REF 20 050 07615H - GB - 2003/10

®
20 NE IVD

Identification system for non-fastidious, non-enteric Gram-negative rods

SUMMARY AND EXPLANATION Material :

API 20 NE is a standardized system for the identification - Pipettes or PSIpettes
of non-fastidious, non-enteric Gram-negative rods - Ampule protector
(e.g. Pseudomonas, Acinetobacter, Flavobacterium, - Ampule rack
Moraxella, Vibrio, Aeromonas, etc.), combining - General microbiology laboratory equipment
8 conventional tests, 12 assimilation tests and a
database. The complete list of those organisms that it is WARNINGS AND PRECAUTIONS
possible to identify with this system is given in the • For in vitro diagnostic use and microbiological
Identification Table at the end of this package insert. control.
• For professional use only.
PRINCIPLE • This kit contains products of animal origin. Certified
The API 20 NE strip consists of 20 microtubes containing knowledge of the origin and/or sanitary state of the
dehydrated substrates. animals does not totally guarantee the absence of
The conventional tests are inoculated with a saline transmissible pathogenic agents. It is therefore
bacterial suspension which reconstitutes the media. recommended that these products be treated as
During incubation, metabolism produces color changes potentially infectious, and handled observing the usual
that are either spontaneous or revealed by the addition of safety precautions (do not ingest or inhale).
reagents. • All specimens, microbial cultures and inoculated
The assimilation tests are inoculated with a minimal products should be considered infectious and handled
medium and the bacteria grow if they are capable of appropriately. Aseptic technique and usual precautions
utilizing the corresponding substrate. for handling the bacterial group studied should be
The reactions are read according to the Reading Table observed throughout this procedure. Refer to "NCCLS
and the identification is obtained by referring to the M29-A, Protection of Laboratory Workers from
Analytical Profile Index or using the identification software. Instrument Biohazards and Infectious Disease
Transmitted by Blood, Body Fluids, and Tissue;
CONTENT OF THE KIT (Kit for 25 tests) Approved Guideline - December 1997". For additional
- 25 API 20 NE strips handling precautions, refer to "Biosafety in
- 25 incubation boxes Microbiological and Biomedical Laboratories, HHS
- 25 ampules of API AUX Medium Publication No. (CDC) 93-8395, 3rd Edition (May
- 25 result sheets 1993)", or to the regulations currently in use in each
- 1 package insert country.
• Do not use reagents past the expiration date.
• Before use, check that the packaging and components
COMPOSITION
are intact.
Strip • Do not use strips which have been damaged : cupules
The composition of the API 20 NE strip is given in the deformed, etc.
Reading Table of this package insert. • Open ampules carefully as follows :
- Place the ampule in the ampule protector.
Medium - Hold the protected ampule in one hand in a
API AUX Ammonium sulphate 2g vertical position (white plastic cap upper-
Medium Agar 1.5 g most).
7 ml Vitamin solution 10.5 ml - Press the cap down as far as possible.
Trace elements 10 ml - Cover the flattened part of the cap with the
Monosodium phosphate 6.24 g upper part of the thumb.
Potassium chloride 1.5 g - Apply thumb pressure in an outward motion
Demineralized water to make 1000 ml to the base of the flattened part of the cap to
Final pH : 7.0-7.2 snap off the top of the ampule inside the
cap.
- Take the ampule out of the ampule protector
REAGENTS AND MATERIAL REQUIRED BUT NOT and put the protector aside for subsequent
PROVIDED use.
Reagents : - Carefully remove the cap.
- API NaCl 0.85 % Medium, 2 ml (Ref. 20 070) • The performance data presented were obtained using
- Reagents : JAMES (Ref. 70 542) the procedure indicated in this package insert. Any
NIT 1 + NIT 2 (Ref. 70 442) change or modification in the procedure may affect the
Zn (Ref. 70 380) results.
- Oxidase (Ref. 55 635*) • Interpretation of the test results should be made taking
* reference not sold in certain countries : use an into consideration the patient history, the source of the
equivalent reagent. specimen, colonial and microscopic morphology of the
- Mineral oil (Ref. 70 100) strain and, if necessary, the results of any other tests
- McFarland Standard (Ref. 70 900) No. 0.5 performed, particularly the antimicrobial susceptibility
- API 20 NE Analytical Profile Index (Ref. 20 090) or patterns.
identification software (consult bioMérieux)

