Novobiocin Susceptibility Test Principle, Procedure, Results

  • The novobiocin (antibiotic) differentiation disc can be used in the Novobiocin Susceptibility Test to help make a preliminary distinction between Staphylococcus saprophyticus and other coagulase-negative staphylococci (CoNS) in clinical (urinary) specimens.
  • Streptomyces nivens is the actinomycete responsible for producing novobiocin.
  • Staphylococcus saprophyticus was once thought to be a skin contaminant, but recent research has shown that it is a major uropathogen, even in healthy individuals.
  • Cystitis and acute UTIs are caused by this bacteria almost as often as by E. coli in young, healthy women.
  • It is thought that the organism’s frequent role in urinary tract infections is due in part to its ability to adhere to uroepithelial cells more frequently and effectively than other staphylococcal species.


  • To find out if a bacterium can be killed by the antibiotic novobiocin.

Principle of  Novobiocin Susceptibility Test

  • Uropathogenic S. saprophyticus is intrinsically resistant to the novobiocin and causes acute, uncomplicated urinary tract infections, especially in young and middle-aged female patients.
  • Antibiotic novobiocin prevents bacteria from completing their cell cycle by interfering with DNA replication. It inhibits adenosine triphosphatase (ATPase) activity by binding to DNA gyrase.
  • S. saprophyticus can be reliably presumed to be present when screening organisms from urine cultures for novobiocin resistance based on the following characteristics: non hemolytic on blood agar; coagulase-negative; Gram response; presence of Gram-positive cocci in single, pair, and cluster.
  • A novobiocin disc is allowed to reach ambient temperature. A pure 18-24 hour culture is used to generate a suspension in Tryptic Soy Broth, Sterile Water, or Brain Heart Infusion (BHI) Broth that is equivalent to a McFarland 0.5 opacity standard.
  • Confluent growth is achieved by inoculating a Mueller Hinton agar plate with a sterile swab containing the organisms of interest.
  • An inoculated agar disc is placed on top and gently pressed down to make full contact with the medium.
  • At the end of the 18-24 hour aerobic incubation at 35-37 degrees Celsius, the size of the zone of inhibition around the disc can be measured and recorded as either susceptible or resistant.
  • A sensitive zone of inhibition is larger than 16 mm, while a resistant zone of inhibition is smaller than or equal to 16 mm.

Test organism

  • Isolated colonies of gram-positive cocci in clusters that are catalase-positive and coagulase-negative should be tested. Usually, these colonies are found in the urine of sexually active young women.
  • All of the colonies should be taken from a plate of blood agar, and the growth should be less than 24 hours old, ideally between 15 and 18 hours.

Requirements for Novobiocin Susceptibility Test 

  • Gram-positive cocci in the cluster, a positive catalase test, a negative coagulase test, and usually nonhemolytic
  • Tryptic Soy broth, sterile water, or brain-heart infusion (BHI) Broth
  • Getting wire ready
  • Bunsen burner
  • Sterile test tubes
  • McFarland Densitometer ( optional)
  • Cotton swabs
  • Mueller Hinton agar plate (MHA) or Blood agar
  • Novobiocin disc
  • Incubator\sControl Strains: Staphylococcus saprophyticus (ATCC 15305) was used as a positive control (resistant) and a negative control (sensitive) –Staphlococcus epidermidis (ATCC 12228)
  • Sliding calipers/ metric ruler

Novobiocin susceptibility test Procedure

  1. Let the discs get to the same temperature as the room.
  2. Using a pure 18–24-hour culture, make a suspension in Tryptic Soy Broth, Sterile Water, or Brain Heart Infusion (BHI) Broth that is equal to a McFarland 0.5 opacity standard.
  3. To get rid of extra inoculum, dip a sterile swab into the suspension and rub it against the side of the tube above the fluid level.
  4. Inoculate Mueller Hinton Agar (MHA), Blood Agar, 5%, or Tryptic Soy Agar (TSA) plate by smearing the expressed swab over the entire agar surface and repeating in two planes to get confluent growth.
  5. Use a sterile swab to spread growth over the whole plate in three directions and around the edge of the plate to make a lawn.
  6. Before putting a disc on the agar surface, let it dry for 15 minutes.
  7. Using sterile forceps, place one novobiocin disc on the surface of the inoculated agar and lightly press it down to make sure it makes full contact with the medium.
  8. Plate should be kept at 35 to 37°C for 18 to 24 hours with air.
  9. Use sliding callipers or a metric ruler to measure the diameter of the zone of inhibition around the novobiocin disc in millimetres. Write down whether the bacteria are susceptible or resistant.

Expected Results

  • Positive: The organism is sensitive to the antibiotic if the zone of inhibition is larger than 16 mm.
  • Negative: Novobiocin resistance is shown by a zone of inhibition that is less than or equal to 16 mm.
Novobiocin Susceptibility Test Principle, Procedure, Results
Novobiocin Susceptibility Test Principle, Procedure, Results

Uses of Novobiocin Susceptibility Test

  • Staphylococci that are negative for the coagulase enzyme are called coagulase-negative staphylococci (CoNS), and they have been split in two groups depending on their susceptibility to the antibiotic novobiocin. Staph epidermidis, S. haemolyticus, S. hominis subsp. hominis, S. capitis, S. lugdunensis, S. saccharolyticus, S. warneri, and other species all belong to the CoNS group that shows novobiocin susceptibility.
  • Bacteria strains S. cohnii, S. xylosus, and S. pulvereri are all part of the novobiocin-resistant clade.
  • Preliminarily separating Staphylococcus saprophyticus from other CoNS in clinical specimens is facilitated by this method.

Limitation of Novobiocin Susceptibility Test

  • If the novobiocin disc is used on isolates from sources than urine, it will not be beneficial and may produce false positives.
  • It is possible that staphylococci other than Staphylococcus saprophyticus, such as Staphylococcus cohnii, Staphylococcus xylosus, and Staphylococcus pulvereri, are resistant to novobiocin when isolated from human patients.
  • Aerobic, catalase-positive, coagulase-negative, Gram-positive cocci in clusters should be the only test subjects.
  • Additionally, it is suggested that organisms be tested using biochemical and immunological means.
  • Occasionally, novobiocin-resistant human isolates are not S. saprophyticus, S. cohnii subsp., or S. xylosis.

Quality Control

Novobiocin tests for quality control should be run with known organisms per lot/shipment date.

  • Positive control (resistant) = Staphylococcus saprophyticus  (ATCC® 15305)
  • Negative control (sensitive) = Staphylococcus epidermidis (ATCC® 12228)



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