Spot Indole Test – Principle, Procedure, Result, Uses

What is Spot Indole Test?

Spot Indole Test is a rapid biochemical test used in diagnostic microbiology to detect the production of enzyme tryptophanase by bacteria. It is a qualitative test that determines whether the organism can degrade the amino acid tryptophan to produce indole. This test is commonly used for the presumptive identification of enteric bacteria .

The enzyme tryptophanase catalyzes the breakdown of tryptophan into indole, pyruvic acid and ammonia. This reaction occurs when bacteria possessing the enzyme are grown on a tryptophan-rich medium. The indole produced during this reaction is detected by specific reagent.

The reaction is as follows–

Tryptophan → Indole + Pyruvic acid + Ammonia

The test is performed by rubbing a small portion of a fresh bacterial colony on a filter paper or cotton swab which is saturated with 1% p-dimethylaminocinnamaldehyde (DMACA) reagent. If indole is present, a distinct blue to blue-green colour is produced within 1–3 minutes. The rapid appearance of colour indicates a positive result.

This method is useful in differentiating closely related bacterial species. For example, Escherichia coli is indole positive whereas Klebsiella pneumoniae is indole negative. Similarly, Proteus vulgaris gives positive reaction while Proteus mirabilis does not produce indole.

The spot indole test reduces the time required for identification as compared to conventional tube method which require 24–48 hours. It is simple, rapid and helpful in routine laboratory diagnosis.

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 objectives of Spot Indole Test are as follows–

1. To determine the ability of bacteria to hydrolyze tryptophan and produce indole by the action of enzyme tryptophanase .
2. To differentiate the members of Enterobacteriaceae family based on indole production.
3. To provide rapid and presumptive identification of Escherichia coli which is characteristically indole positive.
4. To distinguish closely related species such as indole positive Klebsiella oxytoca from indole negative Klebsiella pneumoniae and indole positive Proteus vulgaris from indole negative Proteus mirabilis.
5. To help in identification of certain anaerobic pathogens by differentiating indole positive organisms from indole negative organisms such as Bacteroides ovatus and Bacteroides fragilis.

Principle of Spot Indole Test

The principle of Spot Indole Test is based on the ability of certain bacteria to produce an intracellular enzyme known as tryptophanase. It is the process by which the amino acid tryptophan is enzymatically degraded into simpler end products. The presence or absence of this enzyme is determined by detecting the formation of indole .

When bacteria possessing tryptophanase are grown in a medium rich in tryptophan, the enzyme hydrolyzes and deaminates tryptophan. Three end products are formed during this reaction, which are indole, pyruvic acid and ammonia. If the enzyme is absent, this breakdown does not occurs and indole is not produced.

The reaction is as follows–

Tryptophan → Indole + Pyruvic acid + Ammonia

In this test, a fresh bacterial colony is smeared on filter paper or cotton swab containing aldehyde based reagent such as p-dimethylaminocinnamaldehyde (DMACA). Under acidic condition, the reagent reacts with indole by condensation reaction. A distinct blue or blue-green colour compound is formed immediately which indicates positive result.

If bacteria does not produce tryptophanase enzyme, indole is not formed and no colour change is observed. The test area may remain colourless or show slight pink colour which indicates negative result. Thus, the production of colour is directly related to the presence of indole and this is the basis of spot indole test.

Requirements for Spot Indole Test

The following are the requirements for Spot Indole Test–

1. Fresh bacterial culture – The bacterial colonies must be fresh and actively growing (18–24 hours). Old culture may not gives proper indole reaction.
2. Pure culture – Only pure culture is used. Mixed cultures may show false positive result because indole can diffuse in surrounding media and contaminate indole negative colonies.
3. Tryptophan rich medium – The medium must contain adequate amount of tryptophan such as blood agar or trypticase soy agar (TSA). This is necessary for production of indole.
4. Glucose free medium – The medium should not contain glucose. Glucose repress the synthesis of tryptophanase enzyme.
5. Dye free medium – Selective media containing dyes like MacConkey agar or EMB agar should be avoided. The dyes may interfere with colour interpretation.
6. Mueller Hinton Agar (MHA) – MHA is not suitable because tryptophan content is destroyed during acid hydrolysis of casein during preparation.
7. Spot indole reagent – A special detection reagent is required such as 1% p-dimethylaminocinnamaldehyde (DMACA) or 5% p-dimethylaminobenzaldehyde (DMAB) in hydrochloric acid solution.
8. Sterile applicator – Sterile inoculating loop or wooden applicator stick is used for picking colony.
9. Filter paper or cotton swab – Whatman No.1 filter paper or sterile cotton tipped swab is required for applying reagent and smearing the colony.
10. Quality control organisms – Known reference strains are used to check the reagent and media. Escherichia coli is used as positive control while Proteus mirabilis or Pseudomonas aeruginosa is used as negative control.