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STORAGE CONDITIONS Inoculation of the strip

The strips and media should be stored at 2-8°C until the • Inoculate tests NO3 to PNPG by distributing the saline
expiration date indicated on the packaging. suspension into the tubes (and not the cupules) using
the same pipette. To avoid the formation of bubbles at
SPECIMENS (COLLECTION AND PREPARATION) the base of the tubes, tilt the strip slightly forwards and
API 20 NE is not for use directly with clinical or other place the tip of the pipette or PSIpette against the side
specimens. of the cupule.
The microorganisms to be identified must first be iso- • Open an ampule of API AUX Medium as indicated in the
lated on a suitable culture medium (e.g., Trypticase Soy paragraph "Warnings and Precautions" and add
agar) according to standard microbiological techniques. approximately 200 µl of the remaining saline suspension
to the ampule. Homogenize well with the pipette,
avoiding the formation of bubbles.
INSTRUCTIONS FOR USE
• Fill the tubes and cupules of tests GLU to PAC with
Oxidase test the suspension. Take care to leave a flat or slightly
The oxidase test must be performed according to the convex, but not concave, meniscus. Cupules under or
manufacturer's instructions for use. The result should be overfilled may give incorrect results.
recorded on the result sheet as it is an integral part of the • Add mineral oil to the cupules of the 3 underlined tests
final profile (21st identification test). (GLU, ADH and URE) until a convex meniscus is
formed.
Selection of colonies • Close the incubation box and incubate at 29°C ± 2°C for
API 20 NE should only be used with non-fastidious Gram- 24 hours (± 2 hours).
negative rods which do not belong to the Entero-
bacteriaceae. READING AND INTERPRETATION
NOTE 1 : Some non-enteric Gram-negative rods are Reading the strip
oxidase negative (S. maltophilia, Acinetobacter...). These
• After the incubation period, read the strip by referring to
microorganisms may also be identified with API 20 NE but
the Reading Table.
their selection must be based on other bacteriological or
• Record all spontaneous reactions (GLU, ADH, URE,
clinical criteria.
ESC, GEL and PNPG) on the result sheet.
NOTE 2 : Fastidious organisms having demanding • The reading of the two tests NO3 and TRP should be
nutritional requirements and requiring appropriate performed whilst protecting the assimilation tests from
handling precautions (i.e. Brucella and Francisella) are airborne contamination. To do this, cover the
not included in the API 20 NE database. Alternative assimilation tests with the incubation box lid during the
procedures must be used to exclude or confirm their reading of the NO3 and TRP tests.
presence.
• NO3 test :
Preparation of the strip - Add 1 drop of NIT 1 and 1 drop of NIT 2 reagents to
• Prepare an incubation box, tray and lid, and distribute the NO3 cupule.
about 5 ml of distilled water or demineralized water - After 5 minutes, a red color indicates a positive
[or any water without additives or chemicals which may reaction to be recorded on the result sheet.
release gases (e.g. Cl2, CO2, etc.)] into the bottom of the - A negative reaction may be due to the production of
tray to create a humid atmosphere. nitrogen (indicated by the presence of tiny bubbles) :
• Record the specimen number on the elongated flap of add 2-3 mg of Zn reagent to the NO3 cupule.
the tray. (Do not record the number on the lid as it may - After 5 minutes, a cupule remaining colorless
be misplaced during the procedure.) indicates a positive reaction to be recorded on the
• Remove the strip from its individual packaging. result sheet. If the cupule turns pink-red, the reaction
• Place the strip in the incubation box. is negative as nitrates were present in the tube and
were reduced to nitrite by the zinc.
Preparation of the inoculum The reaction used for the identification of the bacterium
• Open an ampule of API NaCl 0.85 % Medium (2 ml) as is the reduction of nitrates. It is positive when either of
indicated in the paragraph "Warnings and Precautions" the above reactions (production of NO2 or N2) is
of the package insert for this product, or use any tube positive.
containing 2 ml of 0.85 % physiological saline without The production of N2 may, however, be useful alone as
additives. a supplementary test (refer to the Analytical Profile
• Using a pipette or PSIpette, pick up 1-4 colonies of Index).
identical morphology from the agar plate, either by
suction or by successive touches. It is recommended to • TRP test :
use young cultures (18-24 hours old). Add 1 drop of JAMES reagent. The reaction takes place
• Prepare a suspension with a turbidity equivalent to immediately : a pink color which develops in the whole
0.5 McFarland. This suspension must be used cupule indicates a positive reaction to be recorded on
immediately after preparation. the result sheet.

