API 20E Test System: Results and Interpretations

API (Analytical Profile Index) 20E is a biochemical panel from bioMerieux, Inc. for identifying and differentiating members of the family Enterobacteriaceae. The plastic strip holds twenty mini-test chambers containing dehydrated media having chemically defined compositions for each test.

A bacterial suspension is used to rehydrate each of the wells. After incubation, some of the wells will have color changes due to pH differences: others produce end products that must be identified with reagents. A profile number is determined from the sequence of positive and negative test results, then looked up in a codebook to identify the bacterial isolate.

API 20E Biochemical Test Strip
API 20 E Biochemical Test Strip

These include:

  1. ONPG: test for β-galactosidase enzyme by hydrolysis of the substrate o-nitrophenyl-b-D-galactopyranoside
  2. ADH: decarboxylation of the amino acid arginine by arginine dihydrolase
  3. LDC: decarboxylations of the amino acid lysine by lysine decarboxylase
  4. ODC: decarboxylations of the amino acid ornithine by ornithine decarboxylase
  5. CIT: utilization of citrate as only carbon source
  6. H2S: production of hydrogen sulfide
  7. URE: test for the enzyme urease
  8. TDA (Tryptophan deaminase): detection of the enzyme tryptophan deaminase: Reagent to put- Ferric Chloride.
  9. IND: Indole Test-production of indole from tryptophan by the enzyme tryptophanase. Reagent- Indole is detected by the addition of Kovac’s reagent.
  10. VP: the Voges-Proskauer test for the detection of acetoin (acetyl methylcarbinol) produced by fermentation of glucose by bacteria utilizing the butylene glycol pathway
  11. GEL: test for the production of the enzyme gelatinase which liquefies gelatin
  12. GLU: fermentation of glucose (hexose sugar)
  13. MAN: fermentation of mannose (hexose sugar)
  14. INO: fermentation of inositol (cyclic polyalcohol)
  15. SOR: fermentation of sorbitol (alcohol sugar)
  16. RHA: fermentation of rhamnose (methyl pentose sugar)
  17. SAC: fermentation of sucrose (disaccharide)
  18. MEL: fermentation of melibiose (disaccharide)
  19. AMY: fermentation of amygdalin (glycoside)
  20. ARA: fermentation of arabinose (pentose sugar)

Setting up an API20E Biochemical Test Strip

  1. Pick up a single isolated colony (from a pure culture) and make a suspension of it in sterile distilled water.
  2. Take the API20E biochemical test strip which contains dehydrated bacterial media/biochemical 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).
  3. Take a Pasteur pipette and fill up (up to the brim) these compartments with the bacterial suspension.
  4. Add sterile oil into the ADH, LDC, ODC, H2S, and URE compartments.
  5. Put some drops of water in the tray and put the API Test strip and close the tray.
  6. Mark the tray with an identification number (patient ID or organism ID), date, and your initials.
  7. Incubate the tray at 37oC for 18 to 24 hours.

Results and Interpretation

  1. 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.
  2. Add the following reagents to these specific compartments
    1. TDA: Put one drop of ferric chloride
    2. IND: Put one drop of Kovacs reagent
    3. 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).
  3. Get the API reading scale (color chart)
    1. Mark each test as positive or negative on the lid of the tray
    2. The wells are marked off into triplets by black triangles, for which scores are allocated as follows:

      API 20 E 124

  4. 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. (https://glasshousefarms.org)
Numbering in API 20E Test Strip
Numbering in API 20E Test Strip
  1. The profile for this combination of reactions is therefore 7031645 (7 digit code)
  2. Identify the organism by using API catalog or apiweb
    VIDEO: Reading an API20E using the online database


  1. Shayegani, M., Maupin, P. S., & McGlynn, D. M. (1978). Evaluation of the API 20E system for identification of nonfermentative Gram-negative bacteria. Journal of clinical microbiology, 7(6), 539–545. https://doi.org/10.1128/jcm.7.6.539-545.1978 
  2. O’Hara, C. M., Rhoden, D. L., & Miller, J. M. (1992). Reevaluation of the API 20E identification system versus conventional biochemicals for identification of members of the family Enterobacteriaceae: a new look at an old product. Journal of clinical microbiology, 30(1), 123–125. https://doi.org/10.1128/jcm.30.1.123-125.1992 
  3. Butler, D. A., Lobregat, C. M., & Gavan, T. L. (1975). Reproducibility of the analytab (API 20E) system. Journal of clinical microbiology, 2(4), 322–326. https://doi.org/10.1128/jcm.2.4.322-326.1975

Acharya Tankeshwar

Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. As an asst. professor, I am teaching microbiology and immunology to medical and nursing students at PAHS, Nepal. I have been working as a microbiologist at Patan hospital for more than 10 years.

Recent Posts