Analytical Profile Index

The API tests (kit) can identify a wide range of microorganisms. The API's comprise plastic strips that generally contain 20 miniature tubes. Almost all bacterial groups and over 550 different species can be identify used these API's tests. The identification is quite easy way and the API's tests give accurate identification results. The identification system have extensive databases of characteristic biochemical reactions of micro-organisms and are standardized.

(photo: www.biomerieux-usa.com) There are commercial products for bacteria identification: Product API 20E API 20NE API Rapid 20E API NH RAPIDEC Staph API 20 Strep API Staph API Coryne API 20A and others Characterisation of test Species and subspecies identification of Enterobacteriacae and groups and species identification of non-fermenting Identification of gram-negative non-Enterobacteriaceae Identification of Enterobacteriaceae Identification of Branhamella catarrhalis and Neisseria haemophillus Identification of the commonly occurring staphylococci Identification of streptococci and enterococcs Identification of clinical staphylococci and micrococci Identification of Corynebacteria and coryne-like organisms Identification of anaerobes

As per manufacturer's directions, the plastic sterile strip (API test) is inoculated with isolated a pure culture of microorganism suspension. This process also rehydrates the dessicated medium

the color reactions are read (some of the tubes will have color changes due to pH differences and some with the aid of added reagents to detect end metabolic products). The code is into the manufacturer's database (codebook). And some probes (tubes) are overlaid with sterile mineral oil (anaerobic reactions .agar plates of bacterial species . multi-test technique for bacterial identification (by: Jackie Reynolds.API test strip incubation chamber AFTER INCUBATION: 10% FeCl3. There are possibilities to get the information about the identification of microorganism from API test producer via touch-tone telephone system. Holding the strip at a slight angle up from the table top. Inoculate a large colony (2-3mm diameter) of the bacterium (pure culture) into the 0.85% NaCl solution. Kovac's reagent PROCEDURE: Step 1. Learn how to perform and interpret the miniaturized. Step 2. which gives the identification. some documentations from BioMerieux and some modification by autors of webside) MATERIALS NEEDED: . and the reactions (plus the oxidase reaction done separately) are converted to a seven-digit code (see Table B). you will now inoculate the bacterial suspension into each well with the sterile pipette (see Fig. Prepare a suspension of the bacteria in the saline tube 1. allowing capillary action to draw the fluid into the well as you slowly squeeze the bulb. URE (see Table B) The results are read after incubation (24 hours or less . Barrett's reagents A and B.85% NaCl solutions (5ml) .depending which API test is used) in a humidity chamber.sterile mineral oil . Use a McFarland barium sulfate standard #3 to quantitate the suspension. 2. ODC.ADH. usually as genus and species of microorganisms. making sure that the suspension is homogenous and without clumps of floating bacteria.API 20E test strip (for oxidase . A). 2. This should eliminate any bubbles .gram negative rods) .in the miniature tubes. Touch the end of the pipette to the side of the cupule.sterile Pasteur pipettes + bulbs . Inoculate the API strip 1. H2S. Richland College. LDC.0.

which can then be looked up in the codebook. and GEL have boxes around their names. The bottom of the incubation chamber has small indented wells in the bottom: fill it with water just enough to fill these indentations. B). CIT. 3. C and Photo A. Record results on the diagram handed out to you in lab (see Fig. but they will then be filled up to the top with sterile mineral oil. . These test wells will be filled all the way up to the top of the well. Table A. Each well should be filled up to the neck (see Fig. INTERPRETATION: 1. and label it. Add the proper reagents to the compartments: o 1 drop of Kovac's to the IND (read within a couple of minutes) o 1 drop of Barritt's A and B to VP (a + reaction may take up to 10 minutes) o 1 drop of FeCl3 to TDA 2. 2. Place the strip into this bottom. Incubate the strip in its chamber Figure B 1. Three test reactions are added together at a time to give a 7-digit number.*negative* reaction for first. and is added as the last test result! 4. There should not be so much water that it slops onto the API strip. Place the top of the incubation chamber over the bottom. Figure A Step 3. Place the strip at 37° C for 18-24 hours. 3. LDC. and URE are filled as described in point 2 (Step 2). second and third tube in the each triad. 4. or 4 points for + *positive* reaction for first.forming in the wells. 3. ADH. 0 points for . 2. H2S. ODC. !The oxidase test reaction should be negative. Read all other tests as described without reagents (see point 3). VP.) 1. 4. second and third tube in the each triad).

