1. API 20

2. PRINCIPLE:
The API 20E Strip,For Identification Of TheEnterobacteriaceae and other Gram negative rods,Consists of 20 microtubules containing dehydrated substrates. These tests are inoculated with a bacterial suspension that reconstitutethe media.

3. PRINCIPLE:
. During incubation, the metabolism of the organism produces colour changes that are either spontaneous or realed by the addition of reagent.

4. 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.

5. The strips may then be kept for up to 10 months at 2-8 C or
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.

6. STORAGE:
Whiever is sooner. Reagent used in this proedure should also be strored at 2 -8 C and before their expiry date.

7. 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.

8. REAGENT
TDA reagent (API )
James reagent (API )
VPI reagent (API )
VP2 reagent ( API )

9. 2-Record the strain refrence on the tab of the tray.
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.

10. 3- Place the API 20 E strip in 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.

11. 6-Using same pipette fill only the tubes of the other test.
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.

12. 8-Inoculate and streak amacConkey purity plate.
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.

13. 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.

15. 2-Reveal the test which require the addition of reagent as follows:
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:

16. TDA test: add one drop of TDA reagent.
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.

17. INTRPRETATION
3- Read the API strip according to the interpretation table, and record the result on the report sheet.

18. 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.

19. 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.

21. Triad I II
III IV V VI
VII
Tube 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
oxidase
Reaction + -
Point
Add
7-digital Code

22. READING THE API 20
TESTS
SUBSTRATE
REACTION TESTED
- RESULTS *negative*
+ RESULTS *positive*
ONPG
beta-galactosidase
colorless
yellow
ADH
arginine
arginine dihydrolase
red/orange
LDC
lysine
lysine decarboxylase
ODC
ornithine
ornithine decarboxylase
CIT
citrate
citrate utilization
pale green/yellow
blue-green/blue
H2S
Na thiosulfate
H2S production
colorless/gray
black deposit
URE
urea
urea hydrolysis
TDA
tryptophan
deaminase
brown-red
IND
indole production
red (2 min.) VP
Na pyruvate
acetoin production
pink/red (10 min.)
GEL
charcoal gelatin
gelatinase
no diffusion of black
black diffuse
GLU
glucose
fermentation/oxidation
blue/blue-green
MAN
mannitol
INO
inositol
SOR
sorbitol
RHA
rhamnose
SAC
sucrose
MEL
melibiose
AMY
amygdalin
ARA
arabinose OX
oxidase
colorless/yellow
violet

23. 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.

24. 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.

25. 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.