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

What is API (Analytical Profile Index) 20E Test?

  • The API (Analytical Profile Index) 20E Test is a well-established and widely used biochemical panel for the identification and differentiation of bacteria belonging to the family Enterobacteriaceae. It is a miniaturized and standardized gallery of biochemical tests designed for manual microorganism identification at the species level.
  • The API 20E Test kit consists of a plastic strip containing 20 individual test mini-chambers (Wells), each filled with dehydrated media containing chemically defined compositions for specific tests. These tests primarily focus on detecting enzymatic activities related to the fermentation of carbohydrates or the catabolism of proteins or amino acids by the inoculated bacteria.
  • The test procedure involves rehydrating the dehydrated media in each well with a bacterial suspension. After rehydration, the test strip is incubated, allowing the bacteria to metabolize the media components. As the bacteria grow and metabolize, they produce characteristic color changes in the media, which are indicative of specific metabolic reactions.
  • The API 20E Test kit is known for its quick, safe, and easy-to-perform method of bacterial identification. It utilizes extensive databases to provide accurate identifications based on the observed metabolic profiles. The test results are then compared with reference databases to determine the specific bacterial species.
  • API strips, including the API 20E Test, have become a standard tool in microbiological laboratories worldwide, with approximately 10 API strips being used every minute. These strips are also employed to assess the performance of other identification products, making them a reliable and valuable resource in the field of microbial identification and classification.

Principle of APT Test for Bacteria

  • The APT (Analytical Profile Index) test for bacteria, specifically the API 20E Test kit, is a method used to identify and differentiate bacteria belonging to the Enterobacteriaceae family based on their biochemical characteristics.
  • The API range offers a standardized and miniaturized version of traditional identification techniques that were once complex and challenging to interpret. The API 20E test strip consists of twenty mini-test chambers, each containing dehydrated media with chemically defined compositions for individual tests. These tests primarily focus on detecting enzymatic activity, particularly related to carbohydrate fermentation or protein and amino acid catabolism performed by the inoculated bacteria.
  • To initiate the test, a bacterial suspension is used to rehydrate the media in each well of the test strip. The strips are then incubated under suitable conditions. During incubation, the metabolic activity of the bacteria induces color changes in the media. These color changes may occur spontaneously or become apparent upon the addition of specific reagents.
  • After completion of the incubation period, the results of all the tests (both positive and negative) are compiled to generate a profile number, which represents the specific pattern of metabolic reactions exhibited by the bacterial strain. This profile number is then compared with the profile numbers provided in a commercial codebook or logbook (or through an online database) associated with known bacterial species.
  • By matching the profile number with the corresponding identification code, the bacterial species can be determined and identified with a high level of accuracy. The API 20E test simplifies and streamlines the process of bacterial identification, making it a valuable tool in clinical and research settings for precise microbial classification.
API (Analytical Profile Index) Test
API (Analytical Profile Index) Test | Source:

API 20E Test Kit

API 20E is a specialized kit designed for bacterial identification, consisting of 20 distinct tests. These tests aid in characterizing various metabolic activities and enzymatic reactions of the organisms being examined. Each test provides valuable information to differentiate between different bacterial strains. Let’s explore the individual tests in the API 20E kit:

