ENTERO WELL D-ONE®
REF. MS01286 (10 TESTS)
REF. MS01329 (20 TESTS)
Rev. 01.UN – 29/09/2016
ENG
ENTERO WELL D-ONE®
System for the identification of Gram-negative, oxidase negative enterobacteria
1. INTRODUCTION
ENTERO WELL D-ONE® is a system composed by 21 wells containing biochemical substrates for the identification Gramnegative, oxidase negative enterobacteria, with an identification sector for Escherichia coli.
ENTERO WELL D-ONE® has also a well for growth control of Gram negative bacteria.
The system is inoculated with a bacterial suspension of the under study organism and incubated at 36 ± 1 ° C for 12-18-24
hours. The microorganism is identified with the composition of a numerical code obtained with the color change of the
biochemical tests, after the growth of this in selective media in each well of the system.
Additionally, the panel has a sector ID for identifying strains of Escherichia coli, including E. coli inactive. This sector
can differentiate strains belonging to this species and it is a useful help when numeric codes give equivocal results for
identification. It also provides an internal quality control about the functioning of the panel.
2. CONTENT OF ENTERO WELL D-ONE® KIT
REF. MS01286 (10 TEST)
10 Identification panels.
10 x 10 mL Sterile Saline Solution.
TABLE OF RESULTS sheets
1 Instruction sheet (IFU)
REF. MS01329 (20 TEST)
20 Identification panels.
20 x 10 mL Sterile Saline Solution
TABLE OF RESULTS sheets
1 Instruction sheet (IFU)
3. REAGENTS REQUIRED BUT NOT PROVIDED
• ENTERO WELL D-ONE® Reagent
• (Vaseline oil, KOVACS Reagent (Indol Test), Reagents for Voges-Proskauer test).
• Formula of Voges-Proskauer reagents:
Reagent A: is prepared by dissolving 5 g of alpha-naphthol in 100 mL of ethyl alcohol (alpha-naphthol 5%).
Reagent B: is prepared by adding 40 g of NaOH or KOH in 100 ml of distilled water.
• IDENTIFICATION CODEBOOK
• GRAM COLOR KIT
• OXIDASE TEST STICK
Note: The codebook will be delivered to customers upon request in the form (hard copy or digital) applicable to each
laboratory conditions.
4. COMPOSITION OF THE IDENTIFICATION PANEL The
system has the following configuration:
Table 1: Identification panel configuration
WELL
1
ONPG
2
LDC
3
ODC
4
ADC
5
PD
6
CIT
7
UR
8
H2S
9
MLN
10
VP*
BIOCHEMICAL REACTION FOR ENTEROBACTERIA IDENTIFICATION
ortho-nitrophenyl-β-D-galactopyranoside Test
Lysine decarboxylase Test
Ornithine decarboxylase Test
Arginine decarboxylase Test
Phenylalanine deaminase Test
Citrate Test
Urease Test
Hydrogen sulfide production Test
Malonate Test
Voges-Proskauer Test
Page 8
ENTERO WELL D-ONE®
REF. MS01286 (10 TESTS)
REF. MS01329 (20 TESTS)
Rev. 01.UN – 29/09/2016
11
IND*
12
GLU
13
MAN
14
INO
15
SOR
16
SAC
17
ARA
18
RAF
vaseline oil -
Indole Test
Glucose fermentation Test
Mannitol fermentation test
Inositol fermentation Test
Sorbitol fermentation Test
Saccharose fermantation test
: Add
Arabinose fermentation Test
Raffinose fermentation Test
* : Add the reagent indicated for the performance of the tesT
E.coli Identification Sector
Presumptive test for E. coli
Presumptive test for E. coli
GC
Gram negative bacteria Growth Control
Well
Physiological solution (g/L): Sodium chloride 0,9 g; 1000 mL distilled water; pH 7.0 +/- 0.2
5. ASSAY METHOD
ENTERO WELL D- allows the identification of gram negative, oxidase negative enterobacteria of clinical
significance. Identification is based on biochemical tests performed on specific culture media in the
wells. The combination of positive and negative reactions allows the identification of the organism.