.+ 1 2 4 0 0 0 1 2 0 1 7 0 3 READING THE API 20 Table B.+ + . not according to real Photo A) Triad Tube Reaction Point Add 7-digital Code I II III IV 0 1 0 0 V + + 2 6 7031645 4 0 VI 0 4 + + 4 1 0 5 VII + 4 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 oxidase + + + .Figure C Photo 1 Table A (for sample.RESULTS *negative* colorless yellow yellow + RESULTS *positive* yellow red/orange red/orange .. TESTS SUBSTRATE REACTION TESTED ONPG ADH LDC ONPG arginine lysine beta-galactosidase arginine dihydrolase lysine decarboxylase .

+ + + - .) pink/red (10 min.) black diffuse yellow yellow yellow yellow yellow yellow yellow yellow yellow violet Characteristic reactions of some common bacterial species used API E20 test TESTS ONPG ADH LDC ODC CIT H2S URE TDA IND Escherichia coli + + + + Klebsiella pneumoniae + + + + Proteus vulgaris + + + + Salmonella sp.ODC CIT H2S URE TDA IND VP GEL GLU MAN INO SOR RHA SAC MEL AMY ARA OX ornithine citrate Na thiosulfate urea tryptophan tryptophan Na pyruvate charcoal gelatin glucose mannitol inositol sorbitol rhamnose sucrose melibiose amygdalin arabinose oxidase ornithine decarboxylase citrate utilization H2S production urea hydrolysis deaminase indole production acetoin production gelatinase fermentation/oxidation fermentation/oxidation fermentation/oxidation fermentation/oxidation fermentation/oxidation fermentation/oxidation fermentation/oxidation fermentation/oxidation fermentation/oxidation oxidase 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 blue/blue-green blue/blue-green blue/blue-green blue/blue-green blue/blue-green colorless/yellow red/orange blue-green/blue black deposit red/orange brown-red red (2 min.

J. Pulverer. (1986) Constructing a database for low cost identification Gram negative rods in clinical laboratories.. Journal of Applied Microbiology 90. J.ac. R.som.K. P. and Zai. B. S. and Priest.J. Journal of Clinical Pathology 39. 798-802.www.G.H. P.A.www. Letters in Applied Microbiology 10.. 257-259.. 6. C.N. (1984) A general-purpose system for . 2. pp. Mitchell.uk References 1.. Stuggart: Gustav Fisher Verlag. G.jlindquist.T.soton.edu . and Donachie.edu . L.VP GEL GLU MAN INO SOR RHA SAC MEL AMY ARA + + + + + + + + + + + + + + + + + + + + + - + + + + + + Links: More information about API's tests and micro-organisms identification: .H. 190-200..T. Carson. Journal of General Microbiology 136.net . Wilson.com . R. Zentralblatt für Bakteriologie Supplement 21.. and Sneath.www.A. E.www. G.biomerieux-usa. 367-376. Alexander. (1990) Computer-aided identification of lactic acid bacteria using the API 50 CHL system. (1990) Numerical classification and identification of Bacillus sphaericus including some strains pathogenic for mosquito larvae.P.K. In: Jeljaszewicz/Ciborowski.www.dcccd.K. Feltham. R.indstate. 4. (1991) Recent advances in the classification and identification of the genus Micrococcus. Wagner. 3. Clayton. F. (Ed.A. 5. P. P. Alderson.. (2001) Miniaturized tests for computer-assisted identification of motile Aeromonas species with an improved probability matrix.) The Staphylococci. Cox.web. and Thomsen. Wood.A.rlc. 103-109. Amadi.A. Feltham. and Sneath.

M. Vickers. R. S. Journal of General Microbiology 135.A. characterizing medically important bacteria to genus level. 121-133.. and Kroppenstedt.H. (1989) Construction of a database to identify Staphylococcus species. C. J.. 1681-1689.. Sneath. (1991) Probabilistic identification of streptomyces using miniaturized physiological tests. Journal of General Microbiology 131. Journal of General Microbiology 137. Sneath.A.M.A.M.. C.H.T.. Journal of Applied Bacteriology 57. (1985) Probabilistic identification of Streptoverticillium species.6.C. P. 7..H. Journal of Clinical Pathology 42. C. A.. (1989) New probability matrices for identification of Streptomyces.M. G. . 279-290. 289-294. Geary. A. and Mortimer.D. Schofield. S. and Mortimer. 8. and Mitchell. Langham. Williams. P. Stevens.J. P. Kämpfer. Williams. P. 9. 1893-1902.T. R. Sneath. Locci.. M.

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