrigerator Bunsen burner Marker pen
NIT 2
5ml N,N-dimethyl-1-naphthylamine Acetic acid 5N
0.5% 99.5%
CORROSIVE R34 : Causes burns. S2 : Keep out of reach of children. S23 : Do not breathe vapours. S26 : In case of with eyes, rinse immediately with plenty of water and seek medical advice.
Oxidase Test Reagent
Oxidase Reagent: see Oxidase Test Kit Package Insert.
1
00
api 20 E
Zinc dust 10 g
0008040-6 02/02
Zinc dust FLAMMABLE R15 : with water liberates highly flammable gases. R17 : Spontaneously flammable in air. S7/8 : Keep container tightly closed and dry. S43 : In case of fire caused by metals, use powder extinguishers. 'Never use water'.
STORAGE OF THE STRIPS The strips are supplied in an aluminum pouch with desiccant sachets. Once opened, the pouch should be re-sealed using the clip seal (included in the kit) to preserve the remaining strips with the desiccant sachets : place the open end of the pouch along the seal and carefully clamp between the two parts. The strips may then be kept for up to 10 months after the pouch has been opened, at 2-8°C (or until the expiration date indicated on the packaging, if this comes before).
STORAGE OF THE REAGENTS Kovacs’ reagent, VP1, NIT 1 and NIT 2 should be stored at 15-30°C until the expiration date indicated on the packaging. Ferric chloride should be stored at 2-30°C until the expiration date indicated on the packaging. Zinc dust should be stored at 8-30°C until the expiration date indicated on the packaging. VP2 should be stored in the dark at 2-8°C until the expiration date if this comes first. Record the date opened on the bottle label. See Oxidase Test Kit Package Insert for storage instructions.
USE OF THE REAGENTS Allow reagents to come to room temperature (20-30°C) before using.
1. Ferric chloride, VP1, NIT 1, NIT 2 and Kovacs’:
• Dispense one drop of reagent. • Carefully close the bottle after use and store it as indicated in the paragraph "Storage of the Reagents".
2. VP2 :
• Open the ampule of reagent as indicated in the paragaraph "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".
3. Zinc dust :
• Open the bottle • Take up an aliquot of powder (approximately 2-3 mg) using the spatula fixed to the cap and deposit this quantity in the reaction cupule. • Carefully close the bottle after use and store it as indicated in the paragraph "Storage of the reagents".
4. Oxidase Test Kit:
• Please note that the oxidase test must be performed as it is an integral part of the st final profile (21 identification test .) See the Oxidase Test Kit Package Insert for the use of the oxidase reagent.
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 allow reagents to come into with skin, eyes, or clothing. • Do not use reagents past the expiration date. • Upon removal from refrigerator, allow reagents to come to room temperature (2030°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 to the flattened part of the cap to snap off the top of the ampoule 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 in a vertical position. - Squeeze on the cap to transfer all the reagent into the dropperbottle. • 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. • 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. • The performance data presented were obtained using the procedure indicated. Any changes or modifications in the procedure may affect results.
INSTRUCTIONS FOR USE
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 Diseases 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.
Preparation of the strip • 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, CO2, 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. • Place the strip in the tray. • Perform the oxidase test on a colony identical to the colony which will be tested. Refer to the Oxidase Test Kit Package Insert. This reaction should be recorded on the result sheet as it constitutes the 21st test. NOTE : API 20 E should only be used with non-fastidious Gram-negative rods. Fastidious organisms having demanding nutritional requirements and requiring appropriate handling precautions (i.e., Brucella and Francisella) are not included in the API 20 E database. Alternative procedures must be used to exclude or confirm their presence.
Preparation of the inoculum • Open an ampule of NaCl 0.85 % Medium (5 ml) or an ampule of Suspension Medium (5ml) as indicated in the paragraph "Warnings and Precautions" or use any tube containing 5 ml of sterile NaCl 0.85% Medium, pH 5.5 - 7.0, or sterile distilled water, without additives. • With the aid of a pipette, remove a single well-isolated colony from an isolation plate. • Carefully emulsify to achieve a homogeneous bacterial suspension. NOTE : Most Vibrio species are halophilic. If Vibrio is suspected, suspend the bacteria in NaCl 0.85 % Medium.
