ENTEROTESTID_FRONT_eng4 .pdf 1 22/01/2013 15:20:06
Rapid, accurate, simple,
convenient and economic.
Advanced System for efficient identification
of 27 Enterobacteriaceae.
Indole production
ONPG (hydrolysis) degradation for the
detection of β-D-glucosidase activity
Glucose fermentation (acidity without gas)*
C
Glucose fermentation with CO2 gas formation*
M
Y
Hydrogen Sulfide production
CM
MY
Urea (hydrolysis) degradation for the ureasa activity
CY
Motility
CMY
K
ENTEROTESTID
Enterobacteriaceae is a large family of Gram-negative
catalase positive and oxidase negative1 (mostly, as
Pleisomonas family has been included in oxidase
positive1) bacteria, that includes, along with many
harmless symbionts, many of the more familiar
pathogens.They are distributed worldwide and may be
found in soil, water, plants and animals. Most species
grow well at 37°C, although some species grow better
at 25-30°C. There is a large number of species included
in the family, more than 170, but the clinical and more
important specimens which play a major role as
infectious agents, belong to 20 to 25 species that are
well known for many years. 2-6
The identification of the Enterobacteriaceae is
complicated and has been traditionally based on
biochemical and antigenic characteristics 2-6. With
Enterotest ID, using a method well known by
microbiologists worldwide, stabbing the butt and
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.
smearing a slant, six biochemical reactions are performed in a simple tube as well
as bacteria growth motility for further investigations.
The media tube contains two layers and a paper cap for indol
determination.
Upper layer(3 ml): a butt and a slant with dextrose, ONPG, enrichments, ferrous
sulphate, sodium thiosulfate, agar and phenol-red
Lower layer (2 ml): semi-solid media, containing urea, enrichments, sodium
chloride, agar and phenol-red
Paper cap: soaked with a solution of 4-dimethyl-aminobenzaldehyde for the
indol test.
Warning: The prepared medium is red-peachy colour, slightly opalescent in the upper layer and light
peachy in the lower layer. If there are any physical changes, discard the medium. Dehydration or
liquefaction conditions may also interfere with the accuracy of Enterotest ID:
* If the glucose is not fermented (the medium remains red-orange after inoculation and incubation),
the strain does not belong to the Enterobacteriacea. 2-3
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Indole cap
1
2
3
4
18 - 24 h at 35 ± 2°C
5
6
1,5 cm
0,6’’
Slant for bacterial growth
ONPG
Enterotest ID Record Chart
DATE
STOPPER
Indol
Slant
UPPER
LAYER
LOWER
LAYER
Butt
-
ONPG
+
+
Glucose
AG
H2S
Urea
aeroge
nes
-
-
+
D
AG
-
AG
+
+
+
+
-
-
+
A
AG
-
cloa
cae
agg
lom
erans
+
-
-
H2S
Indol
Urease
ONPG
Glucose
Gas CO2
RESULT
Glucose with or
without gas
-
coli
tard
a
+
+  positive in one day
‹+›  positive weekly
Motility
Enterobacter
+
AG
+
Motility
Edwardsiella
dive
rsus
freu
ndii
Citrobacter
Reference
-
+
+
+
AG
H2S
AG
-
-
+
+/-
+
+/-
+
-
‹+›
‹+›
-
-
-  negative
D  different
[+]
[+]
H2S
LOT NUMBER
Urea, Motility
EXPIRY DATE
Store at room temperature in a dark place.
1
Pick a typical suspected ISOLATED colony from an appropriate plate
culture. Appropiated medium could be MacConkey agar (cat.1052),
Hektoen Enteric agar (cat.1030), Eosin Methylene Blue (EMB) (cat.
1039), Salmonella Shigella (SS) (cat.1064) or nonselective blood agar
media. Only an isolated colony must be chosen.
2
Jab the tube to the bottom and seed in the slant.
3
Place the indol test cap.
4
Incubate for 18-24 hours at 35±2ºC.
5
Read the results and compare with differentiation chart included.
Results can be interpreted by using the differentiation chart,
comparing with known species or using the digital interpretation
guide (codebook) provided. When further tests are needed (two or
more organisms are listed with the same result), the codebook will
inform you as well as the results will be obtained in them.
