Nitrocefin test: Principle, procedure, uses and limitations

The nitrocefin biochemical test is a sensitive technique for detecting beta-lactamase producing strains of N. gonorrhoeae, H. influenzae, Staphylococcus spp, Enterococcus spp and Moraxella (Branhamella) catarrhalis. Nitrocefin is the only reliable test for detecting beta-lactamase producing Enterococcus spp.

It provides the most sensitive test for most β-lactamases, exceptions being staphylococcal penicillinase and ROB-1, an uncommon plasmid-mediated enzyme of haemophili. Another chromogenic cephalosporin, PADAC, exists but is less sensitive and is not readily available. Rapid beta-lactamase tests can yield clinically relevant information earlier than an MIC or disk diffusion test.

Nitrocefin is a chromogenic cephalosporin which changes from yellow to read when the amide bond in beta-lactam ring is hydrolyzed by beta-lactamase.   It is sensitive to hydrolysis by all known lactamases produced by Gram-positive and Gram-negative bacteria.

Although the test can be performed by using a nitrocefin solution, nitrocefin is very expensive, light sensitive, and not easily obtained. Most laboratories find it more convenient to use a commercially available nitrocefin test kit.

Beta-lactase detection using Nitrocefin solution (Powder):

  1. A 0.5 mM nitrocefin solution is prepared by dissolving 2.58 mg of pure powder in 0.5 mL of dimethylsulphoxide (DMSO) then diluting with 9.5 mL of 0.1 M phosphate buffer, pH 7.0.
  • This solution is stable for 10 days at 4°C in a foil-wrapped bottle.
  • Glass containers should be used, since DMSO degrades plastics.
  1. Colonies of the test isolates are scraped from nutrient agar plates and are suspended in 20 µL volumes of 0.1 M phosphate buffer pH 7.0, to produce a dense suspension on a glass slide, and 20 µL amounts of the nitrocefin solution are added.
  2. β-Lactamase activity is indicated by a red colour within 1-2 min.
  3. Weak activities may take longer to appear, but reactions taking >10 min should be treated with scepticism, as they may reflect the secondary ß-lactamase activity of those penicillin-binding proteins that form unstable acyl complexes.

Beta-lactase detection using Nitrocefin disk:

  1. Using sterile forceps, remove a Nitrocefin disk from the vial and place it on an empty petri dish or microscopic slide. Immediately place the remaining unused disks into the freezer.
  2. Prior to inoculation, allow the Nitrocefin disk to equilibrate to room temperature.
  3. Moisten each disk with one drop of sterile deionized water. Do not over saturate the disk, which could dilute the reagent.
    1. Note:Water is critical to the development of the color reaction, if the disk begins to dry out it may be necessary to rehydrate the reaction area of the Nitrocefin disk with a small amount of water.
  4. With a sterilized loop or applicator stick remove a well-isolated colony and spread it on the disk surface.
  5. Observe the inoculated disk for the development of an orange/red color


Fig: Nitrocefin test result (Left Positive), Right (Negative).
Fig: Nitrocefin test result (Left Positive), Right (Negative).

You can purchase Nitrocefin disk here:

Test Results:

  1. Left (H. influenzae): The orange/red color development was indicative as beta-lactamase positive.
  2. Right (M. catarrhalis): The yellow color development was indicative as beta-lactamase negative.

Other techniques:

Rehydrate the Nitrocefin as directed, and use this solution in the following ways:

  1. Direct Plate Method
    Add one drop of the Nitrocefin solution on to the surface of the colony. If the isolate is a high beta-lactamase producer then the colony and the surrounding area will quickly turn red. To detect a weak beta-lactamase producer the plate should then be incubated for 30 minutes before being reported as negative.
  2. Slide Method
    Add one drop of the Nitrocefin solution on to a clean glass slide. Using a sterile loop, pick one colony from the plate and emulsify into the Nitrocefin drop. Report as positive if the colour changes from yellow to red within 30 minutes (protect the slide from desiccation during the waiting period).
  3. Broth Method
    Add four drops of Nitrocefin solution to 1ml of the grown culture. Report as positive if the colour changes to red within 30 minutes.
  4. Broken Cell Method
    Sonicate 1ml of the culture in order to break open the cells. Add 4 drops of Nitrocefin solution. Report as positive if the colour changes to red within 30 minutes.
  5. Paper Disc Spot Test
    A Whatman No.1 filter paper disc (diameter 7cm) is placed in a petri dish and impregnated with Nitrocefin solution (0-5ml). This impregnated paper is generally usable for one day, but should be kept away from light to avoid spontaneous degradation. An isolated colony is applied to the impregnated paper with a loop; a pink to red reaction developing within 15 minutes indicates beta-lactamase presence.


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