Inoculation of the strip 2
00
api 20 E
0008040-6 02/02
• With the same pipette, fill both tube and cupule of test CIT , VP and GEL with the bacterial suspension. • Fill only the tubes (and not the cupules) of the other tests. • Create anaerobiosis in the tests ____ ADH, ____ LDC , ____ ODC, ____ H2S and URE ____ by overlaying with mineral oil. • Close the incubation box. • Incubate at 35-37°C for 18-24 hours. • Weekend incubation : The biochemical reactions of the API 20 E strip should be read after 18-24 hours of incubation. If the strips cannot be read after 24 hours of incubation at 35-37°C, the strips should be removed from the incubator and stored at 2-8°C (refrigerator) until the reactions can be read.
Reading the strip • After 18-24 hours at 35-37°C, read the strip by referring to Interpretation of Reactions (Table 2.) • Record all spontaneous reactions on the result sheet. • If the glucose is positive or 3 or more tests are positive, reveal the tests which require the addition of reagents : - TDA Test : add 1 drop of Ferric chloride. A dark brown color indicates a positive reaction to be recorded on the result sheet. - IND Test : add 1 drop of Kovacs’ reagent. Wait 2 minutes. A red ring indicates a positive reaction to be recorded on the result sheet. - VP Test : add 1 drop of each of VP1 and VP2 reagents. Wait at least 10 minutes. A pink or red color indicates a positive reaction to be recorded on the result sheet. If a slightly pink color appears within 10 to 12 minutes, the reaction should be considered negative. NO2 Test : add 2 drops of each of NIT 1 and NIT 2 reagents to the GLU tube. Wait 2 to 3 minutes. A red color indicates a positive reaction. A negative reaction (yellow) may be due to the reduction to nitrogen (as sometimes evidenced by gas bubbles) : add 2 to 3 mg of Zinc dust to the GLU tube. After 5 minutes, if the tube remains yellow this indicates that (N2) is positive and is to be recorded on the result sheet. If the test turns pink-red, this is a negative reaction : the nitrates still present in the tube have been reduced by the Zinc. This reaction is useful when testing Gram-negative, oxidase positive rods. NOTE : The nitrate reduction and indole production tests must be performed last since these reactions release gaseous products which interfere with the interpretation of other tests on the strip. The plastic incubation lid should not be replaced after the addition of these reagents. • If the glucose is negative and the number of positive tests is less than or equal to 2, do not add reagents : - Inoculate 2 ampules of API OF Medium to confirm the metabolism of glucose. - Streak a MacConkey agar plate. - Check for motility : inoculate 1 ampule of API M Medium or observe between slide and cover slip under a microscope. - Reincubate the strip for 24 hours. - Add the reagents as described above. - Record the strip and supplementary test results on the result sheet by referring to Interpretation of Reactions (Table 2).
Identification Identification can be obtained : • 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 for the 20 tests of the API 20 E strip. The oxidase reaction constitutes the 21st test and has a value of 4 if it is positive. • using the identification software to obtain information on any profile not listed in the Analytical Profile Index, call the Voice Response System at 800-645-7056. In some cases, the 7-digit profile is not discriminatory enough and the following supplementary tests need to be carried out : -
Reduction of nitrates to nitrites (NO2) Reduction of nitrates to N2 gas (N2) Motility (MOB) Growth on MacConkey agar medium (McC) Oxidation of glucose (OF-O) Fermentation of glucose (OF-F)
These supplementary tests, indicated in the Analytical Profile Index, may be used to form a 9-digit profile. Identification is then obtained using the identification software.
5 315 173 (57) Enterobacter gergoviae
DISPOSAL OF USED MATERIAL After use, all ampules, pipettes, strips and incubation boxes should be autoclaved, incinerated, or immersed in a disinfectant for decontamination prior to disposal.
RANGE OF EXPECTED VALUES Consult the Identification Table at the end of this package insert for the range of expected values for the various biochemical reactions.
LIMITATIONS • The API 20 E system is intended uniquely for the identification of those nonfastidious, Gram-negative rods 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. • Discrepancies with respect to conventional methods may be observed due to the different principles of the reactions used in the API technique. • If Salmonella spp., Shigella spp., are identified, serological testing must be performed to confirm the bacterial identification.
QUALITY CONTROL The media, strips, and reagents are systematically quality controlled at various stages of their manufacture. For those who wish to perform their own quality control tests with the strip, it is recommended that the following stock cultures be used, to obtain the results below : Table 1 ONPG ____ ADH ____ LDC ODC ____ 1. 2. 3. 4. 5.
