Download Enterobacteriaceae Identification API 20E System • (Analytical

Enterobacteriaceae Identification
API 20E System
 (Analytical Profile Index System for Identification of Enterobacteriaceae).
 The API 20E System is a miniaturized version of conventional tests that is
used for the identification of members of the family Enterobacteriaceae and
other gram-negative bacteria.
 This system is utilizes a plastic strip with 20 separate compartments, each
compartment consists of a cupule and a small tube that contains a specific
dehydrated medium and contain 20 biochemical tests.
 To inoculate each compartment, it is necessary to make up a saline
suspension of the unknown organism then fill the compartments with it .
The cupule receives the suspension and allows it to flow into the tube of
medium .The dehydrated medium is reconstituted by the saline .
 To provide anerobic conditions it is necessary to add sterile mineral oil to
some reactions .
 After incubation for 18-24 h , the reactions are receded , test reagents are
added to some comportments, the metabolism of the organism produces
color change, and test results are tabulated .then a profile number (7
digits) is computed.
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 By finding the profile number in a code book , the Analytical Profile Index,
one is able to determine the name of the organism .
 Materials
o API 20 E Strips
o Incubation boxes (tray and cover )
o Report sheets
o Disposable Plastic pipettes
o Disposable plastic inoculating loop
o 5 ml sterile distilled water
o 5 ml of 0.85% sterile saline
o Mineral Oil
o MacConkey agar plate
 Procedure
1. Prepare a saline suspension from the center of the colony on an agar
plate to a tube of 0.85% saline solution, then vortex it.
2. Dispense about 5ml of sterile water into the tray. Note that the bottom
of the tray has numerous depressions to accept the water.
3. Remove the API 20 E strip from the sealed pouch and place it into the
tray .
4. Vortex mix saline, fill the micropipette and inoculate all the tubes on
the strip .
5. Slightly underfill ADH, LDC ,ODC, H2S,and URE , to provide
anaerobic conditions for these compartments ,dispense mineral oil to
the cupules .
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6. Fill both the tube and cupule of the test CIT, VP and GGL with the
bacterial suspension, these compartments require oxygen .
7. Inoculate and streak Mackoncey purity plate.
8. place a lid on the incubation tray and incubate both the strip and the
MacKoncey plate at 37 C for 18 – 24 hours
 Results
Evaluation of tests ,all reactions will be recorded on the laboratory report
and test reagents will be added to some compartments then seven –digit
profile number will be determined so the unknown organism can be looked
up in the API 20E analytical profile index.
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1-Check the MacKoncey purity plate to ensure. If the culture is mixed
repeat the test
2-Reveal the test which require the addition of reagent as follows:
Vp test: add one drop of VPI and VP2 reagent and wait for 10 min for
color development. The pale pink color that occurs immediately has no
significance .A positive reaction is dark pink or red.
TDA test: add one drop of TDA reagent. A positive reaction brown –red
will occur immediately. A negative reaction color is yellow.
IND test: add one drop of James reagent. Look for the positive red ring
reaction within 2 minutes. After several minutes the acid in the reagents
reacts with the plastic cupule to produce a color change from yellow to
brownish –red, which consider negative .
3- Read the API strip according to the interpretation table, and record the
result on the report sheet
4- On the report sheet, the test are separated into groups of three and
number 1, 2 or 4 is allocated for each test. By adding the numbers
corresponding to the positive reaction within each group, a7- digit profile
number is obtained for 20 tests of the API 20E strip.
5- The 7- digit profile is then compared with the numerical profile in the
API 20 E analytical profile index book to obtain the organism
identification.
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Triad
Tube
I
II
III
V
VI
VII
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 oxidase
Reaction
+ + + - - - + + - +
-
-
-
+ +
-
-
+ +
-
+
Point
1 2 4 0 0 0 1 2 0 1
0
0
0
2
0
0
4
0
4
Add
7
0
3
7-digital Code
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IV
1
6
4
1
4
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5
READING THE API 20
TESTS SUBSTRATE REACTION TESTED
- RESULTS
*negative*
+ RESULTS
*positive*
ONPG
ONPG
beta-galactosidase
colorless
yellow
ADH
arginine
arginine dihydrolase
yellow
red/orange
LDC
lysine
lysine decarboxylase
yellow
red/orange
ODC
ornithine
ornithine decarboxylase
yellow
red/orange
CIT
citrate
citrate utilization
pale green/yellow
blue-green/blue
H2S
Na thiosulfate
H2S production
colorless/gray
black deposit
URE
urea
urea hydrolysis
yellow
red/orange
TDA
tryptophan
deaminase
yellow
brown-red
IND
tryptophan
indole production
yellow
red (2 min.)
VP
Na pyruvate
acetoin production
colorless
pink/red (10 min.)
GEL
charcoal gelatin
gelatinase
no diffusion of black
black diffuse
GLU
glucose
fermentation/oxidation
blue/blue-green
yellow
MAN
mannitol
fermentation/oxidation
blue/blue-green
yellow
INO
inositol
fermentation/oxidation
blue/blue-green
yellow
SOR
sorbitol
fermentation/oxidation
blue/blue-green
yellow
RHA
rhamnose
fermentation/oxidation
blue/blue-green
yellow
SAC
sucrose
fermentation/oxidation
blue/blue-green
yellow
MEL
melibiose
fermentation/oxidation
blue/blue-green
yellow
AMY
amygdalin
fermentation/oxidation
blue/blue-green
yellow
ARA
arabinose
fermentation/oxidation
blue/blue-green
yellow
OX
oxidase
oxidase
colorless/yellow
violet
The amino acids tested are (in order) arginine, lysine and ornithine (ADH through
ODC) .Decarboxylation is shown by an alkaline reaction (red color of the particular
pH indicator used).
The carbohydrates tested are glucose, mannitol, inositol, sorbitol, rhamnose, sucrose,
melibiose, amygdalin and arabinose. Fermentation is shown by an acid reaction
(yellow color of indicator).
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Hydrogen sulfide production (H2S) and gelatin hydrolysis (GEL) result in a black
color throughout the tube.
A positive reaction for tryptophan deaminase (TDA) gives a deep brown color with
the addition of ferric chloride; positive results for this test correlate with positive
phenylalanine and lysine deaminase reactions which are characteristic of Proteus,
Morganella and Providencia.
O
AL OCHUT I
GGMI S R S MA A
culture N
V
D D D I 2 R D N E L A N O H A E M R identification
no. P
P
HC C T S E AD L UN OR AC L Y A
G
8101 + – + + – – – – + – – + + – + + + + – +
Escherichia
coli
+ – – – + – – – – + – + + + + + – – + +
Enterobacter
agglomerans
8P44 – – + + – + – – + – – + + – – – + – – +
Edwardsiella
hoshinae
5B
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