Spot Indole Test
A bench guide to the rapid Spot Indole test, detecting tryptophanase activity within 20 seconds to quickly differentiate Escherichia coli from Klebsiella.
Spot Indole reaction cards A-B
Organisms are rubbed onto filter paper saturated with spot indole reagent (paradimethylaminocinnamaldehyde). A blue color shift is positive.
Development of a distinct blue/blue-green color within 20 seconds. Tryptophanase hydrolyzed tryptophan to indole, which coupled with cinnamaldehyde.
No color change or a faint pink color on the filter paper. The organism does not produce tryptophanase.
Development of a distinct blue/blue-green color within 20 seconds. Tryptophanase hydrolyzed tryptophan to indole, which coupled with cinnamaldehyde.
No color change or a faint pink color on the filter paper. The organism does not produce tryptophanase.
What to look for
| Visual clue | Meaning | Student shorthand |
|---|---|---|
| Blue/blue-green color | Indole coupled with paradimethylaminocinnamaldehyde to form a blue compound | Positive reaction (within 20s) |
| No color / faint pink | No indole detected; slightly pink is the natural color of the reagent | Negative reaction |
| Rapid timing | Must be read within 20-30 seconds of inoculation | Immediate reaction |
Spot Indole Interpretive Matrix
| Pattern | What to call it | How to think about it |
|---|---|---|
| Blue or blue-green spot (≤20s) | Positive | Organism produces tryptophanase. Presumptive Escherichia coli. |
| Colorless or faint pink | Negative | Organism lacks tryptophanase. Presumptive Klebsiella pneumoniae. |
- The spot indole test is a rapid, 20-second alternative to the traditional Kovac's tube test.
- It utilizes paradimethylaminocinnamaldehyde, which is more sensitive than the p-dimethylaminobenzaldehyde in Kovac's reagent.
- It is a major diagnostic shortcut for confirming E. coli from urinary tract infections.
Blue = Positive (E. coli). Colorless/Pink = Negative (Klebsiella).