+ + – + +
– + – – –
V – – + +
– + + – +
CIT V + V + –
1. Stenotrophomonas maltophilia 2. Enterobacter cloacae 3. Proteus mirabilis
H2S URE ____ TDA IND VP – – + – –
– – + V –
– – + – –
– – – – +
– + – V –
ATCC 51331 ATCC 13047 ATCC 35659
GEL GLU MAN INO SOR RHA SAC MEL AMY ARA NO2 N2 + – V – –
– + + + +
– + – + +
– V – + –
– + – + +
– + – + +
– + V + –
– + – + +
– + – + –
– + – + +
– + + + +
– – – – –
4. Klebsiella pneumoniae ssp pneumoniae ATCC 35657 5. Escherichia coli ATCC 25922
ATCC : American Type Culture Collection, 10801 University Boulevard, Manassas, VA 20110-2209, USA. • Profile obtained after 24-48 hours of incubation for the strain ATCC 51331, using cultures grown on Trypticase Soy agar + blood • Profiles obtained after 18-24 hours of incubation for the other strains, using cultures grown on Trypticase Soy agar + blood • Bacterial suspension prepared in NaCl 0.85 % Medium
PERFORMANCE CHARACTERISTICS The Profile Recognition System has been evaluated by the National Institutes of Health, Bethesda, Maryland. The API 20E System has been evaluated extensively worldwide by institutions such as the Centers for Disease Control, Atlanta, Georgia; the Mayo Clinic, Rochester, Minnesota; the Pasteur 3
00
api 20 E
0008040-6 02/02
Institute, Lyon, ; Sherbrooke University, Sherbrooke, Quebec, Canada. In fact, the API 20E System was one of the original miniaturized biochemical detection systems and has evolved over the years as the standard against which other identification systems have been compared. Recently, the Centers for Disease Control reevaluated the identification capabilities of the API 20 E System and reported that at 24 hours, 20 E correctly identified to genus and species level 87.7% of a clinically weighted assortment of isolates. At 48 hours, 96.3% of the isolates were identified correctly (O’Hara, C.M., D.L. Rhoden, and J.M. Miller, 1992. J. Clin. Microbiol. 30: 123-125). These percentages reflect the product’s identification capabilities for taxa that are identified routinely on the 20 E System. In addition, the API 20E System permits recognition of biotypes within each species. Delineation of biotypes may be of value in determining the effectiveness of treatment and the existence of nosocomial infections as related to of the family Enterobacteriaceae, as well as for control of the latter.
bioMérieux sa
4
api 20 E
0
008040-6 02/02
INTERPRETATION OF REACTIONS Table 2 TESTS
SUBSTRATES
QTY
ENZYMATIC ACTIVITY OR REACTION TESTED
RESULTS NEGATIVE
POSITIVE
beta-galactosidase
colorless
yellow (1)
red / orange (2)
ortho-nitrophenyl-‚ -Dgalactopyranoside(ONPG)
0.2 mg
isopropylthiogalactopyranoside (IPTG)
8.0 µg
ADH ____
arginine
2.0 mg
arginine dihydrolase
yellow
LDC ____
lysine
2.0 mg
lysine decarboxylase
yellow
red/orange (2)
ODC ____
ornithine
2.0 mg
ornithine decarboxylase
yellow
red / orange (2)
sodium citrate
0.8 mg
citrate utilization
pale green / yellow
blue-green / blue (3)
H2S ___
sodium thiosulfate
