Novobiocin susceptibility test


The speciation of coagulase negative Staphylococcus is necessary in certain culture specimens. Coagulase negative staphylococci can either be resistant or susceptible to novobiocin. The one most commonly isolated novobiocin resistant coagulase negative staphylococcus from urine is Staphylococcus saprophyticus. It is one of the most common causes of urinary tract infections in sexually active young women. So, when coagulase negative staphylococcus is isolated from urine of a woman of this age novobiocin susceptibility test can be performed for presumptive identification of Staphylococcus saprophyticus.

Staphylococcus saprophyticus is identified on the basis of

  • Absence of hemolysis on blood agar
  • Gram stain
  • Catalase test
  • Coagulase test and
  • Novobiocin susceptibility test.

Test should be performed from the colonies that are catalase-positive, coagulase-negative gram positive cocci. Colonies that are less than 24 hrs old should be picked from a blood agar plate.

Procedure of Novobiocin test

  1. Prepare a suspension equivalent to a McFarland 0.5 opacity standard from a pure colonies on sterile water.
  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. Put a 5 µg novobiocin disk aseptically onto the inoculated agar plate and lightly press down to ensure full contact with the medium.
  4. Incubate the agar plate aerobically for 18 to 24 hours at 35°C.
  5. Measure the zone size around the novobiocin disk.

Interpretation of Novobiocin test

  1. Resistant – zone size less than 12 mm
  2. Sensitive – zone size greater or equal to 16 mm.

Expected results:

  1. Staphylococcus saprophyticus – growth < 12mm or uniform growth up to the edge of the disk (resistant)
  2. Staphylococcus epidermidis – Zone of inhibition >16 mm (sensitive)


Other occasional coagulase negative staphylococcus isolated from human that are not Staphylococcus saprophyticus,  Staphylococcus cohnii or Staphylococcus xylosus may also be resistant to novobiocin.

Quality Control of Novobiocin disk:

Quality control should be performed per lot/shipment date with known organisms.

  1. Positive control = Staphylococcus saprophyticus
  2. Negative control = Staphylococcus epidermidis


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