Novobiocin Susceptibility Test- Principle, Procedure, Results, Uses

Objectives of Novobiocin Susceptibility Test

  • To determine the susceptibility pattern of a bacterium to novobiocin.
  • To distinguish or differentiate Staphylococcus saprophyticus from other coagulase-negative staphylococci.

Principle of Novobiocin Susceptibility Test

Novobiocin is an antibiotic interfering with the unpackaging and repackaging of DNA during DNA replication and the bacterial cell cycle. Novobiocin binds to DNA gyrase and blocks adenosine triphosphatase (ATPase) activity.  Laboratory identification of S. saprophyticus is made on the basis of the absence of hemolysis, coagulase, and resistance to novobiocin. Staphylococcus saprophyticus, Gram-positive coagulase-negative Staphylococci, is a uropathogenic bacterium that causes acute uncomplicated urinary tract infections, particularly in young, middle-aged female patients.  Unlike most other CONS, S. saprophyticus is rarely resistant to most antibiotics active against gram-positive organisms. S. saprophyticus is innately resistant to the antibiotic novobiocin. Therefore, screening coagulase-negative staphylococci from urine cultures for novobiocin resistance is reliable presumptive identification of S. saprophyticus. Susceptibility to novobiocin is determined by placing a novobiocin-impregnated paper disk on an agar plate seeded with the organism under investigation. As the organism multiplies during incubation to produce a lawn of confluent growth, cells are exposed to the antibiotic diffusing into the agar from the paper disk. If the bacteria are susceptible to novobiocin, there will be a formation of a visible zone of inhibition around the disk, representing an area where the antibiotic concentration has prevented bacterial growth. No zone of inhibition around the disk represents that organism is resistant to the antibiotic.

Procedure of Novobiocin Susceptibility Test

  1. Using a pure 18-24 hour culture, prepare a suspension of the organism; equivalent to a McFarland 0.5 opacity standard; to be identified in Tryptic Soy Broth, Sterile Water,  or Brain Heart Infusion (BHI) broth.
  2. With a sterile swab, prepare a lawn of growth over the entire plate by swabbing over the entire plate in 3 directions and around the edge of the plate.
  3. Aseptically apply one 5ug novobiocin disk onto the inoculated agar surface and lightly press down to ensure full contact with the medium.
  4. Incubate plate aerobically for 18 to 24 hours at 35 to 37°C.
  5. Measure the zone of inhibition around the novobiocin disk, and record as susceptible or resistant.

Result Interpretation of Novobiocin Susceptibility Test

Result Interpretation of Novobiocin Susceptibility Test

Sensitive: zone of inhibition greater than 16 mm
Resistant: zone of inhibition less than or equal to 16mm


  1. The novobiocin disk is not helpful and can give misleading results if it is performed on isolates other than those from urinary specimens. 
  2. Other occasional coagulase-negative Staphylococcus isolated from humans that are not Staphylococcus saprophyticus such as Staphylococcus cohnii, Staphylococcus pulvereri, or Staphylococcus xylosus may also be resistant to novobiocin.

Quality Control

  1. Positive control (resistant) Staphylococcus saprophyticus (ATCC 15305)
  2. Negative control (sensitive)  Staphylococcus epidermidis (ATCC 12228)

References and Sources

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