API 20
PRINCIPLE:
The API 20E Strip,For •
Identification Of
TheEnterobacteriaceae and other
Gram negative rods,Consists of
20 microtubules containing
dehydrated substrates. Theese
tests are inoculated with
abacterial suspension that
reconstitutethe media.
PRINCIPLE:
During incubation, the metabolism .
of the organism produces colour
changes that are either spontaneous
or realed by the addition of
reagent.
STORAGE:
API 20E are contained in an
aluminum pouch. Once opened
the pouch should be re-sealed
using the clip seal supplied with
the kit.
STORAGE:
The strips may then be kept
for up to 10 months at 2-8 C
or
untill the expiry date indicated
on the packaging.
STORAGE:
Whiever is sooner. Reagent
used in this proedure should
also be strored at 2 -8 C and
before their expiry date.
MATERIALS:
API 20 E Strips
Incubation boxes
Report sheets
Disposable Plastic pipettes
Disposable plastic
inoculating loop
5 ml sterile distilled water
Mineral Oil
MacConkey agar plate.
REAGENT
TDA reagent (API 70 400)
James reagent (API 70 540)
VPI reagent (API 70 420)
VP2 reagent ( API 70 430)
PROCEDURE
1-prepare an incubation tray and
lid and distribute 5 ml of sterile
distilled water into the tray to
create ahumid chamber.
2-Record the strain refrence on
the tab of the tray.
PROCEDURE
3- Place the API 20 E strip in the
tray.
4-Using a sterile disposable loop
remove asingle well-isolated colony
from an isolation plate and
carefully emulsify this in 5 ml of
sterile distilled water.
PROCEDURE
5-With asterile disposable pipette
fill both the tube and cupule of
the test CIT, VP and GGL with the
bacterial suspension.
6-Using same pipette fill only the
tubes of the other test.
PROCEDURE
7-Creat an aerobiasis in the tests
ADH , LDC , ODC , URE and H2 S
by overlaying with mineral oil.
8-Inoculate and streak
amacConkey purity plate.
PROCEDURE
9- place a lid on the incubation
tray and incubate both the strip
and the macConkey plate at 37 C
for 18 – 24 hours.
INTRPRETATION
1-Check the macConkey purity
plate to ensure.If the culture is
mixed repeat the test
2-Reveal the test which require
the addition of reagent as follows:
INTERPRETION
Vp test: add one drop of VPI and
VP2 reagent and wait for ten min for
colour development.
TDA test: add one drop of TDA
reagent.
IND test: add one drop of
James reagent.
INTRPRETATION
3- Read the API strip according to
the interpretation table, and
record the result on the report
sheet.
INTRPRETATION
4- On the report shhet, 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
groupe, a7- digit profile number is
obtained for 20 tests of the API
20E strip.
INTRPRETATION(CONT)
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.
Triad
I
II
III
IV
V
VI
VII
Tube
1
2
3
4
5
6
7
8
9
1
0
1
1
1
2
1
3
1
4
1
5
1
6
1
7
1
8
1
9
2
0
oxid
ase
Reactio
n
+
+
+
-
-
-
+
+
-
+
-
-
-
+
+
-
-
+
+
-
+
Point
1
2
4
0
0
0
1
2
0
1
0
0
0
2
4
0
0
4
1
0
4
Add
7-digital
Code
7
0
3
1
6
4
5
5461307
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
QUALITY CONTROL
Refer to laboratory Quality
Control Manual for all Quality
Control procedure.
SAFETY
Standard microbiological technique
should be used in the performance
of this test. After use, the strips
should be placed in red plastic
disposal bag.
LIMITATIONS
The API 20 E system is intended
for the identification of those
non-fastidious , Gram negative
rods included in the analytical
profile index and exclusively for
them. It cannot be used to
identify any other organism or to
exclude their presence.
LIMITATIONS
Interpretation of the test result
should be made by competent
microbiology technologist.
Any difficulties with interpretation
should be referred to a Senior
Technologist or Supervisor.