NOTE : It is very important that the density of the

inoculum be adjusted to 0.5 McFarland ; the API 20 NE
strip tests may otherwise not function correctly. In
particular, a weaker inoculum may lead to false negative
results. Do not touch the cupules while working with the
strip and do not leave the strip exposed to air for a long
period of time after inoculation.

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• Assimilation tests : Interpretation

Observe the bacterial growth. An opaque cupule Identification is obtained with the numerical profile.
indicates a positive reaction.
• Determination of the numerical profile :
Occasionally, a cupule may show weak growth. In this
– or + by On the result sheet, the tests are separated into groups
case, the results should be recorded as + – of 3 and a number 1, 2 or 4 is indicated for each. By
comparing the intensity to that of the other tests on the
adding the values corresponding to positive reactions
strip.
within each group, a 7-digit number is obtained ; the
Once these readings have been made, identification
oxidase reaction constitutes the 21st test and has a
should be possible as indicated in the paragraph
value of 4 if it is positive.
"Interpretation". However, in the following cases, the
strip must be reincubated : • Identification :
- if the profile cannot be found in the API 20 NE This is performed using the database (V6.0)
Analytical Profile Index * with the Analytical Profile Index :
- if the following note is indicated for the profile - Look up the numerical profile in the list of profiles.
obtained : * with the identification software :
IDENTIFICATION NOT VALID - Enter the 7-digit numerical profile manually via the
BEFORE 48-HR INCUBATION keyboard.
Using a pipette or PSIpette, remove the NIT 1, NIT 2
and JAMES reagents by suction and immediately cover
tests NO3 and TRP with mineral oil so that a convex
meniscus is formed. Reincubate the strip at 29°C ± 2°C
for a further 24 hours and read the all the tests again,
except the first 3 (NO3, TRP and GLU) which should 1 154 575 Pseudomonas aeruginosa
only be read once at 24 hours.

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 preferable to use the strain
1. Sphingobacterium multivorum ATCC 35656 or else one of the following strains :
2. Aeromonas hydrophila ATCC 35654 4. Alcaligenes faecalis ATCC 35655
3. Pseudomonas aeruginosa ATCC 27853
ATCC : American Type Culture Collection, 10801 University Boulevard, Manassas, VA 20110-2209, USA.

NO3 TRP GLU ADH URE ESC GEL PNPG GLU ARA MNE MAN NAG MAL GNT CAP ADI MLT CIT PAC OX
1. – – – – + + – + + + + – + + – – – – – – +
2. + + + + – + + + + + + + + + + + – + –* – +
3. + – – V V – + – + – – + + – + + + + + – +
4. – – – – – – – – – – – – – – – + – + + + +

* Weak reactions may occur.

Profiles for tests ADH to PAC obtained after 48 hours of incubation after culture of the colonies on Trypticase Soy agar.
It is the responsibility of the user to perform Quality Control in accordance with any local applicable regulations.

