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API 20 E Biochemical Test Strip These include: •: test for β-galactosidase enzyme by hydrolysis of the substrate o-nitrophenyl-b-D-galactopyranoside • ADH: • LDC: decarboxylations of the amino acid lysine by lysine decarboxylase • ODC: decarboxylations of the amino acid ornithine by ornithine decarboxylase • CIT: as only carbon source • H2S: production of hydrogen sulfide • URE: • TDA (Tryptophan deaminase): detection of the enzyme tryptophan deaminase: Reagent to put- Ferric Chloride. • IND: -production of indole from tryptophan by the enzyme tryptophanase. Reagent- Indole is detected by addition of Kovac’s reagent. Excelsior accordion serial numbers. • Pick up a single isolated colony (from a pure culture) and make a suspension of it in sterile distilled water. • Take the API20E Biochemical Test Strip which contains dehydrated bacterial media/bio-chemical reagents in 20 separate compartments. API20E Biochemical Test Strip is commercially available. (Bacteria will react with them and will give different colors which will help to identify bacteria to the species level).

• Take a pasteur pipette and fill up (up to the brim) these compartments with the bacterial suspension. • Add sterile oil into the ADH, LDC, ODC, H2S and URE compartments. • Put some drops of water in the tray and put the API Test strip and close the tray. • Mark the tray with identification number (Patient ID or Organism ID), date and your initials. • Incubate the tray at 37 oC for 18 to 24 hours. Results and Interpretation • For some of the compartments, you can just read the change in color straightway after 24 hours but for some you have to put reagents before reading.

• Add following reagents to these specific compartments • TDA: Put one drop of Ferric Chloride • IND: Put one drop of Kovacs reagent • VP: Put one drop of 40% KOH (VP reagent 1) & One drop of VP Reagent 2 (α-Naphthol) (you have to wait for 10 minutes before telling it negative). • Get the API Reading Scale (color chart) • Mark each test as positive or negative on the lid of the tray • The wells are marked off into triplets by black triangles, for which scores are allocated as follows: • Add up the scores for the positive wells only in each triplet. Supplementary tests, e.g.: oxidase may also be included in the profile. The highest score possible for a triplet is 7 (the sum of 1, 2 and 4) and the lowest is 0. Numbering in API 20E Test Strip • The profile for this combination of reactions is therefore 7031645 (7 digit code) • Identify the organism by using API catalog or apiweb VIDEO: Reading an API20E using the online database • Identify the organism using api web: • Start Internet Explorer or Firefox web browser • Go to: • Login: your login name • Password: your password • Select the correct test (e.g. • Enter the numerical profile to obtain the identity.

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• Record the identity along with comments (% ID and T value) on the results sheet.