Biochemical Test

API (Analytical Profile Index) 20E Test Result, Principle, Procedure

API test strip (analytical profile index) is a small, standardised collection of biochemical tests that can be used with complete identification databases....

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API (Analytical Profile Index) 20E Test Result, Principle, Procedure
API (Analytical Profile Index) 20E Test Result, Principle, Procedure
  • API test strip (analytical profile index) is a small, standardised collection of biochemical tests that can be used with complete identification databases. The api 20E is the most well-known of these tests (20 characters for Enterobacteriaceae).
  • BIOMERIEUX says that 10 API® strips are used every minute around the world. They are also used to test how well other identification products work (reference technique)
  • Analytical Profile Index, or API, are commercially available miniaturised biochemical test panels that cover a large number of clinically important bacterial groups as well as microorganisms found in food and water.
  • API is a system for grouping bacteria based on tests, which makes it easy to identify them (rapid identification)
  • The system is made to make it easy to find bacteria that are important to medicine.
  • Bacteria that are already known can be identified.
  • API test strips are made up of microtubes (called “cupules”) that contain dehydrated substrates to measure the activity of enzymes or the fermentation of sugars by the organisms that were added to the test strip.
  • During incubation, metabolism causes colour changes that can happen on their own or be seen when reagents are added.
  • When the carbs are fermented, the pH inside the cupule changes, which can be seen with an indicator.

Principle of APT Test for Bacteria

  • Biochemical tests are used in the API Test for Bacteria (API 20E Test kit) to identify and tell apart bacteria in the family Enterobacteriaceae.
  • The API range offers a standard, smaller version of the existing identification methods, which were hard to use and hard to read before.
  • In the API 20E, the plastic strip has twenty mini-test chambers that hold dehydrated media with different chemical compositions for each test.
  • Most of the time, they look for enzyme activity, which is usually caused by fermentation of carbohydrates or breakdown of proteins or amino acids by the organisms that were injected.
  • Each well is rehydrated with a bacterial suspension, and the strips are put in an incubator.
  • During incubation, metabolism causes colour changes that happen on their own or when reagents are added.
  • All positive and negative test results are put together to get a profile number, which is then compared with profile numbers in a commercial codebook or logbook (or online) to determine and identify the bacterial species.
API (Analytical Profile Index) Test
API (Analytical Profile Index) Test | Source: microbiologie-clinique.com

API 20E Test Kit

This kit is made for 20 tests and they are-

  • ONPG ( o-nitrophenyl-b-D-galactopyranoside) test: ONPG is a colorless substrate, similar in structure to lactose, used in this test as the substrate for b-galactosidase. If the organism possesses b-galactosidase, the enzyme will split the b-galactoside bond, releasing galactose and o-nitrophenol.
  • ADH (arginine dihydrolase): decarboxylation of the amino acid arginine by arginine dihydrolase
  • LDC (lysine decarboxylase) test: decarboxylation of the amino acid lysine by lysine decarboxylase
  • ODC( ornithine decarboxylase) test: decarboxylation of the amino acid ornithine by ornithine decarboxylase
  • CIT (citrate) utilization test: utilization of citrate as only carbon source
  • H2S( hydrogen sulfide) test: production of hydrogen sulfide
  • URE(urea) hydrolyzation test: test for the enzyme urease
  • TDA (Tryptophan deaminase): detection of the enzyme tryptophan deaminase: Reagent- Ferric Chloride.
  • IND( Indole)test: Indole Test-production of indole from tryptophan by the enzyme tryptophanase. Reagent- Indole is detected by the addition of Kovac’s reagent.
  • VP (Voges-Proskauer) test: the Voges-Proskauer test for the detection of acetoin (acetyl methylcarbinol) produced by fermentation of glucose by bacteria utilizing the butylene glycol pathway
  • GEL(gelatin) hydrolysis test: test for the production of the enzyme gelatinase which liquefies gelatin
  • GLU ( glucose) fermentation test: fermentation of glucose (a hexose sugar)
  • MAN ( mannitol) fermentation : fermentation of mannose (hexose sugar)
  • INO( inositol) fermentation test: fermentation of inositol (cyclic polyalcohol)
  • SOR( sorbitol) fermentation test: fermentation of sorbitol (alcohol sugar)
  • RHA( rhamnose ) fermentation test: fermentation of rhamnose (methyl pentose sugar)
  • SAC ( sucrose) fermentation test: fermentation of sucrose (disaccharide)
  • MEL ( melibiose) fermentation test: fermentation of melibiose (disaccharide)
  • AMY( amygdalin) fermentation test: fermentation of amygdalin (glycoside) and
  • ARA ( Arabinose) fermentation test : fermentation of arabinose (pentose sugar)

