API Test in Microbiology: Systems, Profile Number, and Identification

What is the API test in microbiology?

API stands for Analytical Profile Index. It is a system of miniaturized biochemical tests, made by bioMérieux, that identifies bacteria and yeasts to the species level. Instead of setting up a dozen separate tube tests, the technologist inoculates a single plastic strip holding up to 20 dehydrated biochemical reactions, incubates it, and reads the pattern of color changes. Those reactions are then converted into a numerical profile that is matched against a database to name the organism.

The name captures the method exactly: the profile is the pattern of biochemical reactions, and the index is the database that translates that pattern into an identification. There is not one API test but a whole family of strips, each tuned to a different group of organisms, from Enterobacteriaceae to staphylococci, streptococci, anaerobes, and yeasts.

It is one of the well-established methods for the identification of microorganisms up to the species level. The company runs APIWEB, where laboratories can input numerical profiles of the API strip results to obtain the organism identification. The software database enables reliable automated interpretation of API strip results which can be read on the screen and printed as per the need.

How the API profile number identifies an organism

Every API strip works on the same principle, whichever organism group it is built for. This is the single idea that ties the whole family together, and the one most students miss when they first meet a strip.

After incubation, each test on the strip is read as positive or negative. The tests are arranged in groups of three, marked off by small black triangles on the strip. Within each group of three, the tests are worth 1, 2, and 4. You add up only the positive tests in each group, which gives a single digit from 0 to 7 for that group. Reading the groups in order produces a short string of digits: the numerical profile, or profile number.

That profile number is then looked up, either in the printed API codebook supplied with the kit or in APIWEB, bioMérieux's online database, which returns the most likely organism along with a confidence value. In other words, the strip converts a pattern of biochemical reactions into a number, and the number is the key that unlocks the identification. This is why the reading table and the profile index matter so much: without them, the color changes on the strip mean nothing on their own.

The number of digits depends on how many tests the strip carries. A 20-test strip such as API 20E, read together with a supplementary oxidase test, produces a seven-digit code; a 10-test strip such as API Candida produces a shorter one. The principle is identical in every case.

For a complete worked example, reading a real strip reaction by reaction and building the full seven-digit code, see the fully worked example on the API 20E strip.

API strips, kits, and the wider identification workflow

Physically, an API kit is a plastic strip of small wells, each holding a different dehydrated biochemical substrate, supplied with the reagents, suspension medium, and codebook needed to run and read it. The dehydrated format gives the strips a long shelf life and makes them easy to store and transport, which is part of why they remain widely used in laboratories without automated identification instruments.

The API range covers most groups a diagnostic laboratory encounters: Enterobacteriaceae and other Gram-negative rods, non-fermenters, staphylococci, streptococci and enterococci, anaerobes, corynebacteria, Campylobacter, Listeria, Neisseria and Haemophilus, lactic acid bacteria, Bacillus, and yeasts. The next section lists each system and the organisms it identifies.

Where does API sit relative to newer methods? Automated systems and mass spectrometry, such as MALDI-TOF, now identify organisms faster and with less hands-on work in better-resourced laboratories. API strips remain valuable where those instruments are not available, as a reliable, low-infrastructure way to identify an organism to species level using only an incubator and the reading table. They are also a teaching tool, because the strip makes the biochemical logic of identification visible in a way a one-line automated readout does not.

Identification System and Species Identified

The following table lists the API and RAPID ID systems and the organism group each one identifies.

API 20 A

The API 20 A system which consists of 20 microtubules containing dehydrated substrates. It enables 21 tests to be carried out quickly and easily for the biochemical identification of anaerobes. Other tests such as colonial and microscopic morphology, Gram stain, etc. should be performed. The results are used to confirm or complete the identification.

These microtubes are inoculated with a bacterial suspension which reconstitutes the media. During incubation, metabolism produces color changes that are either spontaneous or revealed by the addition of reagents. The reactions are read according to the reading table. The identification is obtained by referring to the analytical profile index (API) or using the identification software.

API 20 E

API 20 E is a standardized identification system for Enterobacteriaceae and other non-fastidious, Gram-negative rods which uses 21 miniaturized biochemical tests and a database.

API 20E is the most widely used strip in the range. Because it has its own full walk-through, setup, the 21 reactions, reagent and oil wells, and the seven-digit profile, this hub does not repeat it here. Full guide: API 20E Test: Procedure, Reading the 21 Reactions, and the 7-Digit Profile Code

API 20 NE

API 20 NE is a standardized system for the identification of non-fastidious, non-enteric Gram-negative rods (e.g. Pseudomonas, Acinetobacter, Flavobacterium, Moraxella, Vibrio, Aeromonas, etc.) and a database. The API 20 NE strip consists of 20 microtubes (for 8 conventional tests and 12 assimilation tests) containing dehydrated substrates.

1. The conventional tests are inoculated with a saline bacterial suspension which reconstitutes the media. During incubation, metabolism produces color changes that are either spontaneous or revealed by the addition of reagents.
2. The assimilation tests are inoculated with a minimal medium. The bacteria grow if they are capable of utilizing the corresponding substrate.

The reactions are read according to the reading table. Then, the identification is obtained by referring to the analytical profile index (API) or using the identification software.

API 50 CHB/ CHE

API 50 CHB/E medium is used for the identification of Bacillus and related genera, as well as Gram-negative rods belonging to the Enterobacteriaceae and Vibrionaceae families. It is a ready-to-use medium that allows the fermentation of the 49 carbohydrates on the API 50 CH strip.

A suspension is made in the medium with the microorganism to be tested and each tube of the strip is then inoculated with the suspension. During incubation, fermentation of carbohydrates is detected by the color change of the pH indicator. The results make up the biochemical profile which is used by the identification software to identify the strain. The API 20 E strip may be used in association with the API 50 CH strip to provide supplementary tests for Enterobacteriaceae and Vibrionaceae.

