Oxidase Test (Kovac's Method)
A bench guide to Kovac's oxidase test, detecting cytochrome c oxidase activity to separate Pseudomonas (positive) from Enterobacteriaceae (negative).
Oxidase reaction cards A-B
Bacterial colonies are rubbed onto filter paper saturated with Kovac's reagent. A dark purple color within 10 seconds is positive.
Development of a deep purple/violet color within 10 seconds. Cytochrome c oxidase oxidized Kovac's reagent to indophenol.
Absence of color change after rubbing the colony onto the filter paper. The organism is oxidase-negative.
Development of a deep purple/violet color within 10 seconds. Cytochrome c oxidase oxidized Kovac's reagent to indophenol.
Absence of color change after rubbing the colony onto the filter paper. The organism is oxidase-negative.
What to look for
| Visual clue | Meaning | Student shorthand |
|---|---|---|
| Dark purple color | Cytochrome c oxidase oxidizes the substrate to purple indophenol | Positive (within 10 seconds) |
| No color change / white paper | Reagent remains reduced (colorless) | Negative reaction |
| 10-second window | Must be read within 10-15 seconds; auto-oxidation can cause false-positive purple coloration later | Timing is critical |
Oxidase Interpretive Matrix
| Pattern | What to call it | How to think about it |
|---|---|---|
| Deep blue or purple (≤10s) | Positive | Cytochrome oxidase present. Typical for Pseudomonas, Neisseria, Campylobacter. |
| No color change | Negative | Cytochrome oxidase absent. Typical for Escherichia, Klebsiella, Salmonella. |
- The oxidase test detects cytochrome c oxidase, a key enzyme in the aerobic respiratory chain.
- A positive result is a deep purple/indigo spot appearing within 10 seconds.
- This test is a cornerstone for separating Enterobacteriaceae (all are oxidase negative) from aerobic Gram-negative rods like Pseudomonas (oxidase positive).
Purple in 10s = Positive. Enterobacteriaceae are all oxidase-negative (like E. coli). Pseudomonas is oxidase-positive.