Spot Indole Test – Principle, Procedure, Result, Uses

What is Spot Indole Test?

  • The Spot Indole Test is a swift method employed for the detection of indole production in bacteria. This test is rooted in the principle that certain bacteria, when cultivated in a medium enriched with tryptophan, have the capability to produce indole. This production is a result of the bacterial metabolism of tryptophan, an essential amino acid.
  • Bacteria that possess the enzyme tryptophanase can enzymatically degrade tryptophan. This degradation process involves the deamination and hydrolysis of tryptophan, leading to the formation of three distinct end products: indole, pyruvate, and ammonium. Among these, indole serves as a key marker, allowing researchers and laboratory professionals to ascertain the metabolic activity related to tryptophan.
  • The significance of the indole test extends beyond mere detection. It plays a pivotal role in the IMViC test series, where it stands as the inaugural test. Furthermore, the indole test is instrumental in distinguishing members within the Enterobacteriaceae family, a diverse group of bacteria with varied metabolic capabilities.
  • Laboratory execution of the indole test can be approached via two primary methodologies: the tube method and the spot indole test method. The former involves the inoculation of sample bacteria into a test tube containing a tryptophan-rich medium.
  • After an incubation period, typically spanning 24 hours under standard conditions, specific reagents are introduced to the medium to ascertain the presence or absence of indole. On the other hand, the spot indole test method offers a more direct approach, wherein bacteria grown in a tryptophan-rich environment are directly subjected to an Indole Reagent, facilitating immediate detection of indole production.
  • In conclusion, the Spot Indole Test stands as a vital tool in the realm of microbiology, offering rapid and precise insights into bacterial metabolism, specifically concerning the enzymatic degradation of tryptophan. Its application is crucial for both academic research and clinical diagnostics, ensuring accurate differentiation and identification of bacteria based on their metabolic profiles.

Definition of Spot Indole Test

The Spot Indole Test is a rapid biochemical method used to detect the production of indole by bacteria, indicating their ability to metabolize the amino acid tryptophan. This test aids in differentiating specific bacterial species based on their tryptophan degradation capabilities.

Objectives of Spot Indole Test

The Spot Indole Test, a pivotal biochemical assay, is underpinned by specific objectives that guide its application in microbiological research and diagnostics. These objectives are:

  1. Tryptophan Hydrolysis Differentiation: One of the primary objectives of the Spot Indole Test is to discern bacteria based on their enzymatic capability to hydrolyze the amino acid tryptophan. This hydrolysis results in the production of indole, a key metabolic marker.
  2. Identification within Enterobacteriaceae: The test is also instrumental in distinguishing and pinpointing specific members of the Enterobacteriaceae family. This family encompasses a broad spectrum of bacteria, and the ability to produce indole serves as a distinguishing metabolic trait for certain members.

In essence, the Spot Indole Test offers a precise and efficient means to categorize bacteria based on specific metabolic activities, particularly those related to tryptophan degradation. This categorization is crucial for both academic research and clinical diagnostic endeavors, ensuring accurate bacterial identification and differentiation.

Principle of Spot Indole Test

The Spot Indole Test is a diagnostic method grounded in the biochemical capabilities of certain bacteria to produce an enzyme named tryptophanase. This enzyme facilitates the enzymatic hydrolysis of the amino acid, tryptophan, which is typically found in specific culture mediums. As a consequence of this enzymatic reaction, there is a resultant production of three primary compounds: indole, pyruvic acid, and ammonia.


A salient feature of this test is the detection of the produced indole. When indole interacts with an aldehyde present in the diagnostic reagent, a characteristic color change is observed. Specifically, the formation of a green to blue hue is indicative of the presence of the cinnamaldehyde reagent. Conversely, the manifestation of a pink to violet-red color suggests the utilization of an aldehyde reagent.

The precision of the Spot Indole Test is rooted in its ability to identify bacteria based on their capability to produce tryptophanase and, subsequently, indole. The observed color shifts provide reliable and instantaneous results, making it an invaluable tool in the domain of microbiological research and diagnostics. The test embraces a balance between scientific rigor and pragmatic simplicity, ensuring accurate results devoid of ambiguity. This method not only elucidates the metabolic pathways of bacteria but also serves as an efficient means of distinguishing between tryptophanase-producing and non-producing bacterial strains.


