Furazolidone Susceptibility Test: The Nitrofuran Disk That Confirms Staphylococcus Over Micrococcus

Why It Matters

A catalase-positive Gram-positive coccus needs to be placed in one of two genera: Staphylococcus, which contains major human pathogens, or Micrococcus, a largely harmless skin and environmental commensal. They look alike on routine media, and no single test settles it with certainty, so the laboratory uses a small battery of quick tests that each exploit a genuine biological difference.

Furazolidone susceptibility is one of the most reliable of them. Staphylococci are killed by furazolidone; micrococci are not. Place a furazolidone disk on a lawn of the organism, and a staphylococcus produces a clear zone of inhibition around it, while a micrococcus grows right up to the disk edge, untroubled. In one published series, 99% of staphylococci were susceptible and all micrococci were resistant, which is why either furazolidone or the oxidase test is recommended as a primary separator.

The reason the two organisms respond so differently comes down to how furazolidone works, and here most descriptions (including the older version of this article) get it wrong. Furazolidone is a nitrofuran, and its antibacterial action has nothing to do with the monoamine-oxidase inhibition it causes in humans. This article covers the real mechanism, the reading breakpoints, and how furazolidone fits with the other Staphylococcus-versus-Micrococcus tests.

Figure: An area of growth inhibition of staphylococci around the furazolidone disk

Principle

Furazolidone (Furoxone) is a synthetic nitrofuran with antibacterial and antiprotozoal activity. Its antibacterial action works by reductive activation: inside the bacterial cell, nitroreductase enzymes reduce the drug's nitro group into highly reactive intermediates that damage bacterial DNA and other essential macromolecules, inhibiting and killing the organism.

(Furazolidone is also a monoamine oxidase inhibitor in humans, which is the basis of its dietary interactions when used as a drug, but that is a separate effect and is not how it kills bacteria.)

Occasionally, coagulase-negative staphylococci resistant to furazolidone are encountered, so the test is read alongside other differentiating tests.

Furazolidone (furoxone) susceptibility test is performed as a disk susceptibility procedure using commercially available furazolidone disks. Staphylococci are inhibited by furazolidone, but micrococci and related species are resistant. Micrococci usually grow right up to the edge of the 6-mm furazolidone disk.

Reagents

1. Furazolidone disks, 100 μg
2. Mueller-Hinton agar plate

Quality Control

Known strains of Staphylococcus (either S. aureus or a coagulase-negative strain) and Micrococcus species should be tested with each new lot of disks or weekly.

Procedure

1. Prepare a suspension of the organism to be tested in sterile distilled water or broth. The suspension should be equivalent to a 0.5 McFarland turbidity standard.
2. With a swab, spread the organism suspension onto one-half of a Mueller-Hinton agar plate.
3. Aseptically place a furazolidone disk in the center of the inoculated area and gently tap the disk so that it adheres to the agar surface.
4. Incubate the plate at 35°C in an ambient air incubator for 18–24 hours.

Figure: Furazolidone disk test

Results and Interpretations

1. Furazolidone resistant: zone of inhibition ≤ 9 mm (growth up to or near the disk). Example: Micrococcus luteus
2. Furazolidone sensitive: zone of inhibition > 15 mm. Example: Staphylococcus aureus, Staphylococcus saprophyticus
3. Intermediate (9-15 mm): an indeterminate zone in this range should not be forced into a call; repeat the test or confirm with another Staph/Micrococcus test (modified oxidase, bacitracin). Most true staphylococci give clearly larger zones and most micrococci clearly smaller, so an intermediate result warrants a second look.

