Analytical Profile Index (API) Test: Procedure, Principle & Numerical Profile Explained

Table of Content:

• Introduction to Analytical Profile Index (API) Test
• The Principle of Analytical Profile Index (API) Test
• Common Types of API Systems
• Biochemistry Tests in API Strips
• Procedure (Example: API 20E)
• Interpretation and Reading
• The Numerical Profile (The Coding System)
• Advantages and Disadvantages
• Mock Calculation Exercises
• A. API 20E (Enterobacteriaceae Example)
• B. API 20NE (Non-Enteric Example)
• C. API 10S (Rapid Screening Example)
• Conclusion

Introduction to Analytical Profile Index (API) Test

• The Analytical Profile Index (API) is a standardized, miniaturized system used for the biochemical identification of bacteria and some yeasts.
• It consists of a series of small, dehydrated biochemical substrates arranged in a plastic strip, each designed to test a specific metabolic or enzymatic property of the microorganism.
• The API system was developed by bioMérieux, a leading company in diagnostic microbiology, and is now a globally used tool for microbial identification.
• This system provides a rapid, reliable, and cost-effective alternative to traditional biochemical testing methods, which often require multiple media, larger volumes, and longer incubation times.
• Each API strip is tailored to a specific microbial group (e.g., Enterobacteriaceae, non-fermenters, Gram-positive cocci, anaerobes, or yeasts), ensuring precise and targeted identification.
• The tests are interpreted based on color changes after incubation, and the resulting profile is converted into a numerical code known as an API profile number.
• This profile number is matched against an extensive database to accurately determine the genus and species of the microorganism.
• API systems are commonly used in clinical diagnostics, food microbiology, water testing, pharmaceutical quality control, and research laboratories.
• They require only a small inoculum, making them suitable when sample quantity is limited.
• The entire process is user-friendly, reproducible, and can deliver final identification results within 18–24 hours depending on the strip type.
• Because of its simplicity and standardized nature, the API system is widely used for teaching microbiology and training laboratory personnel.

 

The Principle of Analytical Profile Index (API) Test

• The Analytical Profile Index (API) test works on the principle of evaluating multiple biochemical reactions performed simultaneously in a compact plastic strip.
• Each API strip contains a series of micro-cupules (tiny wells) filled with dehydrated biochemical substrates, each designed to test a specific metabolic or enzymatic activity of the organism.
• Rehydration: When a standardized bacterial suspension is inoculated into these wells, the dehydrated substrates become rehydrated, allowing biochemical reactions to begin.
• Metabolism: If the inoculated microorganism produces the enzymes required to utilize, degrade, or modify the substrates in a specific well, a metabolic reaction occurs. This can involve carbohydrate fermentation, amino acid breakdown, enzyme production, or utilization of specific compounds.
• Some tests may require anaerobic conditions, which are achieved by overlaying certain wells with mineral oil to block oxygen exposure.
• Visualization: As the reactions proceed during incubation, they produce visible color changes that indicate positive or negative results.
• In some wells, reagents must be added after incubation to reveal the final color reaction (e.g., Kovac’s reagent for indole, ferric chloride for phenylalanine deaminase).
• The pattern of color changes forms a unique biochemical profile for the organism and is later converted into a seven-digit or nine-digit API code, depending on the strip type.
• This code is compared with an established database to determine the most probable identity of the microorganism.
• The principle relies on the fact that each bacterial species has a distinctive metabolic fingerprint, making the API system a reliable method for microbial identification.

Common Types of API Systems

• Different API systems are designed to target specific groups of microorganisms based on their biochemical characteristics and metabolic pathways.
• Each strip contains a defined set of tests optimized to identify organisms within a particular taxonomic or physiological group, ensuring more accurate identification.
• Common API systems used in microbiology laboratories include:
• API 20E: Enterobacteriaceae and other non-fastidious Gram-negative rods.
• API 20NE: Non-enteric Gram-negative rods such as Pseudomonas, Acinetobacter, Moraxella.
• API 10S: Simplified version for Enterobacteriaceae, containing 10 essential biochemical tests.
• API Staph: Designed for identification of Staphylococcus and Micrococcus species, focusing on enzymatic and carbohydrate metabolism.
• API Strep: Used for Streptococcus, Enterococcus, and related genera through tests like enzyme activity and carbohydrate utilization.
• API 20A: Targets anaerobic bacteria, including Clostridium, Bacteroides, and others requiring oxygen-free conditions.
• API Candida: Used for the identification of medically important yeasts such as Candida albicans, Candida tropicalis, and Cryptococcus species.
• The choice of API strip depends on the Gram reaction, morphology, and preliminary biochemical traits observed during routine laboratory workup.