Requirements for Spot Indole Test

The Spot Indole Test is a specialized diagnostic approach necessitating particular tools, reagents, and organisms to ensure its precision. This test is imperative for discerning specific bacterial metabolic properties. Below are the comprehensive requirements for conducting this assay:

1. Culture Medium: Contrary to some assays, the rapid spot indole test does not employ any specific culture medium.


2. Reagents:

  • 5% p-Dimethylaminobenzaldehyde: Formed by dissolving 5 grams of p-dimethylamino benzaldehyde in a solution comprising 10 mL of concentrated hydrochloric acid (HCl) and 90 mL of distilled water.
  • 1% p-Dimethylaminocinnamaldehyde: This is prepared by adding 1 gram of p-Dimethylaminocinnamaldehyde to a solution that contains 10 mL of concentrated HCl and 90 mL of distilled water. This reagent is particularly employed for testing both aerobic and anaerobic organisms.

3. Equipment:

  • Filter Paper: Vital for the placement of bacterial samples during the test.
  • Dropper: Utilized to add the necessary reagents to the sample.
  • Cotton Swab: Enables the collection and transfer of bacterial cultures.
  • Inoculating loop: Essential for picking and streaking bacterial colonies.

4. Test Organisms (Sample Bacteria): For the test to be effective, a fresh culture of bacteria, aged between 18 to 24 hours and cultivated on a tryptophan-rich medium, is vital.

5. Control Organisms: To ensure the credibility of the test results, several control organisms are required. These include:

  • Escherichia coli ATCC 25922: A standard control to verify the presence of indole.
  • Pseudomonas aeruginosa ATCC 27853: Typically used as a negative control.
  • Porphyromonas asaccharolytica ATCC 25260: Another control to validate the assay’s specificity.
  • Bacteroides fragilis ATCC 25285: Essential for contrasting with test organisms to determine specific metabolic properties.

In conclusion, the meticulous adherence to these requirements ensures the validity and reproducibility of the Spot Indole Test, reinforcing its position as a reliable diagnostic tool in microbiological studies.

Procedure of Spot Indole Test

The Spot Indole Test, a crucial microbiological assay, can be executed via any of the subsequent three methodologies, each meticulously designed to ascertain the presence of indole, indicative of the bacterial ability to metabolize tryptophan:

1. Filter Paper Method:

  • Begin by moistening a strip of filter paper with the designated indole reagent.
  • Employing a sterile inoculating loop, carefully collect fresh colonies that have been cultured for a duration spanning 18 to 24 hours in a tryptophan-rich medium.
  • Gently rub the acquired colonies over the previously moistened filter paper segment.
  • Within a timeframe of approximately 20 seconds, keenly observe the filter paper for any discernible color alterations.

2. Swab Method:

  • Initiate the procedure by using a sterile swab to obtain fresh colonies, ensuring these colonies have been nurtured for 18 to 24 hours within a tryptophan-rich medium.
  • Proceed to administer a drop of the indole reagent directly onto the colonies.
  • Again, a critical observation phase ensues, wherein any color modifications should be discerned within a 20-second interval.

3. Direct Agar Plate Method:

  • Directly apply the indole reagent over the fresh colonies present on the agar plate, ensuring the colonies have been cultivated in a tryptophan-conducive medium.
  • Within a window of 20 seconds, attentively inspect both the colonies and the adjacent medium for any notable shifts in coloration.

For each methodology, the manifestation of specific color changes serves as a robust indicator of the bacterial strain’s enzymatic capabilities, particularly its proficiency in tryptophan hydrolysis.

Result and Interpretation of Spot Indole Test

The Spot Indole Test, grounded in its biochemical underpinnings, provides clear and definitive results based on the presence or absence of color changes in the test samples. The interpretation of these results is pivotal for accurate bacterial identification, especially concerning their metabolic proficiency related to tryptophan hydrolysis.

  • Positive Test Result: A positive result is evidenced by the emergence of distinct colorations:
    • The manifestation of a blue or pink to red hue on the filter paper.
    • Observation of a blue or pink to red tint on the cotton swab.
    • Development of a blue or pink to red shade over the colonies that have been treated with the indole reagent.
  • The specific color observed is contingent upon the type of indole reagent utilized in the test:
    • Utilization of 5% p-Dimethylaminobenzaldehyde culminates in the evolution of a pink to violet-red shade.
    • Deployment of 1% p-Dimethylaminocinnamaldehyde leads to the appearance of a green to blue coloration.
  • Negative Test Result: A negative outcome is unequivocally signified by the absence of any color alterations. Specifically:
    • No discernible color change in the filter paper.
    • Absence of color development in the cotton swabs.
    • Lack of any color transition in the colonies even after their exposure to the indole reagent.

Such a result suggests the bacterial strain under scrutiny lacks the enzymatic capability to hydrolyze tryptophan, thereby not producing indole.

Quality Control

Ensuring accuracy and reliability in microbiological assays necessitates stringent quality control measures. These measures involve the use of specific microbial strains, both to validate the positive and negative outcomes of a test and to ascertain that the methods employed are both sensitive and specific.