Where the furazolidone test fits

Furazolidone susceptibility is one of several tests that separate Staphylococcus from Micrococcus, and it is worth knowing how it lines up with the others, because two of them point in opposite directions:

• Furazolidone: Staphylococcus susceptible (zone), Micrococcus resistant (no zone)
• Bacitracin (Taxo A): Staphylococcus resistant (no zone), Micrococcus susceptible (zone), the exact reverse
• Modified oxidase (microdase): Micrococcus positive (blue), Staphylococcus negative
• Lysostaphin: Staphylococcus susceptible (lysed), Micrococcus resistant

The furazolidone and bacitracin results are mirror images, which is a useful check: a true Staphylococcus should be furazolidone-susceptible and bacitracin-resistant, while a Micrococcus is the opposite on both. For the full comparison and when to reach for each test, see the Staphylococcus vs Micrococcus article.

How to remember

Furazolidone favors finding Staph. The alliteration helps: Furazolidone kills Fstaph (staphylococci are susceptible). Micrococci shrug it off and grow to the disk edge. Zone = Staph; no zone = Micrococcus.

Furazolidone and bacitracin are mirror images. This is the cleanest way to remember both: Staphylococcus is furazolidone-susceptible but bacitracin-resistant; Micrococcus is the reverse (bacitracin-susceptible, furazolidone-resistant). If an organism is susceptible to one and resistant to the other, the pattern itself names the genus. If it is susceptible or resistant to both, something is off, recheck.

It's a nitrofuran, not an MAO story (for bacteria). Furazolidone kills bacteria as a nitrofuran: bacterial enzymes reduce it into DNA-damaging intermediates. Its monoamine-oxidase-inhibiting effect is a human drug interaction, not the antibacterial mechanism. Do not confuse the two.

Read the zone, confirm the genus. A clear zone (>15 mm) points to Staphylococcus, but because rare coagulase-negative staphylococci are resistant, pair furazolidone with modified oxidase or bacitracin before finalizing.

Key exam facts in one table

Where students get confused

Thinking furazolidone kills bacteria by inhibiting monoamine oxidase. It does not. That is furazolidone's effect in humans (the reason for its dietary tyramine interactions as a drug). Its antibacterial mechanism is as a nitrofuran: bacterial nitroreductases activate it into DNA-damaging intermediates. Two different mechanisms in two different contexts.

Reversing the result with bacitracin. Furazolidone and bacitracin give opposite results, and it is easy to swap them. Staphylococcus is furazolidone-susceptible and bacitracin-resistant; Micrococcus is furazolidone-resistant and bacitracin-susceptible. Anchor on "furazolidone kills Staph, bacitracin doesn't."

Forcing an intermediate zone into a call. A zone between 9 and 15 mm is indeterminate. Do not round it to susceptible or resistant. Repeat or confirm with another test. Most true results fall clearly above 15 mm (Staph) or below 9 mm (Micrococcus).

Forgetting the resistant-staph exception. A few coagulase-negative staphylococci are furazolidone-resistant, so a resistant result does not completely exclude Staphylococcus. This is why furazolidone is read alongside modified oxidase or bacitracin, not alone.

Running it outside the right group. Furazolidone susceptibility differentiates Staph from Micrococcus among catalase-positive Gram-positive cocci. It is not a general susceptibility test to guide therapy here, and it is meaningless on catalase-negative cocci or Gram-negative organisms. Confirm Gram stain and catalase first.

Wrong disk potency. The differentiation uses a 100 µg furazolidone disk. Using a different potency changes the zone sizes and invalidates the breakpoints.

References

1. von Rheinbaben KE, et al. Comparison of various methods for differentiation of staphylococci and micrococci. J Clin Microbiol. 1984;19(6):875-879. doi:10.1128/jcm.19.6.875-879.1984
2. Baker JS. Comparison of various methods for differentiation of staphylococci and micrococci. J Clin Microbiol. 1984;19(6):875-879.
3. Tille PM. Bailey and Scott's Diagnostic Microbiology. 15th ed. St. Louis: Elsevier; 2022.
4. Procop GW, Church DL, Hall GS, Janda WM, Koneman EW, Schreckenberger PC, Woods GL. Koneman's Color Atlas and Textbook of Diagnostic Microbiology. 7th ed. Philadelphia: Wolters Kluwer; 2017.