Procedure of Spot Indole Test

The procedure of Spot Indole Test can be performed by different methods. The following are the common methods–

A. Filter Paper Method (Most widely used)

1. A clean piece of absorbent filter paper (Whatman No.1) is placed in a clean petri dish lid or on a glass slide.
2. 1–2 drops of spot indole reagent (1% DMACA) is added on the filter paper to saturate a small area.
3. A small portion of pure 18–24 hours old isolated colony is picked up by sterile inoculating loop or wooden applicator stick from a tryptophan rich non selective agar medium.
4. The bacterial paste is rubbed firmly on the reagent saturated area of the filter paper.
5. The paper is observed for colour change within 1–3 minutes. Development of blue to blue-green colour indicates positive result.

B. Swab Method

1. 1–2 drops of spot indole reagent is dispensed directly on the tip of sterile cotton swab.
2. The saturated swab tip is touched directly on the surface of actively growing isolated bacterial colony.
3. The swab is examined for development of blue or blue-green colour within 3 minutes.

C. Direct Agar Plate Method (Not recommended for routine use)

1. A few drops of indole reagent is added directly over fresh colonies growing on agar medium.
2. The colonies and surrounding medium is observed for colour change within 20 seconds.

This method is generally not recommended because indole may diffuse through agar and contaminate nearby negative colonies giving false positive result. The colonies also gets destroyed and cannot be used for further testing.

Result and Interpretation of Spot Indole Test

The result of Spot Indole Test is interpreted based on development of colour after addition of reagent. The colour formation indicates the presence or absence of indole produced by bacteria .

The following are the results–

1. Positive Result

• Blue to blue-green colour – It is developed within 1–3 minutes when 1% p-dimethylaminocinnamaldehyde (DMACA) reagent is used. This indicates indole production.
• Pink to violet-red colour – It is produced when 5% p-dimethylaminobenzaldehyde (DMAB), Kovacs or Ehrlich’s reagent is used. It indicates positive reaction.
• Providencia alcalifaciens – This organism produces a red-violet colour with DMACA reagent. It is considered as positive result.
• Delftia acidovorans – It produces pumpkin-yellow or orange colour with Kovacs reagent. This indicates production of anthranilic acid instead of indole.

2. Negative Result

• No colour change – The test area remains colourless or shows faint pink colour. It indicates absence of indole production.
• Pink or purple colour with DMACA – When DMACA reagent is used, pink or purple colour on filter paper is considered negative. This usually occurs due to interaction of reagent with tryptophan present in culture media and not due to indole production.

Thus, the development of specific colour within given time is the basis for interpretation of Spot Indole Test.

Quality Control Organisms of Spot Indole Test

Quality control organisms are used to check the performance of reagent and culture media. It is necessary to use known positive and negative strains to ensure the accuracy of test result .

The following are the quality control organisms–

1. Positive Control Organisms (Indole Positive)

• Escherichia coli (ATCC 25922, NCTC 10418, NCTC 12241) – It is commonly used as aerobic positive control organism.
• Bacteroides ovatus (ATCC 8483) – It is used as anaerobic positive control.
• Porphyromonas asaccharolytica (ATCC 25260) – It is used as anaerobic positive control organism.
• Fusobacterium necrophorum (ATCC 25286) – It is also used as anaerobic positive control.

2. Negative Control Organisms (Indole Negative)

• Proteus mirabilis (ATCC 12453, NCTC 10975) – It is used as aerobic negative control organism.
• Pseudomonas aeruginosa (ATCC 27853) – It is used as aerobic negative control.
• Prevotella melaninogenica (ATCC 25845) – It is used as anaerobic negative control.
• Bacteroides fragilis (ATCC 25285) – It is used as anaerobic negative control organism.
• Neisseria gonorrhoeae (ATCC 49226, NCTC 12700) – It is used as negative control.

These control organisms are tested along with unknown sample to confirm the validity of spot indole test result.

Spot Indole Positive Bacteria & Spot Indole Negative Bacteria

Here is a list of spot indole-positive and spot indole-negative bacteria;

Spot Indole-Positive Bacteria

• Escherichia coli
• Klebsiella oxytoca
• Proteus vulgaris
• Citrobacter koseri
• Morganella morganii
• Bacteroides ovatus
• Providencia alcalifaciens (Produces a unique red-violet color instead of blue)
• Porphyromonas asaccharolytica
• Fusobacterium necrophorum
• Aeromonas hydrophila and Aeromonas punctata
• Vibrio species (e.g., Vibrio cholerae)
• Bacillus alvei
• Edwardsiella species
• Flavobacterium species
• Haemophilus influenzae
• Plesiomonas shigelloides
• Pasteurella multocida and Pasteurella pneumotropica
• Enterococcus faecalis
• Lactobacillus reuteri
• Delftia acidovorans (Produces a pumpkin-yellow/orange color when using Kovacs reagent)
• Raoultella planticola (Variable; some strains are positive, others are negative)

