API 20E: Identification of Enterobacteriaceae and Other Gram-Negative Rods

API 20E System

Introduction to API 20E

The API 20E strip is a standardized system designed to identify Enterobacteriaceae and other Gram-negative rods. It utilizes 20 miniature biochemical tests, each contained in a separate microtube, to analyze the metabolic characteristics of bacterial isolates. The results are interpreted using a database to determine the most probable identity of the organism.

• Enterobacteriaceae: A large family of Gram-negative bacteria, including many pathogens such as Escherichia coli and Salmonella.

• Biochemical identification: Based on the ability of bacteria to metabolize specific substrates, producing color changes in the media.

• Applications: Used in clinical, environmental, and food microbiology laboratories for rapid identification of Gram-negative rods.

Supplies Needed for API 20E Testing

To perform the API 20E test, the following materials are required for each bench or group:

• API 20E test strip

• API 20E incubation tray and lid

• Sterile saline (0.85%)

• Inoculating loops or swabs

• Pasteur pipettes

• Oxidase test reagent

• VP reagents 1 and 2

• Indole reagent (Kovac's reagent)

• API 20E results chart

API 20E Procedure

Day 1: Inoculation and Incubation

The API 20E procedure involves inoculating the test strip with a bacterial suspension and incubating it to allow for biochemical reactions to occur.

1. Prepare a bacterial suspension from a pure culture of a Gram-negative rod.

2. Label the API 20E strip with the organism number or code.

3. Dispense the bacterial suspension into each microtube of the strip as directed.

4. Overlay certain tests with mineral oil to create anaerobic conditions (as specified in the instructions).

5. Incubate the strip at 35–37°C for 18–24 hours.

Day 2: Reading and Interpreting Results

After incubation, the results are read by observing color changes in each microtube. Some tests require the addition of reagents before interpretation.

1. Add reagents to specific wells as indicated (e.g., Kovac's for indole, VP reagents for acetoin production).

2. Record the color changes and compare them to the results chart.

3. Interpret the results using the API 20E identification database or codebook.

Interpreting API 20E Results

Positive and Negative Results

Each test in the API 20E strip yields a positive or negative result, typically indicated by a color change. The combination of results produces a numerical profile used for organism identification.

API 20E Results and Interpretations Table

The following table summarizes the main tests in the API 20E strip, the substrate or activity tested, the expected result, and its interpretation:

#	Test	Substrate/Activity	Result	Interpretation
1	ONPG	β-galactosidase production	Yellow	Organism produces β-galactosidase
2	ADH	Arginine dihydrolase	Red/orange	Organism decarboxylates arginine
3	LDC	Lysine decarboxylase	Red/orange	Organism decarboxylates lysine
4	ODC	Ornithine decarboxylase	Red/orange	Organism decarboxylates ornithine
5	CIT	Citrate utilization	Blue	Organism uses citrate as sole carbon source
6	H2S	Hydrogen sulfide production	Black deposit	Organism produces H2S
7	URE	Urease production	Red/pink	Organism hydrolyzes urea
8	TDA	Tryptophan deaminase	Red/brown	Organism deaminates tryptophan
9	IND	Indole production	Red ring	Organism produces indole from tryptophan
10	VP	Acetoin production (Voges-Proskauer)	Red	Organism produces acetoin

Additional info: The table continues with tests for gelatinase, various carbohydrate fermentations, and other enzymatic activities. Each test is interpreted based on a color change or precipitate formation, indicating the presence or absence of specific metabolic pathways.

Summary and Applications

The API 20E system is a rapid, reliable method for identifying Gram-negative rods, especially members of the Enterobacteriaceae family. It is widely used in clinical diagnostics, food safety testing, and environmental microbiology. Accurate identification aids in disease diagnosis, epidemiological studies, and selection of appropriate antimicrobial therapy.