The E.coli identification sector, contains specific chromogenic and selective media for the presumptive
identification of this species (99.9% specificity and 99.8% sensibility).
6. COMPOSITION
Table No.2: Panel well composition
Content
Well
1/ONPG
Culture medium containing ortho-nitrophenyl-β-D-galactopyranoside
2/LDC
Culture medium containing lysine
3/ODC
Culture medium containing ornithine
4/ADC
Culture medium containing arginine
5/PD
Culture medium containing phenylalanine
6/CIT
Culture medium containing citrate
7/UR
Culture medium containing urea
8/ H 2 S
Culture medium to highlight the hydrogen sulfide production
9/ MLN
Culture medium to highlight the use of malonate
10/ VP
Culture medium to highlight acetone production
11/ IND
Culture medium to highlight indole production
12/ GLU
Culture medium to highlight the glucose fermentation
13/ MAN
Culture medium to highlight the mannitol fermentation
14/INO
Culture medium to highlight the inositol fermentation
15/ SOR
Culture medium to highlight the sorbitol fermentation
16/ SAC
Culture medium to highlight the sucrose fermentation
17/ ARA
Culture medium to highlight the arabinose fermentation
18/ RAF
Culture medium to highlight the raffinose fermentation
Culture medium with chromogenic substrate for the presumptive
identification of E. coli
Selective culture medium containing substrates for the presumptive
identification of E. coli
GC Well
Culture medium for the growth control of Gram negative bacteria
Physiological solution (g/L): Sodium chloride 0,9 g; 1000 mL distilled water; pH 7.0 +/- 0.2
E.coli Identification
Sector
Page 9
ENTERO WELL D-ONE®
REF. MS01286 (10 TESTS)
REF. MS01329 (20 TESTS)
Rev. 01.UN – 29/09/2016
7. SAMPLE COLLECTION AND PREPARATION
ENTERO WELL D-ONE® is used for the identification of gram negative- oxidase negative bacteria, isolated
on selective agar media for Enterobacteriaceae such as Mac Conkey Agar, Eosin Methylene Blue Agar
Salmonella e and Shigella Agar, Hektoen Enteric
Agar, or non-selective media.
Note: Before using the system, saline solution and identification panel must be removed from
refrigerator and wait for them to come to room temperature.
7.1 TEST PROCEDURE
PREPARATION OF THE BACTERIAL SUSPENSION
1. The microorganism to identify must have been recently isolated (18-24 hours); culture from more than
48 hours incubation can provide not reliable results.
2. Before inoculating the microorganism to test, it is necessary to perform Gram staining test and oxidase
test. Only Gram-negative, oxidase-negative bacteria can be inoculated on ENTERO WELL D-ONE®. Consult
suitable manuals of bacteriology to correctly perform these tests .
3. Open one vial of sterile physiological solution contained in the kit.
4. Take one or more morphologically similar, well-isolated colonies from the agar medium and suspend in the
physiological solution so to obtain a turbidity equivalent to 0,5 McFarland (Bacterial suspension).
8. Thoroughly homogenize.
INOCULATION OF THE SYSTEM
1 Take a system from its pouch.
2. Note date and origin of the microorganism isolated in the panel.
3. Add 150 µL or three drops of the bacterial suspension in each well and covered with 2 drops of
vaseline oil for laboratory use in wells 2-LDC 3-ODC 4-ADC 7-UR 8-H 2 S.
4. Cover the system with the provided cap and incubate at 36 ± 1 °C for 12-18-24 hours
8. INTERPRETATION OF RESULTS
After the incubation process:
1. Add 2 drops of Reagent A and 1 drop of Reagent B in the well 10-VP. Wait 15-20 minutes and
watch for the appearance of a pink-red color.