In-Depth Look at Key Strips

API 20E:

• One of the most widely used identification systems for Enterobacteriaceae.
• Tests focus mainly on carbohydrate fermentation, enzyme production (e.g., decarboxylases, deaminases), and substrate utilization.
• Commonly used for identifying organisms such as E. coli, Klebsiella, Enterobacter, Salmonella, and Shigella.
• Produces results typically within 18–24 hours.

API 20NE:

• Specially designed for non-enteric, oxidase-positive or oxidase-negative non-fermenting Gram-negative rods like Pseudomonas aeruginosa and Acinetobacter baumannii.
• Includes assimilation tests that determine whether a microbe can use a compound as its sole carbon source, a key feature separating non-fermenters from enteric bacteria.
• Incubation may extend up to 48 hours due to slower metabolic reactions.

API 10S:

• A compact version of API 20E consisting of only 10 essential biochemical tests.
• Often used for rapid screening, teaching, or settings where a full biochemical panel is unnecessary.
• Useful for basic identification of Enterobacteriaceae when time, workload, or resources are limited.

Biochemistry Tests in API Strips

A) Basic Tests

• ONPG (Ortho-nitrophenyl-β-D-galactopyranoside)
• Substrate similar to lactose; does not require permease to enter bacterial cell.
• Reaction: ONPG → galactose + ortho-nitrophenol (yellow/pale yellow).
• Indicator: B-galactosidase activity.
• Result: Positive = yellow; Negative = colorless.
• ADH (Arginine Dihydrolase)
• Reaction: Arginine → Citrulline + NH₃ → Ornithine + carbamoyl phosphate → ATP + CO₂ + NH₃.
• Indicator: Phenol red (orange within first 24h = positive).
• Enzyme: Catabolic arginine carbamoyltransferase, carbamoyl kinase.
• pH Range: 6.8–8.4.
• Result: Positive = orange, Negative = no color change.
• LDC (Lysine Decarboxylase)
• Reaction: Lysine → Cadaverine + CO₂ (pH increase).
• Indicator: Phenol red.
• Result: Positive = color change (usually purple), Negative = no change.
• ODC (Ornithine Decarboxylase)
• Reaction: Ornithine → Putrescine + CO₂ (pH increase).
• Indicator: Phenol red.
• Result: Positive = color change, Negative = no change.
• CIT (Citrate Utilization Test)
• Reaction: Sodium citrate → Oxaloacetic acid → Pyruvate + CO₂ + NH₃ → Alkalinization.
• Indicator: Bromothymol blue (yellow pH 6 → green 6.9 → blue 7.6).
• Enzyme: Citrate lyase, OAA decarboxylase.
• Result: Positive = blue/green, Negative = yellow.
• H₂S Production
• Substrate: Sodium thiosulfate.
• Reaction: H₂S gas + Fe²⁺ → Ferrous sulfide (black precipitate).
• Result: Positive = black precipitate, Negative = no color.
• URE (Urease Test)
• Reaction: Urea → 2 NH₃ + CO₂.
• Works in both aerobic and anaerobic conditions (prefers anaerobic).
• Indicator: Phenol red.
• Result: Positive = pink, Negative = no change.
• TDA (Tryptophan Deaminase)
• Reaction: Tryptophan → Indole-pyruvic acid + NH₃ → Ferric hydrazone with FeCl₃ + HCl.
• Note: FeCl₃ + HCl prevents false positives.
• Result: Positive = reddish-brown precipitate, Negative = no color.
• IND (Indole Test)
• Reaction: Tryptophan → Indole + pyruvic acid + NH₃.
• Reagent: Kovac’s reagent.
• Result: Positive = red/pink ring, Negative = no color.
• VP (Voges–Proskauer Test)
• Reaction: Sodium pyruvate → 2,3-butanediol → Acetoin.
• Reagents: Barritt’s A (α-naphthol) + B (KOH).
• Result: Positive = red color within 10 min, Negative = no change.
• GEL (Gelatin Liquefaction Test)
• Reaction: Gelatin → Amino acids (gelatinase activity).
• Result: Positive = liquefied gelatin, Negative = solid.
• Sugar Fermentation / Oxidation Tests
• Fermentation (Enterobacteriaceae, Aeromonas, Vibrio):
• Reactions start at the anaerobic bottom → read from bottom to top.
• Yellow at bottom = weak/delayed positive.
• Oxidation (Other Gram-negatives):
• Reactions start at the aerobic top → read top to bottom.
• Yellow top + blue bottom = oxidative utilization → positive only for non-Enterobacteriaceae.