80.0 µg
H2S production
colorless / greyish
black deposit / thin line
URE ____
urea
0.8 mg
urease
yellow
red / orange (2)
tryptophane deaminase
Ferric chloride (1 drop) / immediate yellow brown-red
ONPG
CIT
TDA
tryptophane
0.4 mg
Kovacs’ reagent (1 drop) / 2 min IND
VP GEL
tryptophane
0.2 mg
creatine
0.9 mg
sodium pyruvate
2.0 mg
Kohn's charcoal gelatin
0.6 mg
indole production
IND yellow
IND red ring
VP1 (1 drop) + VP2 (1 drop) / acetoin production gelatinase
10 min
colorless
pink / red (5)
no diffusion of black pigment
diffusion of black pigment
GLU
glucose
2.0 mg
fermentation / oxidation (4)
blue / blue-green
yellow / greyish yellow
MAN
mannitol
2.0 mg
fermentation / oxidation (4)
blue / blue-green
yellow
INO
inositol
2.0 mg
fermentation / oxidation (4)
blue / blue-green
yellow
SOR
sorbitol
2.0 mg
fermentation / oxidation (4)
blue / blue-green
yellow
RHA
rhamnose
2.0 mg
fermentation / oxidation (4)
blue / blue-green
yellow
SAC
sucrose
2.0 mg
fermentation / oxidation (4)
blue / blue-green
yellow
MEL
melibiose
2.0 mg
fermentation / oxidation (4)
blue / blue-green
yellow
AMY
amygdalin
2.0 mg
fermentation / oxidation (4)
blue / blue-green
yellow
ARA
arabinose
2.0 mg
fermentation / oxidation (4)
blue / blue-green
yellow
OX
on filter paper
Oxidase reagent / 1-2 min colorless violet
cytochrome-oxidase
NIT 1 (2 drops) + NIT 2 (2 drops) / 2-3 min GLU tube Nitrate Reduction
MOB
yellow
NO2 production Potassium nitrate
API M Medium or microscope
80.0 µl
red Zinc Dust / 5 min
reduction to N2 gas
orange-red
yellow
motility
non-motile
motile
McC
MacConkey medium
growth
absence
presence
OF-F OF-O
glucose (API OF Medium)
fermentation : under mineral oil oxidation : exposed to the air
green green
yellow yellow
(1) A very pale yellow should also be considered positive. (2) An orange color after 36-48 hours incubation must be considered negative. (3) Reading made in the cupule (aerobic).
(4) Fermentation begins in the lower portion of the tubes, oxidation begins in the cupules. (5) A slightly pink color after 10 minutes should be considered negative.
5
api 20 E
0
008040-6 02/02
IDENTIFICATION TABLE % of positive reactions after 24-48 hrs at 35-37° ONPG 100
ADH 0
LDC 0
ODC 85
CIT 25
H2 S 0
URE 0
TDA 0
IND 0
VP 0
GEL 0
GLU 100
MAN 100
INO 0
SOR 1
RHA 99
SAC 0
MEL 92
AMY 99
ARA 100
OX 0
NO 2 100
N2 0
MOB 100
McC 100
OF/O 100
OF/F 100
Cedecea davisae 100
99
89
0
99
75
0
0
0
0
89
0
100
100
10
0
0
100
0
100
1
0
99
0
87
100
100
100
Cedecea lapagei
99
99
0
0
75
0
0
0
0
90
0
100
99
0
0
0
0
1
100
1
0
99
0
87
100
100
100
Citrobacter braakii
50
45
0
99
75
81
1
0
4
0
0
100
100
1
100
100
1
91
99
99
0
100
0
95
100
100
100
Citrobacter freundii
90
24
0
0
75
75
1
0
1
0
0
100
99
25
99
99
99
82
40
99
0
98
0
95
100
100
100
Citrobacter koseri/amalonaticus
99
75
0
100
97
0
1
0
99
0
0
100
100
25
99
99
1
1
98
99
0
100
0
95
100
100
100
Citrobacter koseri/farmeri
99
2
0
100
25
0
1
0
99
0
0
100
100
1
99
99
99
80
99
99
0
100
0
95
100
100
100
