API 20E Test System: Introduction, Procedure Results and Interpretations

API 20 E Biochemical Test Strip

API (Analytical Profile Index) 20E presented is a biochemical panel for identification and differentiation of members of the family Enterobacteriaceae.

In API 20E for identification of members of the family Enterobacteriaceae, the plastic strip holds twenty mini-test chambers containing dehydrated media having chemically-defined compositions for each test.

API 20 E 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 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/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).
  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 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 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.

    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
  3. Identify the organism using apiweb:
    1. Start Internet Explorer or Firefox web browser
    2. Go to: https://apiweb.biomerieux.com
      1. Login:  your login name
      2. Password: your password
      3. Select the correct test (e.g. API 20E).
      4. Enter the numerical profile to obtain the identity.
      5. Record the identity along with comments (% ID and T value) on the results sheet.
About Acharya Tankeshwar 458 Articles
Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. I am working as an Asst. Professor and Microbiologist at Department of Microbiology and Immunology, Patan Academy of Health Sciences, Nepal. If you want me to write about any posts that you found confusing/difficult, please mention in the comments below.


  1. Very interesting post. We did not do it in school and it’s only during my internship that i got aware of it, and i was like confused and anxious to know more about the procedure untill i accidentally felt on this post. Really after reading it i was, and am performing the test in the lab with ease.

    • Thank you for the suggestion, we use API 2OE test system in our laboratory, so i have written about this. I will go through API 20 A method.

          • @nino.api is a product from biomerieux, u can directly buy it to their exclusive distributer in the US. In our country, i work as an sales agent of TNC Everlight, an exclusive dstributder in the Philippines, if and only i could sold an api 20 e kit (consist of 25 test strip) and reagents. I would do.but, it would be an easy for u if u can contact a biomerieux store in the US.thanks!

          • @Nino, Since u are in the US. You can reach through the contact number below:

            bioMérieux, Inc.
            100 Rodolphe Street
            Durham, NC 27712
            Phone: (800) 682-2666
            Fax (General): (800) 968-9494
            Fax (Orders): (800) 432-9682

            bioMérieux Industry
            595 Anglum Road
            Hazelwood, MO, 63042
            Phone: (314) 731-8500
            Fax: (314) 731-8678

            bioMérieux – Lombard, IL
            1105 N. Main Street
            Lombard, IL 60148
            Phone:(630) 628-6055

            bioMérieux – Boston, MA
            355 Main Street, Ste # 802
            Cambridge, MA 02142
            Phone: (617) 679-8000

            bioMérieux – Canada
            7815, Henri-Bourassa West
            H4S 1P7 ST LAURENT, QC
            Phone: (514) 336-7321

  2. hi, What is the principle behind the these tests tests including the biochemical pathway, name of substrate used, name of the product(s) and/or indicator, why certain reagents are added to complete the test. thanks

    • Manoosh, there are miniature test based on the same principle. To read about principle of each test, you can navigate to related posts in microbeonline.

    • Manoosh
      API 20E test system uses miniature version of the same test, so the principle is almost similar. You can find principle of each test in microbeonline.

  3. hi, thanks for your help i found to answer to all my questions. i am just wondering if in GLU test we are just testing the fermentation or F/O both

  4. Hi there. I am second year student and I have used the API 20 E system for the first time recently. I am struggling to understand the concept of %id and T index obtained from the apiweb. Your reply is much appreciated.

  5. I recently performed the API 20E test strip system at my school and the closest match I have been able to find to my results is the test strip listed at the top of this page. What is the organism’s name that shown in the figure above?

  6. Very interesting we don’t use this strip until now! It reduces time and labor so how can we get it?would you please tell us the source? thanks

  7. hi there, i m required to prepare an accepting test for api20e n api20ne in house. thought the related company has provided the acceptance from their company yet i need to show the in house prepared.can u suggest any idea to do it

  8. ok many thanks i work in CPHL loaboratory in Afghanistan I have some problum about same bacteria which I isolated i sent you the code number 5730004 api 20 NE

  9. Thankyou for the info..am from Papua New Guinea, a country in the western Pacific and am wondering how i could acquire the APIweb system for my Microbiology laboratory…

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