 

B) Supplementary Tests (Needed Only with Multi-Taxon Codes)

• Oxidation Test Tube
• Medium: 1% glucose + nutrient agar + bromothymol blue.
• Reaction: Detects oxidative carbohydrate utilization.
• Fermentation Test Tube
• Medium: 1% glucose + nutrient agar + bromothymol blue + sterile mineral oil.
• Reaction: Detects fermentative carbohydrate utilization.

Note: If supplementary tests are used, delay reading of all results to 48 hours to allow complete development of reactions.

Procedure (Example: API 20E)

A. Preparation

• Ensure a well-isolated, pure culture of the organism is obtained on a fresh agar plate, as mixed cultures will produce incorrect biochemical profiles.
• Select a single, well-defined colony using a sterile loop or needle to avoid contamination.
• Prepare a bacterial suspension by emulsifying the colony in sterile saline (0.85% NaCl) or sterile distilled water, ensuring the mixture reaches the recommended turbidity (typically equivalent to a 0.5 McFarland standard).
• Proper turbidity is essential because an overly dense or dilute suspension can alter the intensity of biochemical reactions, affecting the accuracy of the final API code.
• Mix the suspension thoroughly to ensure an even distribution of bacterial cells before inoculation.

B. Inoculation

• Place the API 20E strip into the plastic incubation tray, ensuring that distilled water is added to the bottom of the tray to maintain a humid environment during incubation. This prevents the reactions from drying out.
• Use a sterile pipette to carefully dispense the prepared bacterial suspension into the tube portion of each micro-cupule. Avoid introducing bubbles, as they can interfere with proper rehydration.
• For tests requiring anaerobic conditions, apply a layer of sterile mineral oil over the cupule after filling the tube. These typically include ADH, LDC, ODC, H₂S, and URE tests. The oil prevents oxygen exposure, allowing anaerobic reactions to proceed correctly.
• For tests requiring aerobic conditions, fill only the tube portion and leave the cupule exposed to the air. This allows oxygen-dependent biochemical reactions to occur naturally.
• Ensure all wells are properly filled and that no overflow or cross-contamination occurs between adjacent cupules.
• Gently close the incubation tray lid to maintain a stable microenvironment throughout the incubation period.

C. Incubation

• Incubate the inoculated API strip at 37°C, which is the optimal temperature for most Enterobacteriaceae and other common Gram-negative rods tested with API 20E.
• Maintain the strip inside the closed incubation tray to preserve humidity and prevent evaporation of the biochemical reactions.
• Ensure the tray is placed in an incubator with stable temperature control, as fluctuations may slow down or alter metabolic reactions.
• Standard incubation time ranges from 18 to 24 hours, after which most biochemical reactions will have developed sufficiently to be interpreted.
• Some reactions may appear earlier, but reading the results before the recommended time can lead to false or incomplete reactions.
• If certain tests require delayed reading (such as VP), follow the manufacturer’s instructions for timing and reagent addition after incubation.
• Once incubation is complete, proceed immediately to add required reagents and interpret the results to prevent over-incubation, which may cause color fading or false positives.