2. Add 3 drops of Kovac’s Reagent in the well 11-IND. Wait 1-2 minutes and watch for the appearance of pink-red en in
the well.
3. Observe the color change of the wells and interpret the results using the table No.3.
4. Note the result on the Test Results Form and build up the 6-digit code following the instructions provided in the
paragraph CODE NUMBER FORMING.
5. Identify the microorganism using the CODEBOOK in digital or hard copy supplied by CPM SAS.
Escherichia coli Identification Sector
Reading is visually performed according to the description of the Table No.4 reactions, and these results are not
included in the
code numbers. Escherichia coli Identification Sector is an additional tool for the presumptive
identification of Escherichia coli, including inactive Escherichia coli.
Page 10
ENTERO WELL D-ONE®
REF. MS01286 (10 TESTS)
REF. MS01329 (20 TESTS)
Rev. 01.UN – 29/09/2016
Table No. 3. Reaction Table
Well
1/ONPG
2/LDC
3/ODC
4/ADC
5/PD
6/CI
T
7/UR
8/ H 2 S
9/ MLN
10/ VP
11/ IND
12/ GLU
13/ MAN
14/INO
15/ SOR
16/ SAC
17/ ARA
18/RAF
Well Color
BIOCHEMICAL REACTIONS
Hydrolysis of ONPG
Decarboxylation of lysine
Decarboxylation of ornithine
Decarboxylation of arginine
Deamination of phenylalanine
Utilization of citrate
Hydrolysis of urea
Production of hydrogen
sulphide
Utilization of malonate
Production of acetoine
Test of indole
Fermentation of glucose
Fermentation of mannitol
Fermentation of inositol
Fermentation of sorbitol
Fermentation of saccharose
Fermentation of arabinose
Fermentation of raffinose
Positiv
e
Yellow
Red
Red
Red
Black-Brown
Negativ
e
Colorless
Yellow -Orange
Yellow -Orange
Yellow -Orange
Yellow
Blue-Dark Green
Red-Fuchsia
Black-Brown
Yellow
Yellow
Yellow
Blue-Green
Pink- Red
Pinkish -Red
Yellow
Yellow
Yellow
Yellow
Yellow
Yellow
Yellow
Yellow
Yellow
Yellow
Blue-Green
Blue-Green
Blue-Green
Blue-Green
Blue-Green
Blue-Green
Blue-Green
Page 11
ENTERO WELL D-ONE®
REF. MS01286 (10 TESTS)
REF. MS01329 (20 TESTS)
Rev. 01.UN – 29/09/2016
8.1 CODE NUMBER FORMING
The biochemical tests are divided into 6 groups each containing 3 tests and each one is indicated with a positive value of 1,
2, 4.
 Value 1 : first test positive in each group

Value 2 : second test positive in each group

Value 4 : third test positive in each group

Value 0 : every negative test
Example:
ONPG
Test
Positivi
ty
code
Results
Group 1
Group 2
LDC ODC ADC PD CIT
UR
Group 3
H2S MLN
VP
Group 4
Group 5
Group 6
IND GLU MAN INO SOR SAC ARA RAF
1
2
4
1
2
4
1
2
4
1
2
4
1
2
4
1
2
4
+
+
+
-
-
-
-
-
-
-
+
+
+
-
+
-
+
-
Code
Numerical Code: 700652
7
0
0
6
5
2
Identification: Escherichia coli
Table No.4 . Reaction Table of Escherichia coli Identification Sector
Escherichia coli Identification
Sector
Well
GC
TEST
Escherichia spp.
Colorimetric Reaction
POSITIVE
NEGATIV
E
Aqua Green
Light Yellow / Turbid
Chromogenic culture medium for the
presumptiveidentification of E. coli
Selective culture medium for the presumptive
identification of E. coli
Culture medium for the growth control of
Enterobacteriaceae
ONP
G
1
(+)
Blue
Yellow
Pinkis
h
White Clear / White
Turbid
SIGNIFICANT BIOCHEMICAL REACTIONS FOR ENTEROBACTERIACEAE DIFFERENTIATION
UR
H2S
IND
VP
LDC
CIT
MLN
7
8
11
10
2
6
9
-
-
(+)
-
(+)
-
-
Enterobacter
spp.