Citrobacter youngae
100
50
0
1
80
80
0
0
1
0
0
100
100
0
95
100
1
0
25
100
0
85
0
95
100
100
100
Edwardsiella hoshinae
0
0
100
99
50
94
0
0
99
0
0
100
100
0
0
1
100
0
0
1
0
100
0
100
100
100
100
Edwardsiella tarda
0
0
100
99
1
75
0
0
99
0
0
100
0
0
0
0
0
0
0
0
0
100
0
98
100
100
100
Enterobacter aerogenes
99
0
99
98
82
0
1
0
0
85
0
99
99
99
99
99
99
99
99
99
0
100
0
97
100
100
100
Enterobacter amnigenus 1
99
25
0
99
40
0
0
0
0
75
0
100
100
0
1
100
99
99
99
99
0
100
0
92
100
100
100
Enterobacter amnigenus 2
99
80
0
99
80
0
0
0
0
75
0
100
100
0
99
100
1
99
99
99
0
100
0
100
100
100
100
Enterobacter asburiae
100
25
0
99
80
0
0
0
0
10
0
100
99
25
100
0
99
0
100
100
0
100
0
95
100
100
100
Enterobacter cancerogenus
100
75
0
99
99
0
0
0
0
89
0
100
100
0
1
100
1
1
100
100
0
100
0
99
100
100
100
Enterobacter cloacae
98
82
1
92
90
0
1
0
0
85
0
99
99
12
90
85
96
90
99
99
0
100
0
95
100
100
100
Enterobacter gergoviae
99
0
32
100
75
0
99
0
0
90
0
100
99
23
1
100
99
100
99
100
0
100
0
90
100
100
100
Enterobacter intermedius
99
0
0
99
1
0
0
0
0
2
0
100
97
0
88
99
40
100
99
99
0
100
0
92
100
100
100
Enterobacter sakazakii
100
96
0
91
94
0
1
0
25
91
10
100
100
75
8
99
99
99
99
99
0
100
0
96
100
100
100
Escherichia coli 1
90
1
74
70
0
1
3
0
89
0
0
99
98
1
91
82
36
75
3
99
0
100
0
95
100
100
100
Escherichia coli 2
26
1
45
20
0
1
1
0
50
0
0
99
90
1
42
30
3
3
1
70
0
98
0
5
100
100
100
Escherichia fergusonii
96
1
99
100
1
0
0
0
99
0
0
100
99
1
0
87
0
1
99
99
0
100
0
93
100
100
100
Escherichia hermannii
100
0
1
100
1
0
0
0
99
0
0
100
100
0
0
99
25
0
99
99
0
100
0
99
100
100
100
Escherichia vulneris
100
30
50
0
0
0
0
0
0
0
0
100
100
0
1
95
7
95
95
99
0
100
0
100
100
100
100
Ewingella americana
98
0
0
0
75
0
0
0
0
95
1
99
99
0
0
1
0
1
50
1
0
100
0
60
100
100
100
Hafnia alvei 1
75
0
99
98
50
0
10
0
0
50
0
99
99
0
1
99
0
0
25
99
0
100
0
85
100
100
100
Hafnia alvei 2
50
0
99
99
1
0
1
0
0
10
0
99
98
0
1
1
1
0
0
1
0
100
0
0
100
100
100
Klebsiella ornithinolytica
100
0
99
99
99
0
85
0
100
65
0
100
100
99
100
100
100
100
100
100
0
100
0
0
100
100
100
Klebsiella oxytoca
99
0
80
0
89
0
78
0
99
80
0
100
100
99
100
99
99
100
100
100
0
100
0
0
100
100
100
Klebsiella pneumoniae ssp ozaenae
94
18
25
1
18
0
1
0
0
1
0
99
96
57
66
58
20
80
97
85
0
92
0
0
100
100
100
Klebsiella pneumoniae ssp pneumoniae
99
0
73
0
86
0
75
0
0
90
0
100
99
99
99
99
99
99
99
99
0
100
0
0
100
100
100
1
0
0
0
0
0
0
0
0
0
0
99
100
90
90
75
75
1
99
10
0
100
0
0
100
100
100
Klebsiella terrigena
100
0
99
6
52
0
0
0
0
75
0
99
99
99
99
99
100
100
100
99
0
100
0
0
100
100
100
Kluyvera spp
95
0
25
99
60
0
0
0
80
0
0
100
99
0
25
93
89
99
99
99
0
95
0
94
100
100
100
Leclercia adecarboxylata
99
0
0
0
0
0
1
0
99
0
1
100
99
0
2
100
66
99
99
100
0
100
0
100
100
100
100
Moellerella wisconsensis
97
0
0
0
40
0
0
0
15
1
0
100
1
0
0
0
100
99
0
0
0
90
0
0
100
100
100
1
0
10
98
1
1
99
93
99
0
0
99
0
0
0
0
1
0
0
0
0
88
0
95
100
100
100
Pantoea spp 1
85
1
0
0
13
0
1
0
1
9
1
100
99
1
26
1
98
26
59
61