  1. ONPG (o-nitrophenyl-b-D-galactopyranoside) Test: This test assesses the presence of b-galactosidase enzyme by detecting the release of galactose and o-nitrophenol from the substrate ONPG.
  2. ADH (Arginine Dihydrolase) Test: This test involves the decarboxylation of the amino acid arginine by the enzyme arginine dihydrolase.
  3. LDC (Lysine Decarboxylase) Test: Lysine decarboxylase enzyme catalyzes the decarboxylation of the amino acid lysine, and this test helps detect its presence.
  4. ODC (Ornithine Decarboxylase) Test: The enzyme ornithine decarboxylase facilitates the decarboxylation of ornithine, and this test assists in its identification.
  5. CIT (Citrate) Utilization Test: This test determines if the organism can utilize citrate as its sole carbon source.
  6. H2S (Hydrogen Sulfide) Test: The production of hydrogen sulfide is detected in this test.
  7. URE (Urea) Hydrolyzation Test: This test identifies the presence of the enzyme urease, which hydrolyzes urea.
  8. TDA (Tryptophan Deaminase) Test: This test is used to detect the enzyme tryptophan deaminase’s presence, and it involves the use of Ferric Chloride reagent.
  9. IND (Indole) Test: The Indole test assesses the production of indole from tryptophan by the enzyme tryptophanase, using Kovac’s reagent.
  10. VP (Voges-Proskauer) Test: This test helps detect the presence of acetoin (acetyl methylcarbinol) produced during glucose fermentation via the butylene glycol pathway.
  11. GEL (Gelatin) Hydrolysis Test: The presence of the enzyme gelatinase, which liquefies gelatin, is determined in this test.
  12. GLU (Glucose) Fermentation Test: Fermentation of glucose, a hexose sugar, is examined in this test.
  13. MAN (Mannitol) Fermentation Test: This test evaluates the fermentation of mannose, another hexose sugar.
  14. INO (Inositol) Fermentation Test: Fermentation of inositol, a cyclic polyalcohol, is tested in this procedure.
  15. SOR (Sorbitol) Fermentation Test: The fermentation of sorbitol, an alcohol sugar, is assessed.
  16. RHA (Rhamnose) Fermentation Test: This test checks for the fermentation of rhamnose, a methyl pentose sugar.
  17. SAC (Sucrose) Fermentation Test: Fermentation of sucrose, a disaccharide, is examined in this test.
  18. MEL (Melibiose) Fermentation Test: This test determines the fermentation of melibiose, another disaccharide.
  19. AMY (Amygdalin) Fermentation Test: Fermentation of amygdalin, a glycoside, is tested in this procedure.
  20. ARA (Arabinose) Fermentation Test: This final test evaluates the fermentation of arabinose, a pentose sugar.

These 20 tests in the API 20E kit collectively provide a comprehensive profile of bacterial strains, aiding microbiologists in accurate and efficient identification.


Requirements for API (Analytical Profile Index)

  1. API 20E Test Strip and Reagents: The API 20E test strip, along with related reagents like Kovac’s reagent and ferric chloride, are fundamental components of the testing process.
  2. 40% KOH and α-Naphthol: These chemicals play a crucial role in certain API tests and must be available in appropriate concentrations.
  3. Growth of Test Organism: To perform the tests, the test organism should be cultivated in its pure form, isolated from other contaminants. Pure isolated colonies are necessary to avoid interference with the results.
  4. Oxidase Disks: Oxidase disks are essential for identifying bacteria with cytochrome oxidase activity, which aids in differentiation.
  5. Distilled Water: High-quality distilled water is required for various steps of the API testing procedure, ensuring accurate and reliable outcomes.
  6. Pasteur Pipette: A Pasteur pipette is used for precise and controlled transfer of liquids during the testing process.
  7. Sterile Oil: Sterile oil is used to create a layer over test cultures, preventing dehydration and contamination.
  8. Marker: A marker is necessary for labeling and organizing the API test plates or strips.
  9. API Logbook: An API logbook helps in recording essential information about each test, such as organism details, test results, and observations.
  10. Clean and Grease-Free Slides and Coverslips: Slides and coverslips must be thoroughly cleaned and free from grease or any contaminants before use in microscopic examinations.

API Test Procedure

  1. Confirm Culture Identity: Ensure that the bacterial culture under examination belongs to the Enterobacteriaceae family. To verify this, perform a quick oxidase test for cytochrome c oxidase. Note that all members of Enterobacteriaceae are oxidase negative, except Plesiomonas shigelloides, which is oxidase positive.
  2. Prepare Bacterial Suspension: Select a single isolated colony from a pure culture and create a bacterial suspension by mixing it with sterile distilled water.
  3. API 20E Test Strip Setup: Obtain the API 20E Test Strip, which contains 20 separate compartments, each filled with dehydrated bacterial media or biochemical reagents.
  4. Fill Compartments with Bacterial Suspension: Use a Pasteur pipette to fill each compartment with the bacterial suspension up to the brim.
  5. Add Sterile Oil: In compartments labeled ADH, LDC, ODC, H2S, and URE, add sterile oil.
  6. Prepare the Incubation Tray: Place some drops of water in the incubation tray. Then, carefully position the API Test strip in the tray and securely close it.
  7. Marking and Identification: Label the tray with an identification number (Patient ID), date, and initials to ensure proper tracking of the test.
  8. Incubation: Transfer the sealed tray with the API Test strip to an incubator and allow it to incubate for 18 to 24 hours at a temperature of 37°C.
  9. Record Results: After the incubation period, observe the test strip for color changes and record the results for each test compartment.
  10. Interpretation: Compare the observed results with standardized databases or reference materials to identify the bacterial species based on their metabolic profiles.