Klebsiella spp.
+/-
+/-
-
-
(+)
+/-
(+)
(+)
(+)
+/-
(+)
(+)
(-)
(-)
(-)
+/-
(+)
+/-
+/-
(-)
+/+/-
(+)
Proteus spp.
(-)
+/-
-
-
(-)
(-)
(+)
-
(-)
Salmonella spp.
(-)
(-)
+/-
Citrobacter spp.
(+)
-
(+)
(-)
+/-
Arizona spp.
+/-
(-)
(+)
+/-
-
(+)
+/-
+/-
Yersinia spp.
+/-
+/-
(-)
+/-
+/-
(-)
(-)
+/-
Serratia spp.
+/-
(-)
(-)
(-)
+/+/+/Legend:: + = Positive Reaction, - = Negative Reaction. V = Variable Reaction
+/-
Page 12
ENTERO WELL D-ONE®
REF. MS01286 (10 TESTS)
REF. MS01329 (20 TESTS)
Rev. 01.UN – 29/09/2016
Escherichia coli Identification Sector: All strains belonging to the species Escherichia coli, including
Escherichia coli inactive must correspond to the following positive pattern of reactions:
Variant I: pattern obtained in 99.9% Escherichia coli
Variant II: pattern obtained in 40% inactive Escherichia coli
Variant I
E.coli Growth Control
E.coli
Variant II
Growth Control
9. QUALITY CONTROL
In order to realize the quality control, it is recommended to use the following reference strains:
Enteroracter cloacae
Escherichia coli
Klebsiella pneumoniae
Proteus mirabilis
ATCC 23355
ATCC 25922
ATCC 13883
ATCC 25933
Salmonella typhimurium
Serratia marcescens
Shigella flexneri
Yersinia enterocolitica
ATCC 14028
ATCC 8100
ATCC 12022
ATCC 9610
10. PERFORMANCE
Product performance was studied using reference strains, characterized by internal collections of Tor
Vergata hospital and was compared to a commercially available product with a similar principle and also
with conventional method. For all strains, the behavior was within specifications and 100% concordance
was found in comparing the commercial counterpart and conventional methods.
11. FACTORS THAT MAY INVALIDATE THE RESULTS
Poor standardization of the inoculum.
Unsuitable clinical material.
Use of expired systems and/or reagents.
Non-compliance with incubation temperatures and times.
The use of pure bacterial cultures but incubated longer
than 24 hours. The non-use of pure bacterial cultures.
12. CAUTIONS
The product ENTERO WELL D-ONE®, is not classified as hazardous under current legislation, however refer to the safety
data sheet for a correct use.
ENTERO WELL D-ONE® is a disposable device to be used only for in vitro diagnostic use. It must e used in the laboratory
by properly trained personnel, using approved aseptic and safety methods for handling pathogenic agents.
12. STORAGE
Store at 2-8 °C in its original package.
Keep away from sources of heat and avoid excessive changes of temperature.
In such conditions the product maintains its validity until the expiry date
indicated on the label. Do not use beyond that date. Eliminate without using
if there are signs of deterioration.