0
85
0
85
100
100
100
Pantoea spp 2
99
1
0
0
99
0
1
0
53
62
4
100
99
36
82
90
98
91
99
99
0
85
0
85
100
100
100
Pantoea spp 3
99
1
0
0
21
0
1
0
1
86
15
100
99
34
1
97
93
23
65
97
0
85
0
85
100
100
100
Pantoea spp 4
86
1
0
0
29
0
1
0
59
1
1
99
100
10
32
99
72
89
99
99
0
85
0
85
100
100
100
Proteus mirabilis
1
0
0
99
50
75
99
98
1
1
82
98
0
0
0
0
1
0
0
0
0
93
0
95
100
100
100
Proteus penneri
1
0
0
0
1
20
100
99
0
0
50
99
0
0
0
0
100
0
1
0
0
99
0
85
100
100
100
Proteus vulgaris
1
0
0
0
12
83
99
99
92
0
74
99
1
1
0
1
89
0
66
1
0
100
0
94
100
100
100
Providencia alcalifaciens/rustigianii
0
0
0
0
80
0
0
100
99
0
0
99
1
1
0
0
1
0
0
1
0
100
0
96
100
100
100
API 20 E
V4.0
Buttiauxella agrestis
Klebsiella pneumoniae ssp rhinoscleromatis
Morganella morganii
7
api 20 E 0 008040-6 02/02 ______________________________________________________________________________________________________________________________________________________________________ __ Providencia rettgeri
1
1
0
0
74
0
99
99
90
0
0
98
82
78
1
50
25
0
40
1
0
98
0
94
100
100
100
Providencia stuartii
1
0
0
0
85
0
30
98
95
0
0
98
3
80
0
0
15
0
0
0
0
100
0
85
100
100
100
Rahnella aquatilis
100
0
0
0
50
0
0
1
0
99
0
100
100
0
98
99
100
97
100
98
0
100
0
6
100
100
100
Salmonella arizonae
98
75
97
98
75
99
0
0
1
0
0
100
99
0
99
99
1
78
0
99
0
100
0
99
100
100
100
Salmonella choleraesuis
0
15
99
99
6
64
0
0
0
0
0
100
99
0
98
99
0
20
0
0
0
100
0
95
100
100
100
Salmonella gallinarum
0
1
100
1
0
25
0
0
0
0
0
100
100
0
0
1
0
0
0
100
0
100
0
0
100
100
100
8
api 20 E 0 008040-6 02/02 ______________________________________________________________________________________________________________________________________________________________________ __ ONPG 0
ADH 5
LDC 0
ODC 99
CIT 0
H2 S 1
URE 0
TDA 0
IND 0
VP 0
GEL 0
GLU 100
MAN 99
INO 0
SOR 99
RHA 98
SAC 0
MEL 96
AMY 0
ARA 99
OX 0
NO 2 100
N2 0
MOB 95
McC 100
OF/O 100
OF/F 100
Salmonella pullorum
0
1
75
100
0
85
0
0
0
0
0
100
100
Salmonella typhi
0
1
99
0
0
8
0
0
0
0
0
100
99
0
0
100
0
99
0
0
0
0
75
0
100
0
99
0
0
0
100
0
0
100
100
100
0
97
100
100
Salmonella spp
1
56
82
93
65
83
0
0
1
0
1
99
100
40
99
86
1
90
1
99
1
100
100
0
95
100
100
Serratia ficaria
99
0
0
0
100
0
0
0
0
40
90
100
100
50
99
74
99
99
100
99
100
0
92
0
100
100
100
Serratia fonticola
99
0
73
99
75
0
0
0
0
0
0
100
100
97
100
99
30
99
99
100
99
0
99
0
91
100
100
Serratia liquefaciens
95
1
78
98
80
0
2
0
0
59
65
100
99
80
98
2
99
72
100
97
97
0
100
0
95
100
100
Serratia marcescens
94
0
95
95
96
0
25
0
1
70
87
100
99
85
98
1
99
100
68
97
25
0
95
0
97
100
100
Serratia odorifera 1
95
0
95
99
95
0
0
0
99
50
99
100
99
99
99
99
100
99
99
99
99
0
99
0
100
100
100
Serratia odorifera 2
95
0
96
1
95
0
0
0
99
50
99
100
99
99
99
100
99
1
99
99
95
0
99
0
100
100
100
Serratia plymuthica
99
0
0
0
65
0
0
0
0
65
50
100
90
70
100
70
1
99
85
98
98
0
99
0
50
100
100
Serratia rubidaea
99
0
30
0
92
0
1
0
0
71
82
99
99
100
75
1
3
99
95
99
99
0
100
0
85
100
100
1
0
0
1
0
0
0
