Indole Test: Principle, Procedure, Interpretation & Indole-Positive Organisms

Indole test is used to determine the ability of an organism to split the amino acid tryptophan to form the compound indole. It's one of the four IMViC tests used to differentiate members of the family Enterobacteriaceae — and it's usually the fastest of the four to read.

Why It Matters

Picture a urine culture growing a lactose-fermenting Gram-negative rod on MacConkey agar. Full identification and sensitivity will take another 24–48 hours — but a clinician needs an empiric antibiotic choice today. A 20-second spot indole test, combined with the colony's oxidase result, can presumptively call it E. coli right then: indole-positive, oxidase-negative, pink lactose-fermenter on MacConkey. That presumptive call is often enough to start a sensible empiric regimen while the formal culture finishes.

This is also why indole is one of the oldest tests in clinical bacteriology — it doesn't need expensive reagents or incubators beyond what every lab already has, and it still separates indole-positive E. coli from indole-negative Enterobacter and Klebsiella pneumoniae faster than almost any other single test.

Principle

Tryptophan is hydrolyzed by the enzyme tryptophanase into three end products: indole, pyruvate, and ammonia. Indole, when present, combines with the aldehyde in the test reagent to form a colored compound:

• Pink to red-violet (quinoidal compound) — if a benzaldehyde reagent (Kovac's or Ehrlich's) is used
• Blue to green — if a cinnamaldehyde reagent (DMACA) is used

No tryptophanase → no indole → no color change (indole-negative).

Figure: Indole Test Reaction

Procedure of Indole Test

There are two ways to run this test, and which one you choose matters.

1. Spot (Rapid) Indole Test

Best for: quick presumptive ID directly from an isolated colony.

• Saturate a filter paper with reagent, then rub a colony sample onto it — or
• Swab the colony and add a drop of reagent to the swab — or
• Add reagent directly onto the colony on the agar plate

Read the color change within 20 seconds.

2. Conventional Tube Method

Best for: confirming a negative spot test, or testing weak indole producers that the rapid method misses.

1. Inoculate tryptophan broth with the test organism.
2. Incubate at 37°C for 24–48 hours.
3. Kovac's method: Add 0.5 mL Kovac's reagent down the side of the tube; read the color at the meniscus.
4. Ehrlich's method (for anaerobes/fastidious organisms): Add 0.5 mL xylene, mix and let settle, then add 6 drops of Ehrlich's reagent down the side; read the color below the xylene layer.

Figure: Indole Test Results: Positive (Right Tube)-development of Red-ring

Where students actually get confused

• Reagent choice isn't optional. Ehrlich's (or DMACA/cinnamaldehyde) is more sensitive and is the only reagent suitable for anaerobes and fastidious organisms like Cardiobacterium hominis — Kovac's alone can miss weak producers in this group.
• Don't inoculate from MacConkey or EMB agar. The dyes in these media interfere with color interpretation. Use a non-selective medium for the colony you're testing.
• Don't use Mueller-Hinton agar colonies either — tryptophan is destroyed during the acid hydrolysis used to make the medium, which can produce a false negative.
• A negative spot test isn't the final answer. If it's negative, run the tube method — some organisms are weak producers that only the more sensitive tube test will catch.
• Watch pigmented colonies on a spot test. A naturally pigmented organism (for example the blue-green of Pseudomonas aeruginosa) can tint the filter paper and be misread. Judge the color of the reagent reaction, not the colony's own color, and confirm in a tube if unsure.
• Avoid plates with a nitrate disk nearby. Nitrate can cause a false-negative spot indole reaction.

Result Interpretation

Reporting:

• E. coli, most Enterobacteriaceae, Vibrio, Aeromonas, Plesiomonas, and Pasteurella species — indole positive
• Most strains of P. vulgaris, M. morganii, and Providencia — indole positive
• Fastidious Gram-negatives like Cardiobacterium hominis and Pasteurella bettyae — indole positive
• Propionibacterium acnes — indole positive

The "OK VIP" Trick

Three closely related genus-pairs trip students up every exam season because one species is indole-positive and its close relative is indole-negative:

Remember it as "OK VIP": Oxytoca (Klebsiella), Koseri (Citrobacter), Vulgaris (Proteus) → Indole Positive. Everything else in that trio defaults to negative.

The Klebsiella split is worth more than an exam mark. K. oxytoca, the indole-positive one, is the species behind antibiotic-associated hemorrhagic colitis: it produces a toxin (tilivalline) that damages colonic mucosa, and it classically emerges when penicillins suppress competing flora. So "the indole-positive Klebsiella" is not just a practical-exam distinction; it names the Klebsiella you think of when a patient develops bloody diarrhea after starting an antibiotic. A mnemonic you can attach to a real clinical picture is one you keep.

Multitest Media That Include Indole

In diagnostic labs, indole is rarely tested alone — it's usually read off a combination medium:

Quality Control

• Discard benzaldehyde reagents (Kovac's/Ehrlich's) if the color isn't pale yellow before use.
• QC every new reagent lot before use; QC in-house reagents weekly — they degrade, especially if not kept at 4°C.

Limitations

1. The growth medium must contain adequate tryptophan — avoid Mueller-Hinton agar colonies.
2. Avoid plates with a nitrate disk nearby (false negatives).
3. Only cinnamaldehyde reagent is validated for spot-testing anaerobes — it's more sensitive but less stable than benzaldehyde reagents.
4. A negative spot test should be confirmed with the tube method before reporting indole-negative.
5. Fastidious Gram-negative rods (e.g., C. hominis) may need a heavier inoculum and extraction step.

References and further readings

1. MacWilliams MP. Indole test protocol. American Society for Microbiology, 2009.
2. Leber AL, editor. Clinical Microbiology Procedures Handbook. 4th ed. Washington, DC: ASM Press; 2016. doi:10.1128/9781555818814
3. Tille PM. Bailey and Scott's Diagnostic Microbiology. 15th ed. St. Louis: Elsevier; 2022.