Page 13
ENTERO WELL D-ONE®
REF. MS01286 (10 TESTS)
REF. MS01329 (20 TESTS)
Rev. 01.UN – 29/09/2016
YERSINIAE
PROTEEAE
KLEBSIELLE
AE
SALMONELLEAE
ESCHERICHIEAE
Reaction Table
MICROORGANISM
ONP LDC ODC ADC PD CIT UR H2S MAL VP IND GLU MAN INO SOR SAC ARA RAF
Escherichia coli inactive G V
V
V
V
+
V
V
V
V
V
Escherichia coli
+
V
V
V
V
+
+
+
V
V
V
V
Shigella dysenteriae
V
V
+
V
V
Shigella flexneri
V
V
V
+
V
V
V
V
Shigella boydii
V
V
V
V
+
+
V
V
Shigella sonnei
V
+
+
+
V
V
+
+
V
+
+
+
V
Edwardsiella tarda
+
+
+
+
+
+
+
Edwardsiella tarda tipo +
V
V
V V V
V
V
+
+
V
+
V
+
V
Citrobacter
freundii
1
+
+
V
V V
V
+
+
+
V
V
+
Citrobacter diversus
+
+
V
+ V
+
+
+
+
V
+
+
Citrobacter
Citrobacter
amalonaticus
+
+
V
V
+
+
+
+
+
+
+
amalonaticus type 1
Salmonella spp
+
+
V
V
V
+
+
V
V
V
Salmonella typhi
+
V
V
+
+
+
Salmonella cholerae
V
+
V
V
+
+
V
suis
Salmonella
paratyphi A V
V
V
+
+
+
+
Salmonella arizonae
V
+
+
V
V
+
V
V
+
+
+
+
V
Salmonella gallinarum
V
V
+
+
+
V
V
+
V
V
V
+
+
V
+
Salmonella pullorum
+
V
+ V
V
V
+
+
V
+
+
+
+
Klebsiella pneumoniae
+
+
V V
+
V
+
+
+
+
+
+
+
+
Klebsiella oxytoca
V
V
V
V V
+
+
V
V
V
V
V
Klebsiella ozaenae
Klebsiella
V
+
+
V
V
V
V
rhinosclerom
Klebsiella group 47
+
+
+
+
+
+
V
+
+
+
V
+
+
+
+
atis
Enterobacter aerogenes V
+
+
V
V
+
+
+
V
V
+
+
+
Enterobacter cloacae
V
V
V
V
V V
V
+
+
+
V
V
V
+
V
Enterobact
V
V V
V
V
V
+
+
V
V
V
V
V
er
+
V
+
V
+
+
+
+
+
V
+
+
+
Enterobacter
agglomera gergoviae
+
V
V
V
V
+
V
+
+
V
+
+
+
Enterobacter sakazakii
ns
Enterobacter alvei
V
+
+
V
V
V
+
V
V
V
(Hafnia)
V
V
+
V
V
V
V
+
+
V
+
+
V
V
Serratia liquefaciens
V
+
V
V V
V
+
+
V
V
+
V
Serratia marcescens
+
V
V
V
V
V
+
+
V
+
V
+
+
Serratia rubidaea
V
+
V
V V
V
V
V
+
+
+
+
+
+
+
Serratia odorifera 1
V
+
+
V
V
V
+
+
+
+
+
V
Serratia odorifera 2
+
V
+
V
+
+
+
V
+
V
+
Serratia fonticola
+
V
V
V
+
V
+
V
+
+
V
Serratia plymuthica
+ V V V
V
V
+
V
V
Proteus vulgaris
V
V V V V
V
V
+
V
Proteus mirabilis
+
V V +
V
+
+
Morganella morganii
+
V
+
+
V
V
+
+
Morganella morganii 1
+
+
V
+
+
Proteus penneri
V
+ V +
+
+
V
V
V
V
Providencia rettgeri
V V V
+
+
V
+
V
V
Providencia stuartii
V
+
+
+
+
Providencia
+ V
+
+
V
Providencia
alcalifaciensrustigiani
V
V
V
V
V
V
+
+
V
+
V
V
Yersinia enterocolitica
Yersinia pestis
V
+
+
V
+
Yersinia intermedia
V
+
V V
V
V
+
+
+
V
+
+
+
V
Yersinia
V
+
V
+
+
V
V
pseudotubercol
osis
Legend : + = Positive Reaction V = variable reaction, 10 to 95% positivity; - = Negative Reacti
Page 14