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

in the miniature tubes. And some probes (tubes) are overlaid with sterile mineral oil (anaerobic reactions - ADH, LDC, ODC, H2S, URE (see Table B) The results are read after incubation (24 hours or less - depending which API test is used) in a humidity chamber, 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), and the reactions (plus the oxidase reaction done separately) are converted to a seven-digit code (see Table B). The code is into the manufacturer's database (codebook), which gives the identification, usually as genus and species of microorganisms. There are possibilities to get the information about the identification of microorganism from API test producer via touch-tone telephone system.

Learn how to perform and interpret the miniaturized, multi-test technique for bacterial identification
(by: Jackie Reynolds, Richland College, some documentations from BioMerieux and some modification by autors of webside)

MATERIALS NEEDED: - agar plates of bacterial species - 0.85% NaCl solutions (5ml) - sterile Pasteur pipettes + bulbs - sterile mineral oil - API 20E test strip (for oxidase - gram negative rods) - API test strip incubation chamber AFTER INCUBATION: 10% FeCl3, Barrett's reagents A and B, Kovac's reagent PROCEDURE: Step 1. Prepare a suspension of the bacteria in the saline tube 1. Inoculate a large colony (2-3mm diameter) of the bacterium (pure culture) into the 0.85% NaCl solution, making sure that the suspension is homogenous and without clumps of floating bacteria. 2. Use a McFarland barium sulfate standard #3 to quantitate the suspension. Step 2. Inoculate the API strip 1. Holding the strip at a slight angle up from the table top, you will now inoculate the bacterial suspension into each well with the sterile pipette (see Fig. A). 2. Touch the end of the pipette to the side of the cupule, allowing capillary action to draw the fluid into the well as you slowly squeeze the bulb. This should eliminate any bubbles

forming in the wells. Each well should be filled up to the neck (see Fig. B). 3. CIT, VP, and GEL have boxes around their names. These test wells will be filled all the way up to the top of the well. 4. LDC, ODC, ADH, H2S, and URE are filled as described in point 2 (Step 2), but they will then be filled up to the top with sterile mineral oil.

Figure A Step 3. Incubate the strip in its chamber

Figure B

1. The bottom of the incubation chamber has small indented wells in the bottom: fill it with water just enough to fill these indentations. 2. Place the strip into this bottom. There should not be so much water that it slops onto the API strip. 3. Place the top of the incubation chamber over the bottom, and label it. 4. Place the strip at 37 C for 18-24 hours. INTERPRETATION: 1. 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. Read all other tests as described without reagents (see point 3). 3. Record results on the diagram handed out to you in lab (see Fig. C and Photo A, Table A.) 1, 2, or 4 points for + *positive* reaction for first, second and third tube in the each triad, 0 points for - *negative* reaction for first, second and third tube in the each triad). !The oxidase test reaction should be negative, and is added as the last test result! 4. Three test reactions are added together at a time to give a 7-digit number, which can then be looked up in the codebook.

Figure C

Photo 1 Table A (for sample, 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 + + + - - - + + - + 1 2 4 0 0 0 1 2 0 1 7 0 3

READING THE API 20

Table B. TESTS SUBSTRATE REACTION TESTED ONPG ADH LDC ONPG arginine lysine beta-galactosidase arginine dihydrolase lysine decarboxylase - RESULTS *negative* colorless yellow yellow + RESULTS *positive* yellow red/orange red/orange

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.) 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. + + + -

VP GEL GLU MAN INO SOR RHA SAC MEL AMY ARA

+ + + + + + +

+ + + + + + + + + +

+ + + + -

+ + + + + +

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

6. 7. 8. 9.

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