Positive Controls: Positive controls are essential to affirm that a given test is functioning optimally and is capable of detecting the target microorganism when present. In the context of the indole test:

  • Escherichia coli ATCC 25922 serves as a standard for aerobic culture control, consistently delivering a positive result.
  • Porphyromonas asaccharolytica ATCC 25260 is employed as an anaerobic culture control, and it too yields a positive outcome, confirming the test’s capability to identify anaerobic bacteria proficient in tryptophan hydrolysis.

Negative Controls: Negative controls, on the other hand, are instrumental in validating that the test does not falsely indicate the presence of the target organism when it is not actually present.

  • Pseudomonas aeruginosa ATCC 27853 is utilized as an aerobic culture control, and it invariably produces a negative result, underscoring the specificity of the assay.
  • Bacteroides fragilis ATCC 25285 functions as the anaerobic culture control for negative outcomes, ensuring that the test remains accurate in differentiating anaerobic strains that do not hydrolyze tryptophan.

Incorporating both positive and negative controls in the testing regimen is paramount to achieving a balance between sensitivity and specificity, ensuring that results are both trustworthy and reproducible.

Spot Indole Positive Bacteria & Spot Indole Negative Bacteria

The Spot Indole Test, a fundamental assay in microbiology, aids in the differentiation and identification of bacteria based on their enzymatic capability to hydrolyze tryptophan, thereby producing indole. Herein are the outcomes for several common bacteria:

Indole Positive Bacteria: These bacterial strains possess the capability to produce indole from tryptophan, hence rendering a positive result in the Spot Indole Test.

  • Escherichia coli
  • Klebsiella oxytoca
  • Vibrio cholerae
  • Proteus vulgaris
  • Porphyromonas asaccharolytica
  • Vibrio spp.
  • Flavobacterium spp.
  • Providencia spp.
  • Enterococcus faecalis
  • Haemophilus influenzae
  • Morganella morganii
  • Aeromonas spp.
  • Citrobacter koseri

Indole Negative Bacteria: These bacterial species do not have the ability to hydrolyze tryptophan into indole, resulting in a negative outcome in the Spot Indole Test.

  • Klebsiella pneumoniae
  • Proteus mirabilis
  • Salmonella spp.
  • Shigella spp.
  • Citrobacter freundii
  • Pseudomonas aeruginosa
  • Bacteroides fragilis
  • Staphylococcus aureus

In summary, the Spot Indole Test serves as a robust tool, facilitating the categorization of bacteria into indole-positive and indole-negative groups, thereby streamlining the identification process in microbiological investigations.

Spot Indole Test Results
Indole Positive BacteriaIndole Negative Bacteria
Escherichia coliKlebsiella pneumoniae
Klebsiella oxytocaProteus mirabilis
Vibrio choleraeSalmonella spp.
Proteus vulgarisShigella spp.
Porphyromonas asaccharolyticaCitrobacter freundii
Vibrio spp.Pseudomonas aeruginosa
Flavobacterium spp.Bacteroides fragilis
Providencia spp.Staphylococcus aureus
Enterococcus faecalis
Haemophilus influenzae
Morganella morganii
Aeromonas spp.
Citrobacter koseri


In the realm of microbiology, meticulous adherence to precautions is paramount to ensure the accuracy and reliability of results, especially when conducting the Spot Indole Test. Below are the fundamental precautions to be observed:

  1. Selection of Bacterial Culture: It is imperative to utilize bacteria that have been cultivated in a tryptophan-rich medium. The presence of tryptophan is vital for the enzymatic action leading to indole production, the detection of which forms the crux of this test.
  2. Timeliness in Observation: Once the indole reagent is added to the sample, the results must be diligently read within a precise timeframe of 20 seconds. Delays can potentially compromise the accuracy of the observation, leading to misleading outcomes.
  3. Choice of Medium: Mediums that incorporate dyes or indicators, such as EMB (Eosin Methylene Blue) or McConkey, should be stringently avoided. These dyes might interfere with the color changes observed during the test, rendering the results ambiguous.
  4. Reagent Selection based on Bacterial Aerotolerance: The choice of reagent is contingent upon the oxygen requirements of the bacterial species under scrutiny:
    • For anaerobic bacteria, which thrive in oxygen-devoid environments, the 1% p-Dimethylaminocinnamaldehyde reagent is recommended.
    • Conversely, aerobic bacteria, which necessitate oxygen for their metabolic processes, should be tested using the 5% p-Dimethylaminobenzaldehyde reagent.

By assiduously adhering to these precautions, the Spot Indole Test can yield results of the utmost accuracy, bolstering its significance in microbiological diagnostics.