Figure: API (Analytical Profile Index) test methods for the identification of gram negative bacilli

API STAPH and ID 32 STAPH

API Staph is a miniaturized biochemical test system for the identification of the genera Staphylococcus and Micrococcus. The API Staph strip consists of 20 microtubes containing dehydrated substrates. These microtubes are inoculated with a bacterial suspension, prepared in API Staph medium, that reconstitutes the tests.

During incubation, metabolism produces color changes that are either spontaneous or revealed by the addition of reagents. The reactions are read according to the reading table and the organism is identified by referring to the analytical profile index or using the identification software.

Figure: API (Analytical Profile Index) test for the identification of gram positive cocci

API 20 STREP and Rapid ID 32 STREP

API 20 Strep is a standardized system combining 20 biochemical tests for the identification of most streptococci and enterococci. The API 20 Strep strip consists of 20 microtubes containing dehydrated substrates for the demonstration of enzymatic activity or the fermentation of sugars.

Figure: API 20 Strep test system

• The enzymatic tests are inoculated with a dense suspension of organisms, made from a pure culture. This then used to reconstitute the enzymatic substrates. During incubation, metabolism produces color changes that are either spontaneous or revealed by the addition of reagents.
• The fermentation tests are inoculated with an enriched medium that rehydrates the sugar substrates. Fermentation of carbohydrates is detected by a shift in the pH indicator.

The reactions are read according to the reading table and the identification is obtained by referring to the analytical profile index or using the identification software.

API (Analytical Profile Index) 20C AUX and ID 32 C

API 20 C AUX and ID 32 C is used for the identification of Candida, Cryptococcus, Geotrichum, Kloeckera, Pichia, Rhodoturela and Trichosporon.

Figure: Identification of yeast by API test methods

API CANDIDA

API Candida is a standardized system for the identification of frequently encountered yeasts (Candida, Cryptococcus, Saccharomyces, and Trichosporon) in clinical microbiology. The API Candida strip consists of 10 tubes containing dehydrated substrates which enable testing of 12 sugar acidification or enzymatic reactions. The reactions produced during incubation are revealed by spontaneous color changes.

API (Analytical Profile Index) CAMPY

API Campy is a standardized miniature test system for the identification of Campylobacter. The API Campy strip consists of 20 microtubes containing dehydrated substrates. It is made up of two parts.

1. The first part of the strip (enzymatic and conventional tests) is inoculated with a dense suspension that rehydrates the substrates. During incubation in aerobic conditions, metabolism produces color changes that are either spontaneous or revealed by the addition of reagents.
2. The second part of the strip (assimilation or inhibition tests) is inoculated with a minimal medium and incubated in microaerophilic conditions. The bacteria grow if they are capable of utilizing the corresponding substrate or if they are resistant to the antibiotic tested. The reactions are read according to the reading table.

The organism is identified using the identification profile list in the package insert or by using identification software.

Figure: API for the identification of Gram positive bacilli

API (Analytical Profile Index) CHL

API 50 CHL is a ready-to-use medium that allows fermentation of 49 carbohydrates and is used for the identification of the genus Lactobacillus and related genera.A suspension is made in the medium with the microorganism to be tested and each tube of the strip is then inoculated with the suspension. During incubation, the carbohydrates are fermented to acids which produce a decrease in the pH, detected by the change in color of the indicator. The results make up the biochemical profile which is used by the identification software to identify the strain.

API (Analytical Profile Index) Coryne

API Coryne is a standardized miniature test system for the identification of Corynebacterium in 24 hours. It consists of 20 microtubes containing dehydrated substrates for the demonstration of enzymatic activity or the fermentation of carbohydrates.

• The enzymatic tests are inoculated with a dense suspension of organisms, which reconstitutes the enzymatic substrates. During incubation, metabolism produces color changes that are either spontaneous or revealed by the addition of reagents.
• The fermentation tests are inoculated with an enriched medium (containing a pH indicator) which reconstitutes the sugar substrates. Fermentation of carbohydrates results in acidification which is detected by a spontaneous color change in the pH indicator.

The reactions are read according to the reading table and the identification is obtained by referring to the analytical profile index (API) or using the identification software.

API Listeria

API Listeria is a standardized miniature test system for the identification of Listeria. The API Listeria strip consists of 10 microtubes containing dehydrated substrates which enable the performance of enzymatic tests or sugar fermentations.

During incubation, metabolism produces color changes that are either spontaneous or revealed by the addition of reagents. The reactions are read according to the reading table and the identification is obtained by consulting the profile list in the package insert or using the identification software.

API NH

API NH is a standardized miniature system for the identification of Neisseria, Haemophilus (and related genera), and Moraxella catarrhalis (Branhamella catarrhalis), using a specially adapted database.

The API NH strip consists of 10 microtubes containing dehydrated substrates, which enable the performance of 12 identification tests (enzymatic reactions or sugar fermentation), as well as the detection of a penicillinase. API NH also enables the biotyping of Haemophilus influenzaeand Haemophilus parainfluenzae.

Figure: Gram negative cocci API NH

References

1. Winn WC, Allen SD, Janda WM, Koneman EW, Procop GW, Schreckenberger PC, Woods GL (2017). Koneman's Color Atlas and Textbook of Diagnostic Microbiology. 7th edn. Wolters Kluwer.
2. Tille PM (2022). Bailey and Scott's Diagnostic Microbiology. 15th edn. Elsevier.
3. bioMérieux (n.d.). API and ID 32 identification systems: technical sheets and package inserts. bioMérieux SA. Retrieved from bioMérieux clinical diagnostics website.