The API 20E Test procedure enables quick, safe, and accurate identification of bacterial strains belonging to the Enterobacteriaceae family. It relies on the metabolic activities of the bacteria to generate characteristic color changes in the test compartments, aiding in the precise classification of the microorganisms.

API (Analytical Profile Index) Test
API (Analytical Profile Index) Test | Source:

Result Interpretation of API 20E Test

Result Interpretation of API 20E Test
API 20E positive vs negative | Source:

After completing the incubation period, the API 20E Test results are interpreted as follows:

  1. Color Change Observation: Examine the color changes in each compartment of the API 20E Test strip, except for those requiring additional reagents (TDA, IND, and VP). For TDA, add one drop of ferric chloride; for IND, add one drop of Kovac’s reagent; and for VP, add one drop of 40% KOH (VP Reagent 1) and one drop of VP Reagent 2 (α-Naphthol).
  2. API Reading Scale: On the lid of the incubation tray, create an API Reading Scale (color chart) by marking each test compartment as either positive or negative. The wells are grouped into triplets, indicated by black triangles, and each triplet is assigned a specific score.
  3. Scoring the Positive Wells: Add up the scores only for the positive wells within each triplet.
  4. Generate the 7-Digit Number: Combine the scores from three test reactions at a time, creating a 7-digit number. This number corresponds to a specific identification code.
  5. Codebook Lookup: Refer to the API codebook using the generated 7-digit number. The codebook contains a comprehensive list of identification codes linked to different bacterial species.
  6. Organism Identification: Using the API catalog or the online APIWEB™ service, identify the bacterial organism based on the obtained identification code. The database provides a wealth of information to accurately identify the bacteria based on their metabolic profiles.

The API 20E Test’s interpretive process relies on the metabolic activities of the tested bacteria, which generate unique color patterns in the test compartments. By following the API Reading Scale and referencing the appropriate codebook or online database, the test enables precise identification of the bacterial species within the Enterobacteriaceae family. This method simplifies and streamlines the identification process, making it an invaluable tool in clinical and research settings for accurate microbial classification.

API (Analytical Profile Index) Test
API (Analytical Profile Index) Test | Source:

There are several online tools available for reading your API test results. One such website is UPBM, although it’s worth noting that the database may not be up to date. Alternatively, you can use apiwebTM by biomerieux, which provides an updated and reliable database for interpreting your API test data.


Advantages of API Test 

The API (Analytical Profile Index) test offers numerous advantages for the identification and differentiation of microorganisms, including bacteria and yeasts:

  1. Rapid Identification: The API test provides fast and efficient identification of organisms, especially Enterobacteriaceae and other non-fastidious gram-negative bacteria, within a short incubation period of 18 to 24 hours.
  2. Versatility: In addition to bacterial identification, the API test is also useful for fungal identifications, specifically yeasts, expanding its applicability to various microorganisms.
  3. User-Friendly and Standardized: The API test is known for its ease of use and user-friendly nature. It offers a standardized testing approach, ensuring consistent and reliable results across different laboratories and users.
  4. Long Shelf Life: API strips have a long shelf life, allowing laboratories to keep the test kits readily available for use when needed. This is particularly advantageous for medium-level laboratories that may face challenges with limited access to other advanced identification methods like MALDI-TOF-MS and molecular testing due to the absence of thermocyclers.
  5. Comprehensive Databases: The API test relies on extensive databases containing a wealth of information on various microorganisms. These databases enable accurate and precise identifications, aiding in the correct classification of the tested organisms.
  6. Cost-Effective: The API test is a cost-effective solution for microbial identification compared to some more sophisticated and expensive identification techniques. Its affordability makes it accessible to a wider range of laboratories and healthcare facilities.
  7. Reliable Results: Due to its standardized nature and extensive databases, the API test consistently delivers reliable results, reducing the likelihood of misidentification and ensuring better patient care and infection control.

