TestOxidase™ (PL.390) is a ready-to-use 0.3% TMPD reagent for detecting cytochrome c oxidase. A fresh colony picked with a platinum or plastic loop (never nichrome) produces a deep purple color within 30 seconds on a reagent-saturated filter paper or strip. Pseudomonas, Neisseria, Moraxella, Pasteurella, and Vibrio are positive; Enterobacterales are negative — making the oxidase test one of the first, fastest organism-level decisions in clinical bacteriology.
Key Facts
- Chemistry: Cytochrome c oxidase oxidizes TMPD to indophenol blue — visible as a deep purple color.
- Reagent: 0.3% N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride (more stable than the original 1% Kovacs formulation).
- Read time: Up to 30 seconds — color appearing after 60 seconds is non-specific.
- Positive: Pseudomonas, Neisseria, Moraxella, Pasteurellaceae, Aeromonas, Vibrio, Campylobacter, Helicobacter.
- Negative: Enterobacterales (E. coli, Klebsiella, Salmonella, Shigella, Enterobacter, Proteus, Serratia).
- Loop matters: Iron / nickel from nichrome wire produces false positives — use platinum or disposable plastic.
What the Oxidase Test Actually Measures
The oxidase test is one of the oldest and most reliable biochemical tools in routine bacteriology. It detects the presence of cytochrome c oxidase — the terminal enzyme of the aerobic electron-transport chain in certain bacteria. Organisms that carry this enzyme can transfer electrons directly to molecular oxygen during respiration; organisms that lack it cannot.
Chemically, the test substrate N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride (TMPD) acts as an artificial electron donor. When TMPD encounters cytochrome c oxidase on a viable bacterial colony, it is rapidly oxidized to an intermediate that condenses to indophenol blue, the deep purple chromogen visible at the smear within seconds. No enzyme, no color.
Because the reaction is enzymatic and not just a chemical oxidation, the test is genuinely species-discriminating — provided the technique is right.
Why TestOxidase™ Uses 0.3% TMPD
The original Kovacs' oxidase reagent used a 1% TMPD solution. While effective, 1% TMPD auto-oxidizes quickly in air and turns blue on its own, shortening shelf life and creating background color that can be mistaken for a weak positive. The modern 0.3% formulation in TestOxidase™ is the standard recommended in ASM and PHE laboratory manuals: stable, low background, and bright purple at the colony when the enzyme is present.
Each bottle ships ready-to-use — no weighing, no reconstitution, and no buffer step. Stored at 2–8°C and protected from light, the reagent holds reactivity to the labeled expiry. Once opened, discard if the solution itself develops a blue tint; that indicates auto-oxidation and will give false positives on every subsequent test.
Wet Swab Method vs Strip Method
Both formats deliver the same chemistry; the choice depends on volume and workflow.
The wet swab (filter paper) method applies 1–2 drops of TestOxidase™ to a clean filter paper in a petri dish, then a fresh colony is rubbed onto the saturated paper using a platinum loop, plastic loop, or wooden stick. This is the high-throughput choice — one bottle services many tests, the user controls dose, and it's the most economical option for benches that run dozens of oxidase tests a day.
The strip method uses paper strips pre-impregnated with dried TMPD. The colony is rubbed directly on the strip; the strip is then moistened (or pre-moistened) and read. Strips are convenient for low-volume labs, point-of-care, or environmental field work where carrying a liquid reagent is impractical. They have shorter shelf life once exposed but eliminate any pipetting step.
science Order Now TestOxidase™ PL.390 — 0.3% TMPD ready-to-use 30-second oxidase result, no reconstitution. Direct from Pro-Lab Diagnostics, Georgetown, TX. CE Marked, IVD. arrow_forwardDifferentiating Organisms at the Bench
The reason the oxidase test is performed so often in routine work is that it cleanly separates two large groups of clinically important Gram-negative bacteria: the oxidase-positive non-fermenters and fastidious genera from the oxidase-negative Enterobacterales. Combined with a Gram stain and a glucose-fermentation result, a 30-second oxidase reaction often narrows the identification to a workable short list before any other biochemical has been read.
| Oxidase Positive (+) | Oxidase Negative (−) |
|---|---|
| Pseudomonas aeruginosa | Escherichia coli |
| Neisseria gonorrhoeae, N. meningitidis | Klebsiella pneumoniae |
| Moraxella catarrhalis | Salmonella spp., Shigella spp. |
| Pasteurella multocida, Haemophilus spp. | Proteus, Enterobacter, Serratia |
| Aeromonas, Vibrio spp. | Stenotrophomonas maltophilia (notable negative non-fermenter) |
| Campylobacter, Helicobacter, Brucella | Acinetobacter baumannii |
A positive Gram-negative rod from a wound or respiratory specimen, growing on blood agar but not MacConkey, with a deep-purple oxidase reaction is — until proven otherwise — a likely Pseudomonas. That single observation drives the next plate, the next biochemical, and often the empiric antibiotic decision.
