The catalase test is a quick way to identify whether bacteria produce the enzyme catalase, and it takes less than a minute to perform. You add hydrogen peroxide to a bacterial colony and watch for bubbles. If the bacteria produce catalase, the enzyme breaks down the hydrogen peroxide into water and oxygen gas, and those oxygen bubbles are your positive result.
How the Test Works
Catalase is an enzyme that protects bacteria from hydrogen peroxide, a toxic byproduct of aerobic metabolism. When hydrogen peroxide contacts a catalase-producing organism, the enzyme splits two molecules of hydrogen peroxide into two molecules of water and one molecule of oxygen gas (2H₂O₂ → 2H₂O + O₂). That released oxygen is what creates the visible bubbling on the slide or in the tube.
The reaction happens in two steps. First, the iron center in the catalase enzyme reduces one hydrogen peroxide molecule to water, forming a reactive intermediate. That intermediate then oxidizes a second hydrogen peroxide molecule, releasing oxygen and another molecule of water. The whole process is nearly instantaneous, which is why a positive result appears within seconds.
What You Need
The standard catalase test requires only a few items: hydrogen peroxide (3% is the routine concentration for clinical microbiology labs), glass slides or small test tubes (12 x 75 mm), a sterile wooden applicator stick or disposable plastic loop, a dropper or pipette, and petri dishes or a test tube rack depending on the method you choose. You’ll also want a dark background surface to help you see faint bubbles.
Use colonies from a non-blood-based agar plate that are 18 to 24 hours old. Fresh, isolated colonies give the most reliable results.
The Slide Method
Label two glass slides, one for your positive control and one for your negative control, and place each in an uncovered petri dish. Using a sterile wooden stick or plastic loop, touch the center of an isolated colony and smear it onto a small area of the glass slide. Discard the stick or loop into a sharps container.
With a dropper, add one or two drops of hydrogen peroxide directly onto the smeared colony on the positive control slide. Immediately place the petri dish cover on and look for bubbles against your bench top or a dark background. You may need a magnifying lens to see faint reactions. An immediate burst of bubbles is a clear positive. A weak positive shows just one or two bubbles. Repeat with the negative control slide to verify your reagent is working correctly. No bubbles within 20 seconds confirms a negative result.
The Tube Method
Label two 12 x 75 mm test tubes (positive and negative) and place them in a rack. Add four to five drops of hydrogen peroxide into each tube. Using a sterile applicator stick or plastic loop, pick up a small amount of colony material from your positive control organism and place the stick directly into the tube, leaving it there. Hold the tube against a dark background and look for immediate bubbling. A weak positive produces one or two bubbles. Repeat the process with your negative control.
An alternate version uses a capped tube. Add a few drops of hydrogen peroxide, then transfer colony material onto the inside wall of the tube just above the liquid level. Cap the tube and tilt it so the hydrogen peroxide washes over the colony material. Watch for bubbling as the liquid makes contact.
Reading the Results
The distinction between positive and negative is straightforward:
- Positive: Immediate, vigorous bubbling when the hydrogen peroxide contacts the colony. The oxygen gas is clearly visible as effervescence.
- Weak positive: Only one or two small bubbles form.
- Negative: No bubble formation within 20 seconds.
Timing matters. Bubbles that appear after 20 seconds are not considered a true positive reaction. Always read results promptly after adding the reagent.
Why the Test Matters
The most common use of the catalase test is distinguishing staphylococci from streptococci. Both are gram-positive cocci that can look similar under a microscope, but staphylococci are catalase-positive and streptococci are catalase-negative. This single test narrows down identification quickly and cheaply, making it one of the first steps in many bacterial identification workflows.
Beyond that classic distinction, the catalase test helps differentiate other groups. Listeria (catalase-positive) can be separated from Enterococcus (catalase-negative), for example. It’s a simple screening tool, not a definitive identification on its own, but it efficiently guides which further tests to run.
Avoiding False Results
The biggest source of false positives is blood agar. Red blood cells contain their own catalase, so if you carry over even a small amount of blood agar with your colony material, the red blood cell catalase will produce bubbles and mimic a positive result. When possible, subculture your organism onto a non-blood medium before testing. If you must work from a blood agar plate, pick only from the very top of the colony and avoid touching the surrounding agar.
Metal inoculating loops can also interfere. Some metals catalyze the breakdown of hydrogen peroxide on their own, producing bubbles that have nothing to do with the bacteria. Wooden applicator sticks or disposable plastic loops eliminate this problem entirely.
Old or improperly stored hydrogen peroxide is another pitfall. Hydrogen peroxide degrades over time, especially when exposed to light or warmth. If your positive control organism fails to produce bubbles, your reagent has likely lost potency and needs to be replaced. Always run both a known positive and a known negative control alongside your test organism to confirm the reagent is working.
Controls to Run Every Time
For your positive control, use a known catalase-positive organism such as Staphylococcus aureus. For the negative control, a Streptococcus species works well. Running both controls with each batch of testing confirms that your hydrogen peroxide is active and that your technique isn’t introducing artifacts. If the positive control doesn’t bubble or the negative control does, troubleshoot before interpreting any test results.
Safety Considerations
Routine catalase testing with 3% hydrogen peroxide poses minimal risk, but the solution is still an oxidizer. Avoid contact with your eyes and skin. Some research protocols use 30% hydrogen peroxide, which is far more concentrated and can cause chemical burns on contact. If you’re working with higher concentrations, wear appropriate gloves and eye protection.
Dispose of used slides, tubes, applicator sticks, and pipettes into biohazardous waste containers, since they’ve been in contact with live bacterial cultures. Follow your lab’s standard protocols for handling and discarding materials contaminated with microorganisms.