6
Use slant growth for further serological test if needed.
REACTION
POSITIVE
NEGATIVE
INDOLE
Purple-pink
Colorless
ONPG
Orange
Cherry-Pink
GLUCOSE
Yellow
-
CO2
Bubbles
-
SH2
Black *
Yellow
MOTILITY
Fuzzy
Clear
UREASE
Pink
Yellow
(*) If too much bacterium is taken in the seed, the ferric sulfide may mask other
results. The black precipitate may fade or revert to negative if the enterotest ID
is read after 24 h.
ADDITIONAL INFORMATION
With Enterotest ID Yersinia is not confused with Proteus.
ADVANTAGES OF ENTEROTEST VS OTHER METHODS
When Yersiniae infection is suspected, motility will be observed at 25ºC
and not at 35±2ºC. This characteristic is sufficient to differentiate this
group from all others.7
Since there is no lactose in the media, no direct lactose fermentation is
observed but, as the original colony was transferred from media
containing lactose (SS agar, MacConkey, EMB, etc), this characteristic is
already known before introduction in tube.8
For final identification and differentiation of E.coli and Citrobacter, a spot
test on Simmons Citrate Agar is recommended. Citrobacter is positive in
this test and E. Coli is negative.6,8
For differentiation between Enterobacter species and Serratia species,a
few supplementary test are needed: Lysine; Ornithine, Arabinose, Sorbitol,
Gelatin.6,8
For final identification and differentiation of Yersinia enterocolitica and
Yersinia pseudotuberculosis a lysine decarboxylase test is recommended.
Yersinia enterocolitica will give a positive result and Yersinia
pseudotuberculosis will be negative.6-8
LIMITATIONS OF THE PROCEDURE
Enterotest ID is designed specifically to identify Gram negative rods which
are oxidase negative and should only be used to differentiate members of
the Enterobacteriaceae family.
Identification of Gram negative bacteria should be made with the
consideration of additional characteristics such as source of specimen,
history of the patient, colonial and microscopic morphology, serology and
antimicrobial susceotibility aptterns.
Conventional
methods
API
Enterotest ID
PRICE
$$
$$$
$
NUMBER OF TUBES
/PLATES
3*
Cat20 reactions
N
6 reactions
per tube
hard-complex
complex
easy
MOTILITY
no
no
yes
FLEXIBILITY FOR
ADDITIONAL TEST
no
no
yes
TIME TO RESULT
(LABOR TIME)
slow
medium
fast
INCUBATOR SPACE
high
small
small
yes
yes
no
SIMPLICITY
NEED OF EXTRA
MATERIAL
*Kliger/Triple Sugar iron; urease broth, SIM
1. Steel, K. J. 1961. The oxidase reaction as a taxonomic tool. J. Gen. Microbiol. 25:297-306.
2. Farmer, J.J. III. 2003. Enterobacteriaceae: introduction and identification. In: Murray, P. R., E. J.
Baron, J.H. Jorgensen, M. A. Pfaller, and R. H. Yolken (ed.). Manual of clinical microbiology, 8 th ed.
American Society for Microbiology, Washington, D.C
3. Edwards and Ewing's identification of Enterobacteriaceae. 3rd ed. Burgess Publishing
Co.,Minneapolis, Minnesota.. 1986 pp. ix + 536pp.
4. Kauffmann, f.; m. d. ]. The bacteriology of enterobacteriaceae. Collected studies of the author
and his co-workers. 1966 pp. 400 pp.
5. R. Podschun, U. Ullmann. Klebsiella spp. as Nosocomial Pathogens: Epidemiology, Taxonomy,
Typing Methods, and Pathogenicity Factors. Clin Microbiol Rev. 1998 October; 11(4): 589–603.
6. Brenner, D,J, et al. Bergey’s Manual of Systematic Bacteriology. Family I, Enterobacteriacae
P595-602
7. Brubaker RR. The genus Yersinia: biochemistry and genetics of virulence. Curr Top Microbiol
Immunol 1972;57:111-58
8. Ewing, W. H. 1962. Enterobacteriaceae. Biochemical tests for group differentiation. Public Health
Service Publication no. 734 (revised). U.S. Department of Health, Education and Welfare,
Washington, D.C.