Why the Loop Material Matters
The single most common technique failure on the oxidase test is a false positive caused by the inoculating loop itself. Nichrome and stainless-steel wires contain iron and nickel; both metals chemically oxidize TMPD without any enzyme present, producing a blue color on a paper that has only ever touched the loop. The result is an "oxidase-positive" E. coli that is nothing of the sort.
The fix is straightforward — use a platinum or platinum-iridium loop, a plastic disposable loop, a clean wooden applicator stick, or a sterile swab to transfer the colony. Every modern microbiology reference (Bailey & Scott, ASM Manual of Clinical Microbiology, UK SMI TP 26) makes this the first cautionary note in the oxidase procedure.
Equally important: test only fresh 18–24 h colonies grown on non-selective, glucose-free media (blood, tryptone soya, chocolate). Aged colonies lose enzymatic activity and read as false negatives; glucose-rich selective media (MacConkey, EMB) repress the aerobic respiratory pathway and likewise suppress oxidase expression.
Where TestOxidase™ Fits in a Routine Bench Workflow
For most clinical labs, TestOxidase™ sits next to the Gram-stain reagents and the spot indole bottle as a first-line, point-of-colony test. A typical Gram-negative workup that begins with a fresh subculture is: Gram stain → oxidase (TestOxidase™) → indole (DMACA spot or Kovacs' reagent) → catalase. Four tests, less than five minutes of bench time, and the organism is usually narrowed to a genus before the MALDI or automated ID is queued.
For environmental and food-safety labs, the same reagent supports rapid screening of Pseudomonas contamination in water and process samples; for research labs, it is a routine confirmatory test for bacterial isolates pulled from Microbank® cryopreservation stocks.
Frequently Asked Questions
What does the oxidase test detect?
The oxidase test detects the enzyme cytochrome c oxidase, the terminal electron carrier in the aerobic respiratory chain of some bacteria. TMPD is oxidized by cytochrome c oxidase to indophenol blue, producing a deep purple color at the inoculum within 30 seconds.
Which organisms are oxidase positive?
Pseudomonas, Neisseria, Moraxella, Pasteurellaceae, Aeromonas, Vibrio, Campylobacter, Helicobacter, and Brucella are oxidase positive. Enterobacterales (E. coli, Klebsiella, Salmonella, Shigella, Proteus, Enterobacter, Serratia) are oxidase negative — this is one of the primary differentiation tests at the bench.
Why can't I use a nichrome or stainless-steel loop?
Iron and nickel in nichrome and stainless-steel loops can oxidize TMPD chemically, producing a false-positive blue color even on an oxidase-negative organism. Use a platinum loop, a plastic disposable loop, a sterile wooden stick, or a clean swab to transfer the colony.
What is the difference between TestOxidase™ wet swab and strip methods?
Both methods use the same 0.3% TMPD chemistry. The wet swab method applies reagent to filter paper at the time of testing — preferred for higher-throughput labs running many tests from one bottle. Pre-impregnated strips are pre-dosed with TMPD and dried — ideal for low-volume or point-of-care use where reconstitution would waste reagent.
Why must the colony be fresh and from a glucose-free medium?
Cytochrome c oxidase activity drops sharply after 24 hours, so an aged colony may read as a false negative. Glucose-fermenting media (MacConkey, EMB) suppress aerobic respiration and can also produce false negatives. Always subculture to blood agar, tryptone soya, or chocolate agar and test within 18–24 hours.
How long is TestOxidase™ stable once opened?
Unopened TestOxidase™ (PL.390) is stable to the labeled expiry when stored at 2–8°C protected from light. Once opened, discard the bottle if the solution itself develops a blue tint — that indicates auto-oxidation of TMPD and will give false positives on every subsequent test.
For more information about TestOxidase™, contact info@pro-lab.us or visit the TestOxidase™ product page to order online.