Overall, the API test’s speed, ease of use, and accuracy make it a valuable tool for microbial identification in various laboratory settings, contributing to improved diagnostics and patient outcomes.


Disadvantages of API Test 

  1. Accuracy Limitations: The API 20E test exhibits an accuracy of approximately 90%, indicating a 10% chance of obtaining incorrect results. This margin of error can lead to misidentification and potentially affect patient treatment decisions.
  2. Limited Identification Scope: The API 20E test’s database contains a finite set of bacteria for identification. As a consequence, it cannot identify new or rare bacteria that are not included in its reference database, limiting its applicability in emerging infectious cases.
  3. Time to Results: Obtaining results from the API 20E test can be time-consuming, taking approximately 24 hours. In situations where urgent identification of the bacteria is required, this waiting period may hinder timely intervention and treatment.
  4. Interpretation Challenges: Interpreting API 20E test results may prove challenging, particularly for inexperienced users. Misinterpretation or errors in result analysis can lead to incorrect identification, compromising patient care.
  5. Requirement for Pure Culture: The API 20E test necessitates a pure culture of the bacteria under examination. Isolating a pure culture can be difficult, particularly when the bacteria are present in a mixed culture, potentially impeding accurate testing.
  6. Ineffectiveness for Fastidious Bacteria: The API 20E test is not suitable for identifying fastidious bacteria—organisms with specific and demanding growth requirements. Consequently, certain clinically significant bacteria may remain unidentified, necessitating alternative testing methods.

Despite its advantages, the API test has several limitations that necessitate careful consideration and complementary approaches in clinical and research settings. Users must be aware of these drawbacks to ensure accurate microbial identification and appropriate patient management.

Important Notes

  • Enteric Identification: The API-20E test strip is designed specifically for the identification of Enteric bacteria, which are Gram-negative bacteria belonging to the Enterobacteriaceae family.
  • Limited Identification: The API 20E/NE strip is a rapid identification system that combines certain conventional tests, allowing for the identification of a restricted number of Gram-negative bacteria, both Enterobacteriaceae and non-Enterobacteriaceae.
  • API-Staph Strip: In contrast to the API-20E, the API-Staph strip is tailored for the identification of Gram-positive bacteria, including Staphylococcus species, Micrococcus species, and related organisms.
  • Cost-Effective: The API test strips offer an economical advantage due to their ability to perform multiple tests on a single strip, streamlining the identification process and reducing overall costs.
  • Fungal Identification: API test strips are not limited to bacterial identification alone; they are also applicable to fungi, particularly yeasts. This makes them versatile tools in clinical and research settings for identifying various microorganisms.
  • Standardized Testing: API test strips provide standardized and reliable results, ensuring consistent identification outcomes across different laboratories and users.
  • Complementing Conventional Methods: API test strips complement conventional identification methods, enabling faster and more efficient microbial identification, especially in medium-level laboratories with limited access to advanced techniques.
  • Interpretation Codes: API test strips generate specific identification codes based on the observed metabolic reactions, which can be cross-referenced with extensive databases for accurate microorganism identification.

Uses of API (Analytical Profile Index) 20E Test

The API 20E test serves various important purposes in clinical, healthcare, and research settings, including:

