2PRINCIPLE: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.
3PRINCIPLE:. During incubation, the metabolism of the organism produces colour changes that are either spontaneous or realed by the addition of reagent.
4STORAGE:API 20E are contained in an aluminum pouch. Once opened the pouch should be re-sealed using the clip seal supplied with the kit.
5The 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 oruntill the expiry date indicated on the packaging.
6STORAGE:Whiever is sooner. Reagent used in this proedure should also be strored at 2 -8 C and before their expiry date.
7MATERIALS: API 20 E Strips Incubation boxes Report sheets Disposable Plastic pipettesDisposable plastic inoculating loop5 ml sterile distilled waterMineral OilMacConkey agar plate.
92-Record the strain refrence on the tab of the tray. PROCEDURE1-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.
103- Place the API 20 E strip in the tray. PROCEDURE3- 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.
116-Using same pipette fill only the tubes of the other test. PROCEDURE5-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.
128-Inoculate and streak amacConkey purity plate. PROCEDURE7-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.
13PROCEDURE9- place a lid on the incubation tray and incubate both the strip and the macConkey plate at 37 C for 18 – 24 hours.
152-Reveal the test which require the addition of reagent as follows: INTRPRETATION1-Check the macConkey purity plate to ensure.If the culture is mixed repeat the test2-Reveal the test which require the addition of reagent as follows:
16TDA test: add one drop of TDA reagent. INTERPRETIONVp 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.
17INTRPRETATION3- Read the API strip according to the interpretation table, and record the result on the report sheet.
18INTRPRETATION4- 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.
19INTRPRETATION(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.
22READING THE API 20TESTSSUBSTRATEREACTION TESTED- RESULTS *negative*+ RESULTS *positive*ONPGbeta-galactosidasecolorlessyellowADHargininearginine dihydrolasered/orangeLDClysinelysine decarboxylaseODCornithineornithine decarboxylaseCITcitratecitrate utilizationpale green/yellowblue-green/blueH2SNa thiosulfateH2S productioncolorless/grayblack depositUREureaurea hydrolysisTDAtryptophandeaminasebrown-redINDindole productionred (2 min.)VPNa pyruvateacetoin productionpink/red (10 min.)GELcharcoal gelatingelatinaseno diffusion of blackblack diffuseGLUglucosefermentation/oxidationblue/blue-greenMANmannitolINOinositolSORsorbitolRHArhamnoseSACsucroseMELmelibioseAMYamygdalinARAarabinoseOXoxidasecolorless/yellowviolet
23QUALITY CONTROLRefer to laboratory Quality Control Manual for all Quality Control procedure.SAFETYStandard microbiological technique should be used in the performance of this test. After use, the strips should be placed in red plastic disposal bag.
24LIMITATIONSThe 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.
25LIMITATIONSInterpretation of the test result should be made by competent microbiology technologist.Any difficulties with interpretation should be referred to a Senior Technologist or Supervisor.