Interpretation and Reading

• After completing the incubation period, each micro-cupule is examined for visible color changes that indicate whether the biochemical reaction is positive or negative.
• The color changes reflect specific metabolic activities of the bacterium, such as fermentation, enzyme production, decarboxylation, deamination, or compound utilization.
• Spontaneous Reactions:
• Some tests develop their final color without the need for additional reagents.
• For example, the GLU (glucose fermentation) test turns yellow when acid is produced, indicating carbohydrate fermentation.
• Other spontaneous reactions include ONPG, ADH, LDC, ODC, CIT, H₂S, and URE, each with characteristic color outcomes.
• Reagent-Dependent Reactions:
• Certain tests cannot be interpreted until specific reagents are added after incubation.
• These reagents interact with metabolic end products created by the bacteria to produce the final detectable color.
• TDA (Tryptophan Deaminase):
• Add Ferric Chloride (FeCl₃) to the well.
• A positive reaction appears as a brown to reddish-brown color due to the formation of ferric complexes.
• IND (Indole Test):
• Add Kovac’s reagent.
• A positive result produces a red ring at the surface, indicating degradation of tryptophan to indole.
• VP (Voges–Proskauer Test):
• Add Barritt’s Reagent A (Alpha-naphthol) followed by Reagent B (KOH).
• A positive result develops a pink to red color, confirming acetoin production via the butanediol fermentation pathway.
• Each color reaction should be interpreted within the recommended time frame, as delayed reading may cause colors to fade, intensify, or shift, leading to incorrect identification.
• After all wells are interpreted, the positive and negative reactions are recorded to generate the final numerical profile for organism identification.

The Numerical Profile (The Coding System)

• One of the defining advantages of the API system is its ability to convert numerous biochemical test results into a single, easy-to-interpret numerical code.
• This coding system transforms qualitative outcomes (positive or negative reactions) into quantitative values, creating a standardized identification method.
• The coding is based on the triplet system, in which the 20 biochemical tests are arranged into groups of three consecutive tests.
• Each test within a triplet is assigned a numerical value if the reaction is positive:
• The first test in the triplet has a value of 1.
• The second test has a value of 2.
• The third test has a value of 4.
• If a test is negative, it receives a value of 0.
• The sum of the values for each triplet generates a single digit, and these digits collectively form the seven-digit (or longer) API profile code.
• This conversion system ensures that each unique biochemical pattern corresponds to a specific numerical signature, making microbial identification simple and reproducible.

Example Calculation:

• Tests in one triplet: ONPG (+), ADH (+), LDC (–)
• Values assigned:
• ONPG (+) → 1
• ADH (+) → 2
• LDC (–) → 0
• Summation:
• 1 + 2 + 0 = 3
• This value becomes the first digit of the final API profile code.
• After all triplets are calculated, the resulting numerical code is either checked in the API codebook, compared with the reference index, or entered into the APIweb software.
• APIweb uses a large database of biochemical profiles to provide the most probable bacterial species, along with confidence percentages and alternative identifications if applicable.
• This standardized system significantly reduces human error and enhances accuracy in microbial identification.

 

Advantages and Disadvantages

Advantages

• Standardized:
• API strips are manufactured under strict quality control, ensuring consistent results across different laboratories, countries, and personnel.
• This standardization improves inter-laboratory comparability and reduces variability seen in conventional biochemical tests.
• Fast Identification:
• Most organisms can be identified within 18–24 hours, which is significantly quicker than traditional biochemical tube methods that may take several days.
• Rapid turnaround time is especially useful in clinical diagnostics, food safety testing, and quality control.
• Comprehensive Testing:
• A single API strip evaluates multiple biochemical properties simultaneously, giving a broad metabolic profile of the organism.
• This allows the identification of organisms that require many tests, saving time and resources.
• Long Shelf Life:
• The dehydrated substrates in each cupule remain stable for extended periods without the need for refrigeration.
• This makes storage easy, reduces waste, and ensures strips are ready for use whenever needed.
• User-Friendly and Minimal Equipment Required:
• Only basic tools like saline, a pipette, and an incubator are needed, making it ideal for small labs, teaching labs, and resource-limited settings.
• The interpretation system (numerical coding) simplifies identification.

 

Disadvantages

• Cost:
• API strips are more expensive per test compared to conventional biochemical tubes or in-house media preparation.
• This may be a concern for high-volume laboratories or institutions with limited budgets.
• Requirement for Pure Culture:
• Mixed cultures cannot be tested, as even a small contamination can alter the biochemical profile and produce incorrect identification.
• Additional steps (subculturing, streaking) are needed to ensure purity.
• Database Limitations:
• Identification is restricted to organisms that exist in the API database.
• Rare, newly discovered, or atypical strains may give low confidence scores or no match.
• Subjective Interpretation:
• Some color changes can be faint, ambiguous, or variable depending on incubation time and lighting conditions.
• Misreading colors may lead to incorrect numerical codes and false identifications.
• Certain Organisms Require Longer or Special Conditions:
• Some bacteria (especially non-fermenters or slow-growing organisms) may require extended incubation times or additional confirmatory tests.

