API test | 20E Test – How it works and uses

What is an API 20E test?

The API (Analytical Profile Index) 20E Test is a well-known and commonly used biochemical test for identifying and differentiating bacteria from the Enterobacteriaceae family. It is a standardized biochemical assay designed for species identification of microorganisms.

API 20E kit

The API 20E Test kit is made up of a plastic strip with 20 different test mini-chambers (Wells) filled with dehydrated media with chemically determined compositions for specific tests. These assays are primarily concerned with detecting enzymatic activities associated with carbohydrate fermentation or protein/amino acid catabolism by the bacterial inoculation.

The API 20E Test kit is well-known for its quick, safe, and simple bacterial detection approach. It makes use of large databases to deliver reliable identifications based on metabolic cycles observed. To determine the exact bacterial species, the test results are compared to reference databases.

Importance of API 20E Test

API strips, especially the API 20E Test, have become an essential tool in microbiological laboratories around the world, with about 10 API strips being used every minute. These strips are also used to evaluate the performance of other identification products, making them a dependable and significant resource in microbiological identification and grouping.

Principle of API test

Analytical Profile Index test for bacteria, specifically the API 20E Test kit, is a method for identifying and distinguishing bacteria from the Enterobacteriaceae family based on biochemical properties.

The API sequence provides a standardized version of conventional identifying procedures that were previously complicated and hard to learn. The API 20E test strip is made up of twenty mini-test chambers, each with dehydrated media of chemically determined compositions for various tests. These assays are primarily concerned with detecting enzymatic activity particularly that linked to glucose fermentation or protein /amino acid catabolism by the inoculated bacteria.

A bacterial suspension is used to rehydrate the medium in each well of the test strip to start the test. The strips are then incubated in the appropriate conditions. The metabolic activity of the bacteria causes color variations in the media throughout incubation. Color changes can occur naturally or as a result of the presence of certain chemicals.

Following the incubation period, the results of all tests (both positive and negative) are combined to form a profile number, which shows the bacterial strain’s unique pattern of metabolic processes. This profile number is then matched to the profile numbers associated with recognized bacterial species in a commercial codebook or logbook (or via an online database).

The bacterial species can be determined and identified with great accuracy by comparing the profile number with the relevant identification code. The API 20E test speeds up the process of bacterial identification, making it a helpful tool for precise microbiological grouping in clinical and research contexts.

Requirements of API test

  • 40% KOH and α-Naphthol are important in specific API tests and must be accessible in adequate amounts.
  • Sterile oil is used to form a coating over test cultures to prevent dehydration and contamination.
  • To perform the tests, the test organism should be grown in its purest form, without any contaminants.
  • A marker is required for labelling and organizing API test plates or strips.
  • Oxidase discs are used to identify microorganisms that have cytochrome oxidase activity, which helps in differentiating.
  • Before using slides and coverslips in microscopic investigations, they must be thoroughly cleaned and free of grease or impurities.
  • During the testing process, a Pasteur pipette is utilized for accurate and controlled liquid transfer.
  • An API notebook aids in the recording of important information about each test, such as organism data, test results, and observations.

Procedure of API 20E kit Test

  • To make sure that the bacterial sample culture belongs to the Enterobacteriaceae family, run a fast oxidase test for cytochrome C oxidase. Except for Plesiomonas shigelloides, all members of the Enterobacteriaceae are oxidase negative.
  • Select a single isolated colony from a pure isolated culture and make a bacterial solution by combining it with sterile distilled water.
  • Take the API 20E Test Strip, which has 20 distinct compartments filled with dehydrated bacterial media or biochemical reagents.
  • Fill each chamber to the completely with the bacterial suspension using a Pasteur pipette.
  • Add sterile oil to the compartments labelled ADH, LDC, ODC, H2S, and URE.
  • Fill the incubation tray halfway with water. Then, gently place the API test strip in the tray and close it tightly.
  • To ensure accurate test tracking, label the tray with an identity number (Patient ID), date, and initials.
  • Transfer the sealed tray containing the API Test strip to an incubator and leave it to incubate for 18 to 24 hours at 37°C.
  • After the incubation period, look for color changes on the test strip and record the results for each test compartment.
  • Based on the metabolic profiles of the bacteria, compare the observed results to standardized databases or reference materials to identify the bacterial species.
api test procedure
Picture represents How to get code to match with the database for bacterial identification | source : microbiologie-clinique