Applications of Limitations of Spot Indole Test

  1. Guiding Further Testing: By acknowledging that the Spot Indole Test is not a confirmatory test, one can recognize the need for supplementary biochemical assays. This ensures a comprehensive and definitive bacterial identification, minimizing diagnostic errors.
  2. Improving Methodology: Being aware that the efficiency of the Spot Indole Test is lower than the tube indole test might lead researchers and clinicians to develop more refined or improved versions of the test.
  3. Informed Decisions on Sample Preparation: Understanding the necessity of a fresh culture grown in a tryptophan-rich medium means that preparatory steps can be taken in advance, ensuring that the test’s prerequisites are met.
  4. Avoiding False Positives: Recognizing that indole diffusion in the culture medium can lead to false positives allows laboratory technicians to interpret results with caution, especially when performing the test directly on an agar plate.
  5. Preparation for Fastidious Organisms: Being aware of the need for a heavy inoculum for certain organisms means that adequate sample sizes can be prepared, ensuring accurate testing for these specific bacteria.
  6. Educating Users: The limitations can be used as educational points for new laboratory technicians or students in microbiology to emphasize the importance of understanding the constraints of the methods they employ.
  7. Resource Allocation: In clinical or research settings, knowing the limitations can help in making decisions about resource allocation. For instance, if a lab knows that the Spot Indole Test has limitations in certain scenarios, they might choose to invest in alternative or supplementary testing methods.

Limitations of Spot Indole Test

The Spot Indole Test, while an invaluable tool in microbiological diagnostics, is not devoid of limitations. Acknowledging these constraints is crucial for accurate interpretation and application of the test results. The following encapsulates the primary limitations of this assay:

  1. Comparative Efficiency: The efficacy of the Spot Indole Test is marginally inferior when juxtaposed with the tube indole test. This underscores the fact that while the Spot Indole Test offers rapid results, it may not always be as precise as its tube counterpart.
  2. Culture Specificity: A prerequisite for the test is the use of a fresh bacterial culture that has been nurtured in a tryptophan-rich environment. The absence of such a specific culture medium can compromise the accuracy of the test.
  3. Diffusion of Indole: A notable concern arises from the tendency of indole to diffuse into the surrounding culture medium. When executing the Spot Indole Test directly on an agar plate, this diffusion can inadvertently produce false positive results, potentially leading to misinterpretations.
  4. Necessity of Heavy Inoculum: For bacteria that are deemed fastidious, which are particular about their growth conditions, a heavy inoculum is mandatory. This necessitates a larger sample size for such organisms, which might not always be feasible.
  5. Lack of Confirmatory Nature: The Spot Indole Test, while instrumental in initial screenings, is not definitive in its results. Consequently, it should be seen as a preliminary step, with subsequent biochemical assays required for a comprehensive identification of the bacterial isolate.

Recognizing these intrinsic limitations underscores the importance of holistic diagnostic approaches, ensuring that the Spot Indole Test is employed judiciously in tandem with other corroborative assays for precise bacterial identification.



What is the Spot Indole Test?

The Spot Indole Test is a microbiological assay used to determine if bacteria can produce indole by hydrolyzing tryptophan.

Why is the Spot Indole Test important?

This test assists in the differentiation and identification of various bacteria based on their ability to metabolize tryptophan into indole.

How is a positive result indicated in the Spot Indole Test?

A positive result is indicated by the development of specific colors, typically blue or pink to red, depending on the reagent used.

What does a negative result in the Spot Indole Test mean?

A negative result, denoted by no color change, suggests that the bacteria do not have the capability to hydrolyze tryptophan into indole.

Why is it necessary to use a tryptophan-rich medium for the test?

A tryptophan-rich medium provides the necessary substrate for bacteria to produce indole, which is then detected in the test.

How does the Spot Indole Test compare to the tube indole test?

The Spot Indole Test offers more rapid results but might be slightly less precise compared to the tube indole test.

Can the Spot Indole Test be used as a confirmatory test?

No, the Spot Indole Test is a preliminary step. It should be complemented with other biochemical assays for comprehensive bacterial identification.

Why should the results be read within 20 seconds of adding the indole reagent?

Reading results within this precise timeframe ensures accuracy, as any delay could lead to misleading outcomes or color changes.

Are there any specific precautions to be taken while performing the test?

Yes, it’s crucial to use fresh cultures, avoid medium-containing dyes, choose the right reagent based on bacterial aerotolerance, and ensure timely observation of results.

What are some bacteria that test positive for the Spot Indole Test?

Some indole-positive bacteria include Escherichia coli, Klebsiella oxytoca, and Proteus vulgaris among others.

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