Novobiocin Susceptibility Test: Principle, procedure and interpretations

Novobiocin Test is used to differentiate coagulase-negative staphylococci (CONS) and presumptively identify the isolate as S. saprophyticus  (Novobiocin resistant) .  It is usually performed on urine isolates that are coagulase negative.

Principle of Novobiocin Test

S. saprophyticus is second only to E. coli as the most frequent causative organism of uncomplicated Urinary tract infections in young sexually active women.  So when coagulase negative staphylococcus is isolated from a young sexually active women, laboratory must further identify that isolate and find out is this a true pathogen (Staphylococcus saprophyticus) or a contaminant (i.e. Staphylococcus epidermidis).

Laboratory identification of S. saprophyticus is made on the basis of the absence of hemolysin, coagulase and resistance to novobiocin. Novobiocin susceptibility test results are 100% sensitive and 96% specific.

S. saprophyticus is innately resistant to the antibiotic novobiocin 

Therefore, screening coagulase-negative staphylococci from urine cultures for novobiocin resistance is a reliable presumptive identification of Staphylococcus saprophyticus. Novobiocin susceptibility test can be done using a tube method and it takes around 5 hours to get the results.

Specimen Collection and Preparation

Testing should be performed on isolated colonies of aerobic, catalase-positive, coagulase-negative gram positive cocci. All colonies should be taken from a blood agar plate and growth must be less than 24  hours old, 15-18 hours being optimal.

Procedure of Novobiocin test

  1. Allow containers to come to room temperature before use.
  2. 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
  3. Inoculate Mueller Hinton Agar, 5% Blood Agar, or Tryptic Soy Agar plate with a sterile swab to obtain confluent growth.
  4. Aseptically apply one 5ug novobiocin disk onto the inoculated agar surface and lightly press down to ensure full contact with the medium.
  5. Incubate plate aerobically for 18 to 24 hours at 35 to 37°C.
  6. Measure (in millimeters) the diameter of the zone of inhibition around the novobiocin disk, and record as susceptible or resistant.

 Interpretation of Novobiocin test

  1. Resistant – zone size of < 12 mm
  2. Sensitive – zone size greater or equal to 16 mm.
    Novobiocin sensitivity testing A. Novobiocin-resistant (zone of inhibition 16mm). Staphylococcus epidermidis
    Novobiocin sensitivity testing A. Novobiocin-resistant  B. Novobiocin Sensitive


Expected results:

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

Quality Control of Novobiocin test:

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

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

Limitation of Novobiocin susceptibility testing method:

The novobiocin disk is not helpful and can give misleading results if it is performed on isolates other that those from urinary specimens. Occasional human isolates that are not S. saprophyticus, S. cohnii subsp., or S. xylosis may also be resistant to novobiocin.

5 thoughts on “Novobiocin Susceptibility Test: Principle, procedure and interpretations

    1. Tankeshwar Acharya

      - Edit


      Thank you for your concern and comment. We prepare the agar plates using commercially available dehydrated media and we purchase Novobiocin disc from the suppliers.

    1. Tankeshwar Acharya

      - Edit


      Anonymous Ji
      As novobiocin is not included in the CLSI chart, there is slight variation regarding zone size interpretation criteria among manufacture of the disk.
      As this drug is not used for the therapeutic purpose but only for the identification of species of the genera Staphylococcus designating intermediate to an isolate makes no sense. So, if >16 mm is sensitive; then <16 mm has been designated as resistant in some literature. Remember, While performing the test, please follow zone size interpretative chart provided by the manufacture of the disk.

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