 

Mock Calculation Exercises

• These exercises illustrate how to calculate the final numerical profile for different API strips.

A. API 20E (Enterobacteriaceae Example)

• Scenario: Identification of an Enterobacteriaceae organism.
• Profile: 7 digits (6 triplets + 1 pair)

Triplet Group	Test Name	Result (+/-)	Assigned Value
Triplet 1	ONPG	+	1
	ADH	-	0
	LDC	+	4
Triplet 2	ODC	+	1
	CIT	-	0
	H2S	-	0
Triplet 3	URE	-	0
	TDA	+	2
	IND	+	4
Triplet 4	VP	-	0
	GEL	+	2
	GLU	+	4
Triplet 5	MAN	+	1
	INO	-	0
	SOR	+	4
Triplet 6	RHA	-	0
	SAC	+	2
	MEL	+	4
Pair 7	AMY	-	0
	ARA	+	2

• Calculation:
• Digit 1: 1 + 0 + 4 = 5
• Digit 2: 1 + 0 + 0 = 1
• Digit 3: 0 + 2 + 4 = 6
• Digit 4: 0 + 2 + 4 = 6
• Digit 5: 1 + 0 + 4 = 5
• Digit 6: 0 + 2 + 4 = 6
• Digit 7: 0 + 2 = 2
• Final API 20E Profile: 5 1 6 6 5 6 2 → This suggests E. coli.

 

B. API 20NE (Non-Enteric Example)

• Scenario: Identification of a Pseudomonas-like organism.
• Profile: 7 digits (6 triplets + 1 pair)

Triplet Group	Test Name	Result (+/-)	Assigned Value
Triplet 1	NO3	+	1
	TRP	-	0
	GLU (Ferm)	-	0
Triplet 2	ADH	+	1
	URE	-	0
	ESC	-	0
Triplet 3	GEL	+	1
	PNPG	-	0
	GLU (Assimil)	+	4
Triplet 4	ARA	-	0
	MNE	-	0
	MAN	-	0
Triplet 5	NAG	-	0
	MAL	-	0
	GNT	+	4
Triplet 6	CAP	+	1
	ADI	+	2
	MLT	-	0
Pair 7	CIT	+	1
	PAC	+	2

• Calculation:
• Digit 1: 1 + 0 + 0 = 1
• Digit 2: 1 + 0 + 0 = 1
• Digit 3: 1 + 0 + 4 = 5
• Digit 4: 0 + 0 + 0 = 0
• Digit 5: 0 + 0 + 4 = 4
• Digit 6: 1 + 2 + 0 = 3
• Digit 7: 1 + 2 = 3
• Final API 20NE Profile: 1 1 5 0 4 3 3 → This suggests Pseudomonas aeruginosa.

 

C. API 10S (Rapid Screening Example)

• Scenario: Quick identification of Enterobacteriaceae.
• Profile: 4 digits (3 triplets + 1 single)

Triplet Group	Test Name	Result (+/-)	Assigned Value
Triplet 1	ONPG	+	1
	GLU	+	2
	ADH	-	0
Triplet 2	LDC	-	0
	ODC	+	2
	CIT	-	0
Triplet 3	H2S	-	0
	URE	-	0
	TDA	-	0
Single 4	IND	-	0

• Calculation:
• Digit 1: 1 + 2 + 0 = 3
• Digit 2: 0 + 2 + 0 = 2
• Digit 3: 0 + 0 + 0 = 0
• Digit 4: 0 = 0
• Final API 10S Profile: 3 2 0 0 → This suggests Shigella sonnei.

 

Conclusion

• What does API stand for?
• Analytical Profile Index
• What is the most common API strip?
• API 20E (used for Enterobacteriaceae)
• What is API 20NE used for?
• Identification of non-enteric Gram-negative rods (e.g., Pseudomonas, Acinetobacter)
• What is the main difference in 20NE tests?
• It includes assimilation tests (ability to use compounds as a carbon source), not just fermentation.
• Why is mineral oil used in some wells?
• To create anaerobic conditions for reactions that are inhibited by oxygen.
• How is a bacterium identified using API?
• By generating a numerical profile code based on positive/negative results and comparing it with the API database or APIweb software.