  1. Diagnosis of Infections: The API 20E test is a valuable tool for identifying the specific bacteria responsible for infections. This information is crucial for selecting the appropriate antibiotic therapy, ensuring targeted and effective treatment.
  2. Monitoring Antibiotic Resistance: The API 20E test can be employed to monitor the antibiotic resistance patterns of bacteria. By identifying resistant strains, healthcare professionals can make informed decisions when choosing antibiotics, thereby helping to combat antibiotic resistance and optimize treatment outcomes.
  3. Antibiotic Stewardship: In addition to guiding antibiotic selection, the API 20E test plays a role in antibiotic stewardship programs. By providing accurate and rapid identification of bacteria, it helps in the prudent and responsible use of antibiotics, reducing the risk of unnecessary prescriptions and promoting optimal patient care.
  4. Infection Control: The API 20E test aids in identifying the sources of infections and tracking the spread of specific bacteria within healthcare facilities. This information is vital for implementing effective infection control measures, preventing outbreaks, and safeguarding patient safety.
  5. Taxonomy and Physiology Studies: In the field of research, the API 20E test is utilized to study the taxonomy and physiology of bacteria. The data obtained from these studies improves our understanding of bacterial characteristics, behaviors, and interactions, contributing to advancements in microbiology and the development of new methods for bacterial control.
  6. Microbial Ecology Research: Understanding the microbial ecology of different environments, such as soil, water, and the human body, is essential for various fields, including environmental sciences and medical research. The API 20E test aids in identifying bacterial species in complex ecosystems, shedding light on microbial diversity and ecological interactions.
  7. Epidemiological Studies: In epidemiology, the API 20E test can be employed to identify and track specific bacterial strains in outbreaks or clusters of infections. This helps in investigating the sources and routes of transmission, enabling effective containment and control strategies.

Overall, the API 20E test is a versatile and valuable tool with diverse applications in clinical diagnosis, antibiotic management, infection control, and scientific research. Its rapid and accurate identification capabilities make it an indispensable resource in the fight against infectious diseases and the exploration of the microbial world.

Download Protocols from API Galleries

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API (Analytical Profile Index) 20E Test
API (Analytical Profile Index) 20E Test | Source:

Where You can buy API (Analytical Profile Index) 20E Test Kit?

The API 20E test kit can be purchased from a variety of online and offline retailers. Some of the most popular retailers include:

  • BioMérieux: The manufacturer of the API 20E test kit, BioMérieux, sells the kit directly on their website.
  • VWR: A leading supplier of laboratory products, VWR sells the API 20E test kit on their website. VWR website
  • Fisher Scientific: Another leading supplier of laboratory products, Fisher Scientific sells the API 20E test kit on their website.
  • Sigma-Aldrich: A global supplier of chemicals and laboratory products, Sigma-Aldrich sells the API 20E test kit on their website.
  • Amazon: The API 20E test kit is also available for purchase on Amazon.

When purchasing the API 20E test kit, it is important to make sure that you are buying from a reputable retailer. The kit should come with a valid certificate of analysis, which ensures that the kit has been properly manufactured and tested.

It is also important to note that the API 20E test kit is a medical device. In some countries, you may need a prescription to purchase the kit.


What is the API 20E Test?

The API 20E Test is a biochemical panel designed for the identification and differentiation of bacteria, particularly those belonging to the Enterobacteriaceae family.

How does the API 20E Test work?

The API 20E Test uses a plastic strip with 20 mini-test chambers, each containing dehydrated media and biochemical reagents. Bacterial suspensions are added to the wells, and the strip is incubated to observe metabolic reactions and color changes.

What bacteria can be identified with the API 20E Test?

The API 20E Test can identify a range of Gram-negative bacteria, including Enterobacteriaceae and non-Enterobacteriaceae.

How accurate is the API 20E Test?

The API 20E Test has an accuracy of around 90%. While it is reliable, there is a small chance of obtaining incorrect results.

Can the API 20E Test identify new or rare bacteria?

No, the API 20E Test’s identification is limited to the bacteria present in its reference database. It may not identify new or rare bacterial species.

What is the incubation time for the API 20E Test?

The API 20E Test typically requires incubation for 18 to 24 hours at 37°C to allow bacteria to metabolize the media and produce characteristic color changes.

Is the API 20E Test user-friendly?

Yes, the API 20E Test is designed to be user-friendly and standardized, making it accessible and consistent across different laboratories.

Can the API 20E Test identify fungi as well?

Yes, the API 20E Test can be used for fungal identification, particularly yeasts.

Is a pure culture necessary for the API 20E Test?

Yes, the API 20E Test requires a pure culture of the bacteria for accurate testing, which can be challenging if the bacteria are present in a mixed culture.

What are some alternative tests if API 20E is not suitable for certain bacteria?

For bacteria with specific growth requirements (fastidious bacteria), alternative tests like molecular methods or MALDI-TOF-MS (Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry) can be used for identification.



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