Interpretation of results

  • Examine the color changes in each compartment of the API 20E Test strip, excluding those that require extra reagents (TDA, IND, and VP). Add one drop of ferric chloride to TDA; one drop of Kovac’s reagent to IND; and one drop of 40% KOH (VP Reagent 1) and one drop of VP Reagent 2 (-Naphthol) to VP.
  • Make an API Reading Scale (color chart) on the lid of the incubation tray by marking each test compartment as positive or negative. The wells are divided into triplets, which are represented by black triangles, and each triplet is granted a score.
  • Add up the points just for the positive wells inside each triplet.
  • Add the scores from three test reactions at a time to produce a seven-digit number. This number is associated with a specific identifying code.
  • Lookup the API codebook using the generated seven-digit number. The codebook includes a comprehensive set of identification codes associated with various bacterial species.
  • Based on the received identification code, identify the bacterial organism using the API catalogue or the online APIWEBTM service. The database has a lot of information that can be used to reliably identify microorganisms based on their metabolic characteristics.
  • You may read your API test results using a variety of internet tools. UPBM is one such website. Alternatively, you can use biomerieux’s apiwebTM, which provides an up-to-date and dependable database for understanding your API test data.
Figure 1: this figure represents API 20E test kit typical Salmonella reaction. The color responses were read after 18-24 hours incubation in humidity at 37°C (some with the use of additional reagents supplied by the kit). The data was evaluated using the manufacturer’s software, and positive results were confirmed as Salmonella with an 89% probability (Imen et al. 2012)

What tests can you perform using API 20E kit?

API 20E is a specialized kit for bacterial identification that includes 20 different tests. These tests aid in characterizing the organisms’ diverse metabolic processes and enzymatic reactions. Each test provides useful information for distinguishing between various bacterial strains. Let’s look at the individual tests included in the API 20E kit:

  • ODC Test: Ornithine Decarboxylase (ODC) test aids in the identification of the enzyme ornithine decarboxylase, which aids in the decarboxylation of ornithine.
  • ONPG Test: The presence of b-galactosidase enzyme is determined by detecting the release of galactose and o-nitrophenol from the substrate o-nitrophenyl-b-D-galactopyranoside (ONPG).
  • MAN (Mannitol) Fermentation Test : This test detects the fermentation of mannose, a hexose sugar.
  • CIT (Citrate) Utilization Test: This test examines if the organism can use citrate as its only carbon source.
  • URE (Urea) Hydrolyzation Test: This test detects the presence of urease, an enzyme that hydrolyzes urea.
  • GEL (Gelatin) Hydrolysis Test: This test determines the presence of the enzyme gelatinase, which liquefies gelatin.
  • Arginine Dihydrolase (ADH): This test involves the enzyme arginine Dihydrolase that decarboxylates the amino acid arginine.
  • INO (Inositol) Fermentation Test: This process examines the fermentation of inositol, a cyclic polyalcohol.
  • H2S (Hydrogen Sulphide) Test: This test detects the generation of hydrogen sulphide.
  • LDC (Lysine Decarboxylase) Test: This test detects the presence of the enzyme that catalyzes the decarboxylation of the amino acid lysine.
  • TDA (Tryptophan Deaminase) Test: This test uses Ferric Chloride reagent to determine the presence of the enzyme tryptophan deaminase.
  • SOR (Sorbitol) Fermentation Test: This test evaluates the fermentation of sorbitol, an alcohol sugar.
  • AMY (Amygdalin) Fermentation Test: This process examines the fermentation of amygdalin, a glycoside.
  • GLU (Glucose) Fermentation Test: This test detects the fermentation of glucose, a hexose sugar.
  • VP (Voges-Proskauer) test: This test detects acetoin (acetyl methylcarbinol) generated during glucose fermentation via the butylene glycol pathway.
  • MEL (Melibiose) Fermentation Test: This test evaluates the fermentation of a disaccharide, melibiose.
  • IND (Indole) Test: The Indole test evaluates the enzyme tryptophanase’s synthesis of indole from tryptophan using Kovac’s reagent.
  • ARA (Arabinose) Fermentation Test: This test used to examine arabinose, a pentose sugar, fermentation.
  • SAC (Sucrose) Fermentation Test: This test looks at the fermentation of sucrose, a disaccharide.
  • RHA (Rhamnose) Fermentation Test: This test detects Rhamnose fermentation, a methyl pentose sugar.
Figure 2: API 20E kit test interpretation (Iqbal et al. 2017)

Applications of API 20E test

  • The API 20E test is a useful tool for identifying the specific bacteria that cause infections. This data is essential for identifying the best antibiotic therapy and ensuring targeted and effective treatment.
  • This test can be used to track bacterial antibiotic resistance patterns. By identifying resistant strains, healthcare personnel can make better antibiotic selection decisions, assisting in combating against antibiotic resistance and optimizing treatment outcomes.
  • The API 20E test is used in antibiotic stewardship programs, in addition to guide antibiotic selection. It aids in the sensible and appropriate use of antibiotics by providing precise and speedy identification of bacteria, lowering the risk of prescriptions and improving optimal patient care.
  • This test assists in detecting illness causes and tracking the spread of certain bacteria within healthcare institutions. This data is vital for adopting effective infection control measures, controlling outbreaks, and protecting patient safety.
  • The test is also useful in research to explore the taxonomy and physiology of bacteria. The information collected from these investigations advances our understanding of bacterial identification, behaviors, and interactions, hence contributing to advances in microbiology and the development of new bacterial control approaches.
  • Understanding the microbial ecology of various environments, such as soil, water, and the human body, is essential in a variety of domains, including environmental sciences and medical studies. The API 20E test aids in the identification of bacterial species in complex ecosystems, providing information on microbial diversity and ecological relationships.
  • The API 20E test can be used in epidemiology to identify and track specific bacterial strains in outbreaks. This aids in the investigation of transmission origins and routes, allowing for better containment and control methods.

