API Test for Bacteria: Introduction, Principle, Test Requirements, Test Procedure, Result- Interpretation, Advantages, and Keynotes

Introduction of API Test for Bacteria

API test for bacteria as shown above picture is a 20-jumbo tests kit for a biochemical panel for the identification and differentiation of members of the family Enterobacteriaceae.  API stands for Analytical Profile Index. It is an API 20E Test kit that is quick, safe, and easy to perform and it is thus a well-established method for manual microbial identification to the species level. Modified API test kits are also available for the identification of microbes covering  Gram-positive and Gram-negative bacteria and yeast. API strips give accurate identifications based on extensive databases. They are standard technique, easy-to-use test systems. But here we are concerned with the API 20E test kit.

Principle of APT Test for Bacteria

API Test for bacteria (API 20E Test kit) uses to identify and differentiate bacteria of the family Enterobacteriaceae on the basis of biochemical tests. The API range provides a standardized, miniaturized version of existing identification techniques. But It also until now was complicated to perform and difficult to read. In the API 20E, the plastic strip holds twenty mini-test chambers containing dehydrated media having chemically defined compositions for each test. They usually detect enzymatic activity, mostly related to the fermentation of carbohydrates or the catabolism of proteins or amino acids by the inoculated organisms.

A bacterial suspension is used to rehydrate each of the wells and the strips are incubated. During incubation, metabolism produces color changes that are either spontaneous or revealed by the addition of reagents. All positive and negative test results are compiled to obtain a profile number, which is then compared with profile numbers in a commercial codebook or logbook (or online) for the determination and identification of the bacterial species.

API 20E Test Kit

This kit is made for 20 tests and they are-

1. 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.
2. ADH (arginine dihydrolase): decarboxylation of the amino acid arginine by arginine dihydrolase
3. LDC (lysine decarboxylase) test: decarboxylation of the amino acid lysine by lysine decarboxylase
4. ODC(ornithine decarboxylase) test: decarboxylation of the amino acid ornithine by ornithine decarboxylase
5. CIT (citrate) utilization test: utilization of citrate as only carbon source
6. H₂S ( hydrogen sulfide) test: production of hydrogen sulfide
7. URE(urea) hydrolyzation test: test for the enzyme urease
8. TDA (Tryptophan deaminase): detection of the enzyme tryptophan deaminase: Reagent- Ferric Chloride.
9. 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.
10. 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
11. GEL(gelatin) hydrolysis test: test for the production of the enzyme gelatinase which liquefies gelatin
12. GLU (glucose) fermentation test: fermentation of glucose (a hexose sugar)
13. MAN (mannitol) fermentation : fermentation of mannose (hexose sugar)
14. INO(inositol) fermentation test: fermentation of inositol (cyclic polyalcohol)
15. SOR(sorbitol) fermentation test: fermentation of sorbitol (alcohol sugar)
16. RHA(rhamnose ) fermentation test: fermentation of rhamnose (methyl pentose sugar)
17. SAC (sucrose) fermentation test: fermentation of sucrose (disaccharide)
18. MEL (melibiose) fermentation test: fermentation of melibiose (disaccharide)
19. AMY(amygdalin) fermentation test: fermentation of amygdalin (glycoside) and
20. 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(D/W)
• Pasteur pipette
• Sterile oil
• Marker
• Bunsen Burner/Safety Cabinet
• Waste Bin/ Waste table jar
• Incubator
• API logbook
• Clean and grease-free slides and coverslips

API Test Procedure

1. Confirm the culture is of Enterobacteriaceae. To test this, a quick oxidase test for cytochrome c oxidase may be performed. Note: All the members of the family Enterobacteriaceae are oxidase negative except Plesiomonas shigelloides which is oxidase test positive.
2. Pick a single isolated colony (from a pure culture) and make a suspension of it in sterile distilled water.
3. Take the API 20E Test Strip which contains dehydrated bacterial media/biochemical reagents in 20 separate compartments.
4. Using a Pasteur pipette, fill up (up to the brim) the compartments with the bacterial suspension.
5. Add sterile oil into the compartments having  ADH, LDC, ODC, H2S, and URE.
6. Put some drops of water in the tray and put the API Test strip and close the tray.
7. Mark the tray with an identification number (Patient ID ), date, and initials.
8. Incubate the tray for 18 to 24 hours at 37°C.

Result -Interpretation of API 20E Test

• Read the color change of the compartments except those that need extra reagent like ‘TDA’ -put one drop of ferric chloride, ‘IND’- put one drop of Kovac’s reagent and ‘VP’ need one drop of 40 % KOH (VP reagent 1)  and one drop of VP Reagent 2 (α-Naphthol).
• Get the API Reading Scale (color chart) by marking each test as positive or negative on the lid of the tray. The wells are marked off into triplets by black triangles, for which scores are allocated. Follow the API reading scale as –
• Add up the scores for the positive wells only in each triplet. Three test reactions are added together at a time to give a 7-digit number, which can then be looked up in the codebook. The highest score possible for a triplet is 7 (the sum of 1, 2, and 4) and the lowest is 0 as shown above picture.
• Identify the organism by using the API catalog or the system offers a large and robust database now accessible through the Internet-based APIWEB™ service (online).

Advantages of API Test

1. It is a fast and efficient method of identification and differentiation of organisms (18-24 hour identification of Enterobacteriaceae and other non-fastidious gram-negative bacteria).
2. It is also useful for fungal identifications ( yeasts).
3. It is an easy-to-run, user-friendly, and standardized method of identification and differentiation of microorganisms.
4. API strips have a long shelf life, enabling every laboratory to keep the test kits on hand, and are very useful in medium-level set-up laboratories where identification of organisms is difficult using conventional tests and lack of sources like MALDI-TOF-MS and molecular testing ( absence of thermocyclers).
5. This allows accurate identifications based on extensive databases

Keynotes on API Assay

1. The API-20E  test strip is for the Enteric ( bacteria)  Identification System.
2. The API 20E/NE strip is for a fast identification system that combines some conventional tests and allows the identification of a limited number of Gram-negative Enterobacteriaceae or non-Enterobacteriaceae.
3. API-Staph strip is specific for Gram-positive bacteria, including Staphylococcus species, Micrococcus, species, and related organisms
4. It is economic to run because of having multiple tests in a single strip.
5. It is not only applicable to bacteria but also to fungi (yeasts).

Further Reading

1. https://www.biomerieux-usa.com/clinical/api
2. https://www.jlindquist.com/generalmicro/102bactid2.html
3. https://bio.libretexts.org/Bookshelves/Ancillary_Materials/Laboratory_Experiments/Microbiology_Labs/Microbiology_Labs_I/43%3A_API-20E_multitest_strip
4. https://jcm.asm.org/content/jcm/7/6/539.full.pdf
5. https://microbiologyinfo.com/api-20e
6. https://en.wikipedia.org/wiki/Analytical_profile_index