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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. 1|Page 460MIC REEM ALJOWAIE 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 . 2|Page 460MIC REEM ALJOWAIE 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. 3|Page 460MIC REEM ALJOWAIE 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. 4|Page 460MIC REEM ALJOWAIE 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 5|Page IV 1 6 4 1 4 7031645 460MIC REEM ALJOWAIE 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). 6|Page 460MIC REEM ALJOWAIE 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 7|Page 460MIC REEM ALJOWAIE