0
29
0
0
99
100
63
0
7
7
1
20
0
50
0
100
0
0
100
100
Shigella sonnei
96
0
0
93
0
0
0
0
0
0
0
100
99
99
0
1
75
1
1
0
99
0
100
0
0
100
100
Yersinia enterocolitica
80
0
0
90
0
0
98
0
50
5
100
0
99
99
25
98
1
99
4
75
75
0
98
0
2
100
100
Yersinia frederiksenii/intermedia
99
0
0
75
1
0
99
0
99
100
1
0
100
99
25
99
99
99
1
99
99
0
98
0
5
100
100
Yersinia kristensenii
80
0
0
80
0
0
99
0
100
97
0
0
100
99
10
99
0
0
0
99
99
0
98
0
5
100
100
Yersinia pestis
68
0
0
0
0
0
0
100
0
0
1
0
99
99
0
70
0
0
0
30
30
0
47
0
0
99
100
Yersinia pseudotuberculosis
98
0
0
0
1
0
100
99
0
0
0
0
99
97
0
0
75
0
50
25
50
0
95
0
0
100
100
Aeromonas hydrophila gr. 1
98
90
25
1
25
100
0
0
0
85
25
90
99
99
1
3
5
97
1
75
75
100
97
0
95
99
99
Aeromonas hydrophila gr. 2
99
97
80
1
99
80
0
0
0
85
80
97
97
99
9
9
1
80
1
75
5
100
97
0
95
99
99
Aeromonas salmonicida ssp salmonicida
1
60
1
99
0
0
0
0
0
1
0
75
50
54
0
0
0
0
0
1
0
100
98
0
1
99
99
Photobacterium damsela
1
99
99
75
0
1
0
98
0
0
10
1
50
0
0
0
0
1
0
0
0
100
100
0
25
99
99
Plesiomonas shigelloides
95
99
99
100
100
0
0
0
0
100
0
0
99
0
99
0
0
0
0
0
0
100
99
0
95
99
99
99
0
0
98
75
60
0
1
0
100
10
75
99
100
0
1
0
100
0
10
1
100
47
0
100
99
94
94
Vibrio cholerae
98
1
94
97
75
0
0
0
99
58
92
98
98
0
0
0
94
0
5
0
100
96
0
100
96
99
99
Vibrio fluvialis
95
99
0
0
1
0
0
0
80
0
75
75
80
0
1
0
75
0
36
75
100
100
0
100
99
99
99
Vibrio hollisae
1
0
0
0
0
0
0
0
94
0
0
10
0
0
0
0
0
0
0
0
100
100
0
0
99
99
99
Vibrio mimicus
99
0
99
99
50
0
0
0
99
1
99
99
99
0
0
0
0
0
0
0
100
95
0
100
95
99
99
0
0
100
99
50
0
1
0
100
1
75
100
99
0
0
1
1
0
12
50
100
63
0
100
98
99
99
Vibrio vulnificus
99
0
91
90
25
0
0
0
99
1
99
99
75
0
0
0
1
0
90
0
99
54
0
100
99
99
99
Pasteurella aerogenes
99
0
0
80
0
0
99
0
0
0
0
99
0
97
0
1
99
0
0
75
75
100
0
0
100
100
100
Pasteurella multocida 1
4
0
0
25
0
0
0
0
99
0
0
29
1
0
1
0
75
0
0
0
99
90
0
0
2
23
23
Pasteurella multocida 2
7
0
0
45
0
0
0
0
99
0
0
44
99
0
99
0
99
0
0
0
89
90
0
0
2
23
23
Pasteurella pneumotropica/haemolytica
60
0
1
10
0
0
25
0
15
7
3
35
12
12
12
1
35
1
2
1
80
99
0
0
9
33
33
Acinetobacter baumannii/calcoaceticus
0
0
0
0
51
0
1
0
0
5
5
99
0
0
0
0
0
99
1
99
0
3
0
0
90
98
0
Bordetella/Alcaligenes/Moraxella spp *
0
0
0
0
52
0
14
1
0
25
1
0
0
0
0
0
0
0
0
0
95
62
1
75
75
0
0
50
0
25
16
78
0
0
0
0
1
43
60
1
0
0
0
13
0
7
20
90
40
0
99
88
97
0
0
99
0
0
75
0
0
0
14
0
99
99
0
0
0
0
10
0
0
0
99
75
0
99
99
99
99
86
75
0
0
94
0
0
0
0
25
13
84
0
1
0
1
1
15
1
85
0
30
0
100
91
94
0
5
0
0
0
12
0
90
0
75
0
80
0
0
0
0
0
0
0
0
0
99
20
0
0
57
90
10
API 20 E
V4.0
Salmonella paratyphi A
Shigella spp
Vibrio alginolyticus
Vibrio parahaemolyticus
Burkholderia cepacia Chromobacterium violaceum Chryseomonas luteola Chryseobacterium indologenes Chryseobacterium meningosepticum
77
0
0
0
20
0
1
0
85
0
90
0
0
0
0
0
0
0
0
0
99
6
0
0
48
93
6
Eikenella corrodens
0
0
75
99
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
100
95
0
1
1
49
49
Flavimonas oryzihabitans
0
0
0
0
89
0
0
0
0
25
1
10
0
1
