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

REAGENT TDA reagent (API 70 400)

James reagent (API 70 540)

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 [Link] the culture is mixed repeat the test 2-Reveal the test which require the addition of reagent as follows:

INTERPRETION

VP2 reagent and wait for ten min for colour development. reagent.
TDA test: add one drop of TDA IND test: add one drop of

Vp test: add one drop of VPI and

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

II

III

IV

VI

VII

Tube

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

Add

7-digital Code

5461307

READING THE API 20

TESTS ONPG ADH LDC ODC CIT H2S URE TDA IND VP GEL GLU MAN INO SOR

SUBSTRATE ONPG arginine lysine ornithine citrate Na thiosulfate urea tryptophan tryptophan Na pyruvate charcoal gelatin glucose mannitol inositol sorbitol

REACTION TESTED beta-galactosidase dihydrolase arginine lysine decarboxylase decarboxylase ornithine citrate utilization H2S production urea hydrolysis deaminase indole production acetoin production gelatinase fermentation/oxidation fermentation/oxidation fermentation/oxidation fermentation/oxidation

- RESULTS *negative* colorless yellow yellow yellow pale green/yellow colorless/gray yellow yellow yellow colorless no diffusion of black blue/blue-green blue/blue-green blue/blue-green blue/blue-green

+ RESULTS *positive* yellow red/orange red/orange red/orange blue-green/blue black deposit red/orange brown-red red (2 min.) pink/red (10 min.) black diffuse yellow yellow yellow yellow

RHA
SAC MEL AMY ARA OX

rhamnose
sucrose melibiose amygdalin arabinose oxidase

fermentation/oxidation
fermentation/oxidation fermentation/oxidation fermentation/oxidation fermentation/oxidation oxidase

blue/blue-green
blue/blue-green blue/blue-green blue/blue-green blue/blue-green colorless/yellow

yellow
yellow yellow yellow 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.