Advantages of API 20E test

  • Within a short incubation time of 18 to 24 hours, the API test identifies organisms quickly and efficiently, particularly Enterobacteriaceae and other non-fastidious gram-negative bacteria.
  • The API test is useful for fungal identification, notably yeasts, in addition to bacterial identification, broadening its usefulness to diverse microorganisms.
  • This test is well-known for its simplicity and use. It provides a standardized testing method that ensures consistent and trustworthy results across laboratories and users.
  • API strips have a long shelf life, allowing laboratories to maintain test kits on hand at all times. This is especially beneficial for medium-level laboratories that may face issues due to a lack of thermocyclers and access to other advanced identification methods such as MALDI-TOF-MS and molecular testing.
  • The API test is based on large databases that include an array of information about different microorganisms. These databases allow for reliable and precise identifications, assisting in the right classification of the species investigated.
  • When compared to more advanced and expensive identification techniques, the API test is a more cost-effective approach for microbiological identification. Its low cost makes it available to a broader range of laboratories and healthcare providers.
  • The API test regularly gives trustworthy results due to its standardized design and huge databases, minimizing the probability of misidentification and delivering better patient care and infection management.

Disadvantages of API 20E kit

  • The API 20E test has an accuracy of about 90%, with a 10% possibility of getting inaccurate results. This margin of error can result in misidentification and, as a result, impact patient treatment decisions.
  • The database of API 20E test includes a limited number of bacterial identification. As a result, it can’t identify new or rare bacteria that aren’t in its reference database, limiting its use in emerging infectious cases.
  • Obtaining API 20E test results can be time-consuming, lasting up to 24 hours. This waiting period may delay quick intervention and treatment in circumstances where the bacterium must be identified immediately.
  • API 20E test results might be difficult to interpret, especially for untrained users. Misinterpretation or inaccuracies in result analysis can result in incorrect identification, putting patients’ care at risk.
  • This test requires a pure culture of the bacteria being tested. Isolating a pure culture can be problematic, especially when the bacteria are present in a mixed culture, which can make reliable testing difficult.
  • The API 20E test is ineffective for detecting fastidious bacteria, which have specialized and rigorous growth needs. As a result, certain clinically relevant bacteria may be undetected, necessitating the use of alternate testing procedures.

From where you can purchase API 20E kits?

  • Biomerieux’s
  • VWR
  • Fisher Scientific
  • Sigma-Aldrich 
  • Amazon.


The API 20E kit test is an essential tool for microbiologists and healthcare practitioners. Its capacity to identify bacteria effectively, based on their metabolic responses, is vital for the diagnosis, research, and upholding public health standards. Microbiologists continue to rely on such novel diagnostic solutions as technology progresses to make the world a better and healthier place.


  • https://www.biomerieux-usa.com/clinical/api
  • https://bio.libretexts.org/Demos%2C_Techniques%2C_and_Experiments/Microbiology_Labs_I/43%3A_API-20E_multitest_strip
  • https://www.jlindquist.com/generalmicro/102bactid2.html
  • https://jcm.asm.org/content/jcm/7/6/539.full.pdf
  • Imen BS, Ridha M, Mahjoub AJS-ADFP. 2012. Laboratory typing methods for diagnostic of Salmonella strains, the “old” organism that continued challenges. 349-372.
  • Iqbal R, Ikram N, Shoaib M, Muhammad J, Raja T, Abid A, Aanam A, Bushra I, Faiza NJJABB. 2017. Phenotypic cofirmatory disc diffusion test (PCDDT), double disc synergy test (DDST), E-test OS diagnostic tool for detection of extended spectrum beta lactamase (ESΒL) producing Uropathogens.  3(3): 344-349.
Mubashir Iqbal
Mubashir Iqbal

Mubashir Iqbal is a highly dedicated and motivated Microbiologist with an MPhil in Microbiology from the University of Veterinary and Animal Sciences. Currently, he is researching the efficacy of commercially available SARS Cov-2 vaccines to neutralize the omicron variant in Pakistan. He holds a Bachelor's degree in Microbiology and has experience in chemical and microbiological analysis of water samples, managing SOPs and documents according to standard ISO 17025. Additionally, he has worked as an internee in BSL 3, Institute of Microbiology, UVAS, where he gained experience in RNA extraction, sample processing, and microscopy.

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