Test Requirements

  • API 20E Test strip and related reagents like Kovac’s  reagent, ferric chloride
  • 40 % KOH and α-Naphthol
  • Growth of test organism ( pure form i.e.  pure isolated colones)
  • Oxidase disks
  • Distilled water
  •  Pasteur pipette
  • Sterile oil
  • Marker
  • API logbook
  • Clean and grease-free slides and coverslips

API  Test Procedure

  1. Check to see if the culture contains Enterobacteriaceae. A quick oxidase test for cytochrome c oxidase can be done to check this. All members of the Enterobacteriaceae family are oxidase negative, except for Plesiomonas shigelloides, which has a positive oxidase test.
  2. Choose a single isolated colony from a pure culture and mix it with sterile distilled water to make a suspension.
  3. Take the API 20E Test Strip. It has 20 separate compartments that hold dehydrated bacterial media and biochemical reagents.
  4. Use a Pasteur pipette to fill the compartments with the bacterial suspension all the way to the top.
  5. Put sterile oil in the places where ADH, LDC, ODC, H2S, and URE are written.
  6. Put a few drops of water in the tray, place the API Test strip in the tray, and then close the tray.
  7. Write your initials, the date, and the patient’s ID number on the tray.
  8. Keep the tray at 37°C for 18 to 24 hours.
API (Analytical Profile Index) Test
API (Analytical Profile Index) Test | Source: microbiologie-clinique.com

Result Interpretation of API 20E Test

Result Interpretation of API 20E Test
API 20E positive vs negative | Source: microbiologie-clinique.com
  1. Read the colour changes of the compartments, except for those that need extra reagents, like “TDA,” which needs one drop of ferric chloride, “IND,” which needs one drop of Kovac’s reagent, and “VP,” which needs one drop of 40% KOH (VP reagent 1) and one drop of VP Reagent 2 (-Naphthol).
  2. Mark each test as positive or negative on the lid of the tray. This will give you the API Reading Scale, which is a colour chart. The wells are split into three groups by black triangles, and each group gets a score. Use the API reading scale as a guide:
  3. In each triplet, only add up the scores for the wells that are correct.
  4. Three test reactions are added together at a time to get a 7-digit number that can be looked up in a codebook. The best score a triplet can get is 7, which is the sum of 1, 2, and 4, and the worst is 0, as shown in the picture above.
  5. Use the API catalogue to find the organism, or use the system’s large and reliable database, which is now available through the Internet-based APIWEBTM service ( online).
API (Analytical Profile Index) Test
API (Analytical Profile Index) Test | Source: microbiologie-clinique.com

Advantages of API Test 

  • It is a quick and easy way to tell the difference between organisms and identify them (18-24 hour identification of Enterobacteriaceae and other non-fastidious gram-negative bacteria).
  • It can also be used to identify fungi ( yeasts).
  • It is a standard, easy-to-use way to find out what microorganisms are and how they differ from each other.
  • API strips have a long shelf life, so every lab can keep test kits on hand. They are very useful in medium-level labs, where organisms are hard to identify with traditional tests and there aren’t enough resources like MALDI-TOF-MS and molecular testing ( absence of thermocyclers).
  • This lets correct identifications be made based on large databases.

Important Notes

  • The Enteric (bacteria) Identification System uses the API-20E test strip.
  • The API 20E/NE strip is for a fast identification system that combines some traditional tests and can identify a small number of Gram-negative Enterobacteriaceae or non-Enterobacteriaceae.
  • API-Staph strip is only good for Gram-positive bacteria, like Staphylococcus species, Micrococcus species, and related organisms.
  • It costs less to run because each strip can do more than one test.
  • It can be used not only for bacteria but also for fungi ( yeasts).

Uses

  • API lets people manually identify microorganisms in order to: diagnose infectious diseases
  • Important industrial microorganisms need to be found.

Download Protocols from API Galleries

GalleriesProtocols
API 20E pdfDownload
API NE pdfDownload
API STREPT pdfDownload
API STAPH pdfDownload
API LISTERIA pdfDownload
API CANDIDA pdfDownload
API CH50 pdfDownload
API 20C pdfDownload
API (Analytical Profile Index) 20E Test
API (Analytical Profile Index) 20E Test | Source: microbiologie-clinique.com

References

  • https://www.slideshare.net/zarisaleh/analytical-43473658
  • https://microbiologie-clinique.com/api-for-microorganism-identification.html
  • https://www.biomerieux-usa.com/clinical/api
  • https://universe84a.com/collection/api-test-bacteria/
  • https://wineserver.ucdavis.edu/industry-info/enology/methods-and-techniques/winery-lab-techniques/api-test-strips
  • https://microbeonline.com/api-20e-test-system-introduction-procedure-results-interpretations/
  • https://bacdive.dsmz.de/api-test-finder
  • https://microbiologyinfo.com/api-20e-test/
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