• After E. coli, Staphylococcus saprophyticus is the second most causative organism for the Urinary tract infections (UTIs) in sexually active young women.
• Laboratory identification of Staphylococcus saprophyticus is performed on the basis of haemolysis, coagulase and resistance to novobiocin.
• Thus, after isolation of coagulase negative staphylococcus, laboratory should further identify the isolate and find out if the isolate is sensitive to novobiocin or not.
• Novobiocin is the antibiotic produced by the actinomycete Streptomyces nivens.
• Sensitivity to novobiocin is detected by placing a novobiocin impregnated paper disk on a agar plate seeded with the organism under identification.
• If the bacteria are sensitive to novobiocin, the formation of visible zone of inhibition  is seen around the disk.
• The zone of inhibition represents the area where the growth of organism was inhibited by the antibiotic concentration.
• No zone of inhibition suggests the resistance of organism to the antibiotic.

Requirements:

1. Isolated colonies of an aerobic, catalase positive, coagulase negative gram positive cocci.
2. Mueller Hinton agar
3. Blood agar/ Tryptic Soy agar
4. Incubator
5. Novobiocin disk
6. Distilled water
7. Sliding calipers/ metric ruler

Procedure of Novobiocin susceptibility test

1. The test isolate taken is of 18-72 hours and in pure culture.
2. Prepare a suspension of the test isolate in tryptic soy broth equal to a McFarland 0.5 standard or equivalent.
3. Dip a sterile swab into the suspension and rotate it against the side of the tube above the fluid level in order to remove excess inoculum.
4. Inoculate a blood agar or Mueller Hinton agar plate by streaking the expressed swab over the entire agar surface and repeat in 2 planes.
5. Allow the agar surface to dry for exactly15 minutes before applying a Novobiocin Disk.
6. Prepare a lawn of growth over the entire plate by use of sterile swab, swabbing over the entire plate in 3 directions and around the edge of the plate.
7. Using alcohol-dipped and flamed forceps, aseptically apply a novobiocin antibiotic disc to the surface of each inoculated plate.
8. Use sterile forceps to gently press the discs down to make sure that they adhere to the agar surface.
9. Incubate plate aerobically for 18 to 24 hours at 35 to 37°C.
10. Use sliding calipers or a metric ruler to measure the diameter of the zone of inhibition.

Results interpretation:

• Positive result:
  • The positive test is indicated by the zone of inhibition greater than 16mm suggesting the sensitivity of the organism.
• Negative result:
  • Negative test is indicated by the zone of inhibition less than 16mm which suggests the novobiocin resistance of the organism.

Limitations:

• Only isolated colonies of aerobic, catalase positive, coagulase negative gram positive cocci are to be tested.
• Biochemical, immunological tests are further recommended for the complete identification of the organism.
• Novobiocin disks can mislead the results if the test is performed for isolates other than urinary specimens.

Novobiocin susceptibility test: Principle, Procedure and Results interpretations

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