Spot Indole-Negative Bacteria

• Klebsiella pneumoniae
• Klebsiella variicola
• Proteus mirabilis and Proteus penneri
• Citrobacter freundii
• Bacteroides fragilis
• Prevotella melaninogenica
• Pseudomonas aeruginosa
• Salmonella species
• Shigella species
• Enterobacter species
• Serratia species
• Yersinia species
• Staphylococcus aureus
• Actinobacillus species
• Aeromonas salmonicida
• Alcaligenes species
• Bordetella species
• Neisseria species
• Raoultella terrigena and Raoultella ornithinolytica
• Mannheimia haemolytica
• Pasteurella ureae
• Rhizobium species
• Most Bacillus and Lactobacillus species

Precautions

The precautions of Spot Indole Test are–

• Personal protective equipment – Gloves, lab coat and safety goggles must be worn. The spot indole reagent contains concentrated hydrochloric acid and it is corrosive and poisonous. It may cause skin burn and eye damage .
• Avoid contact and inhalation – The reagent should not be inhaled or allowed to come in contact with skin and eyes. If contact occurs, it must be washed immediately with plenty of water.
• Handling of cultures – All clinical specimens and bacterial cultures are treated as potentially infectious. Standard aseptic technique is followed and materials are properly sterilized after use.
• Check reagent condition – Expired or deteriorated reagent should not be used. If reagent shows turbidity, contamination or unexpected dark colour it must be discarded.
• Selection of reagent – DMACA reagent is preferred especially for anaerobic organisms. Kovacs reagent is less sensitive and not recommended for anaerobes.
• Use of proper medium – Organisms must be taken from tryptophan rich medium. Without tryptophan indole will not be produced.
• Avoid glucose containing media – Media containing glucose should not be used. Glucose metabolism suppress the synthesis of tryptophanase and false negative result may occurs.
• Avoid dye containing media – Colonies from MacConkey agar or EMB agar should not be tested. The dyes may interfere with colour reaction and give false result.
• Mueller Hinton Agar – MHA should never be used because tryptophan is destroyed during acid hydrolysis in its preparation.
• Use pure and fresh culture – Only pure isolated colonies of 18–24 hours old should be tested. Mixed cultures or old cultures may gives false positive or false negative reaction due to diffusion of indole or reduced enzyme activity.
• Reading of result – The colour development must be observed within the specified time. Delayed reading may give incorrect interpretation of result.

Uses of Spot Indole Test

The uses of Spot Indole Test are–

• It is used for identification of indole producing organisms such as Escherichia coli. It gives rapid presumptive identification from primary isolation media .
• It is used in differentiation of Enterobacteriaceae members. The organisms are separated based on the production of indole from tryptophan.
• Indole positive Klebsiella oxytoca is differentiated from indole negative Klebsiella pneumoniae.
• Proteus vulgaris and Proteus mirabilis is differentiated by this test based on indole production.
• It helps in separating indole positive Citrobacter koseri and indole negative Citrobacter freundii.
• It is used as screening test for anaerobic Gram negative rods such as Bacteroides ovatus and Bacteroides fragilis.
• Indole producing Fusobacterium necrophorum can be detected by this method.
• It is used as rapid confirmatory test to verify identification obtained from automated systems such as MALDI-TOF MS.

Limitations of Spot Indole Test

• Lower sensitivity – It is less sensitive as compared to conventional tube indole method. Some weak indole producers may not be detected .
• Reagent limitation – Kovacs reagent can be used but it is less sensitive than DMACA reagent. It is not recommended for testing of anaerobic bacteria.
• Media containing glucose – Media with glucose cannot be used. Acid end products formed from glucose reduces indole production and false negative result may occurs.
• Mueller Hinton Agar (MHA) – It cannot be used because tryptophan is destroyed during acid hydrolysis of casein in preparation of media.
• Dye containing media – Colonies grown on MacConkey agar or EMB agar should not be used. The dyes may interfere with colour interpretation.
• Diffusion of indole – Indole is diffusible molecule. Indole positive colonies may cause nearby indole negative colonies to appear positive if taken from mixed culture plate.
• False negative strains – Some strains of Proteus vulgaris, Providencia and Aeromonas may give false negative reaction with spot indole test.
• Age of culture – Fresh culture (18–24 hours) is required. Older colonies may gives false negative result because indole may diffuse away or enzymatic activity declines.
• Inoculum size – Heavy inoculum is required for proper reaction. Fastidious organisms which do not grow abundantly may not give proper result.
• Presumptive test – It is not a confirmatory test. It only gives presumptive identification and further biochemical tests is required for complete identification.

FAQ

References

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