0
1
0
10
0
45
0
7
0
100
99
99
0
Myroides /Chryseobacterium indologenes
0
0
0
0
50
0
75
0
0
1
75
0
0
0
0
0
0
0
0
0
99
0
0
0
84
2
2
15
0
0
0
30
0
25
1
0
15
0
1
0
0
0
0
0
0
0
10
90
42
60
99
99
47
0
Ochrobactrum anthropi
8
api 20 E 0 008040-6 02/02 ______________________________________________________________________________________________________________________________________________________________________ __ Pseudomonas aeruginosa
0
89
0
0
92
0
25
0
0
1
75
50
0
0
0
0
1
10
1
25
97
12
56
97
100
98
0
Pseudomonas fluorescens/putida
0
75
0
0
75
0
0
0
0
10
27
25
0
0
0
0
0
25
1
20
99
26
0
100
96
93
0
Non-fermenter spp
1
1
0
0
37
0
1
0
0
15
9
9
0
0
0
1
1
1
1
1
93
48
35
99
85
49
0
Shewanella putrefaciens
0
0
0
80
75
75
1
0
0
0
75
1
0
0
0
0
1
0
0
2
99
96
0
100
96
9
0
70
0
75
1
75
1
0
0
0
0
90
1
0
0
0
0
0
0
0
0
1
26
1
100
91
49
0
Stenotrophomonas maltophilia
* Brucella spp possible / möglich / posible / possibile
8
api 20 E 0
008040-6 02/02
BIBLIOGRAPHY 1. APPELBAUM P.C., STAVITZ J., BENTZ M.S., VON KUSTER L.C. Four Methods for Identification of Gram-Negative Nonfermenting Rods : Organisms more Commonly Encountered in Clinical Specimens. (1980) J. Clin. Microbiol. 12, 271-278.
5. McLAUGHLIN J.K., ZUCKERMAN B.D., TENENBAUM S., WOLF B.A. Comparison of the API 20E, Flow, and Minitek systems for the identification of enteric and nonfermentative bacteria isolated from cosmetic raw materials. (1984) J. Soc. Cosmet. Chem. 35, 253-263.
2. BROOKS K.A., JENS M., SODEMAN T.M. A Clinical Evaluation of the API Microtube System for Identification of Enterobacteriaceae. (1974) Am. J. Med. Techn. 40, 55-61.
6. NORD C.E., LINDBERG A.A., DAHLBÄCK A. Evaluation of Five Test-Kits, API, AuxoTab, Enterotube, PathoTec and R/B, for Identification of Enterobacteriaceae. (1974) Med. Microbiol. Immunol. 159, 211-220.
3. CASTILLO C.B., BRUCKNER D.A. Comparative Evaluation of the Eiken and API 20E Systems and Conventional Methods for Identification of of the family Enterobacteriaceae. (1984) J. Clin. Microbiol. 20, 754-757.
7. SMITH P.B., TOMFOHRDE K.M., RHODEN D.L., BALOWS A. API System : A multitube Micromethod for Identification of Enterobacteriaceae. (1972) Applied Microbiol. 24, 449-452.
4. HAYEK L., WILLIS G.W. Identification of the Enterobacteriaceae : a Comparison of the Enterotube II with the API 20E. (1984) J. Clin. Pathol. 37, 344-347.
8. SWANSON E.C., COLLINS M.T. Use of the API 20E System to Identify Veterinary Enterobacteriaceae. (1980) J. Clin. Microbiol. 12, 10-14.
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.
9
api 20 E 0
008040-6 02/02
Fabricant / Manufacturer bioMérieux sa bioMérieux sa
bioMérieux, Inc.
au capital de 77 421 420 F 673 620 399 RCS LYON 69280 Marcy-l’Etoile / tel. (33) 4 78 87 20 00 / fax (33) 4 78 87 20 90 http://www.biomerieux.com
595 Anglum Road Hazelwood, MO 63042-2320 / USA tel. (1) 314-731-8500 / fax (1) 314-731-8800 Imprimé en / Printed in USA
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