LIMITATIONS OF THE METHOD WASTE DISPOSAL

• The API 20 NE system is intended uniquely for the Unused ampules of API AUX Medium may be considered
identification of those non-fastidious, non-enteric Gram- as non hazardous waste and disposed of accordingly.
negative rods included in the database (see Dispose of all used or unused reagents (other than the
Identification Table at the end of this package insert). It ampules of API AUX Medium) as well as any other
cannot be used to identify any other microorganisms or contaminated disposable materials following procedures
to exclude their presence. for infectious or potentially infectious products.
• Only pure cultures of a single organism should be used. It is the responsibility of each laboratory to handle waste
and effluents produced according to their type and degree
RANGE OF EXPECTED RESULTS of hazardousness and to treat and dispose of them (or
Consult the Identification Table at the end of this package have them treated and disposed of) in accordance with
insert for the range of expected results for the various any applicable regulations.
biochemical reactions.
WARRANTY
PERFORMANCE bioMérieux disclaims all warranties, express or implied,
5728 collection strains and strains of various origins including any implied warranties of MERCHANTABILITY
belonging to species included in the database were AND FITNESS FOR A PARTICULAR USE. bioMérieux
tested : shall not be liable for any incidental or consequential
- 92.53 % of the strains were correctly identified (with or damages. IN NO EVENT SHALL BIOMERIEUX’S
without supplementary tests). LIABLITY TO CUSTOMER UNDER ANY CLAIM
- 3.13 % of the strains were not identified. EXCEED A REFUND OF THE AMOUNT PAID TO
- 4.34 % of the strains were misidentified. BIOMERIEUX FOR THE PRODUCT OR SERVICE
WHICH IS THE SUBJECT OF THE CLAIM.

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READING TABLE

QTY RESULTS
TESTS ACTIVE INGREDIENTS (mg/cup.) REACTIONS/ENZYMES
NEGATIVE POSITIVE
NIT 1 + NIT 2 / 5 min
reduction of nitrates to nitrites colorless pink-red
NO3 potassium nitrate 0.136
Zn / 5 min
reduction of nitrates to nitrogen pink colorless
JAMES / immediate
TRP L-tryptophane 0.2 indole production (TRyptoPhane) colorless
pink
pale green / yellow
GLU D-glucose 1.92 fermentation (GLUcose) blue to green yellow
orange / pink /
ADH L-arginine 1.92 Arginine DiHydrolase yellow
red
orange / pink /
URE urea 0.76 UREase yellow
red
esculin 0.56 grey / brown /
ESC hydrolysis (β-glucosidase) (ESCulin) yellow
ferric citrate 0.072 black
gelatin no pigment diffusion of
GEL 0.6 hydrolysis (protease) (GELatin)
(bovine origin) diffusion black pigment
4-nitrophenyl-βD- β-galactosidase (Para-NitroPhenyl-ßD-
PNPG galactopyranoside 0.22 colorless yellow
Galactopyranosidase)

GLU D-glucose 1.56 assimilation (GLUcose) transparent opaque

ARA L-arabinose 1.4 assimilation (ARAbinose) transparent opaque

MNE D-mannose 1.4 assimilation (ManNosE) transparent opaque

MAN D-mannitol 1.36 assimilation (MANnitol) transparent opaque

NAG N-acetyl-glucosamine 1.28 assimilation (N-Acetyl-Glucosamine) transparent opaque

MAL D-maltose 1.4 assimilation (MALtose) transparent opaque

GNT potassium gluconate 1.84 assimilation (potassium GlucoNate) transparent opaque

CAP capric acid 0.78 assimilation (CAPric acid) transparent opaque

ADI adipic acid 1.12 assimilation (ADIpic acid) transparent opaque

MLT malic acid 1.56 assimilation (MaLaTe) transparent opaque

CIT trisodium citrate 2.28 assimilation (trisodium CITrate) transparent opaque

PAC phenylacetic acid 0.8 assimilation (PhenylACetic acid) transparent opaque

OX (see oxidase test cytochrome oxidase (see oxidase test package insert)
package insert) -

• The quantities indicated may be adjusted depending on the titer of the raw materials used.
• Certain cupules contain products of animal origin, notably peptones.

PROCEDURE p. I
IDENTIFICATION TABLE p. II
LITERATURE REFERENCES p. III
INDEX OF SYMBOLS p. IV

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