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Novobiocin Sensitivity Test Procedure

In this Novobiocin sensitivity test procedure post we have briefly explained about Novobiocin sensitivity test principle, objectives, requirements, novobiocin sensitivity test procedure, uses and limitations of novobiocin sensitivity test.

Novobiocin Sensitivity Test

The novobiocin (antibiotic) differentiation disc is used in the novobiocin sensitivity test to presumptively identify Staphylococcus saprophyticus from other coagulase-negative staphylococci (CoNS) in clinical ( urine) specimens.

Streptomyces nivens, an actinomycete, produces novobiocin. Previously thought to be skin pollutants that only affected immunocompromised people, Staphylococcus saprophyticus has now been identified as a serious uropathogen.

In healthy, young adult women, it is only second to E. coli as the most prevalent cause of cystitis and acute urinary tract infection (UTI).

It adheres to uroepithelial cells more frequently and successfully than other staphylococcal species, which is thought to explain why it is so common in urinary tract infections.

Principle

Novobiocin is an antibiotic that interferes with DNA during DNA replication and the bacterial cell cycle. It binds to DNA gyrase and blocks adenosine triphosphatase (ATPase) activity.

Screening of organism on the basis of the following properties-Non hemolytic on blood agar, coagulase-negative, Gram reaction having Gram-positive cocci in single, pairs and clusters from urine cultures for novobiocin resistance is reliable presumptive identification of S.saprophyticus.

Novobiocin disk is allowed to equilibrate to room temperature. A suspension equivalent to a McFarland 0.5 opacity standard is prepared either in Tryptic Soy Broth or Sterile Water or Brain Heart Infusion (BHI) Broth using a pure 18-24 hour culture.

Organisms are inoculated on  Mueller Hinton agar plate with a sterile swab to obtain confluent growth.

A disk is applied onto the inoculated agar surface and lightly press down to ensure full contact with the medium. Incubate aerobically for 18-24 hours at 35-37ºC and finally measure the diameter of the zone of inhibition around the disk, and record as susceptible or resistant.

A zone of inhibition greater than 16 mm is sensitive whereas a zone of inhibition less than or equal to 16 mm is resistant.

Requirements

1. Sterile Water

2. Inoculating wire

3. Bunsen burner

4. Sterile test tubes

5. Swab sticks

6. Novobiocin disk

7. Incubator

Procedure

1. Allow the discs to reach room temperature. Prepare a suspension, comparable to a McFarland 0.5 opacity standard, from a pure 18-24 hour culture in Tryptic Soy Broth, Sterile Water, or Brain Heart Infusion (BHI) Broth.

2. To remove excess inoculum, dip a sterile swab into the suspension and rotate it against the side of the tube above the fluid level.

3. To achieve confluent growth, streak the expressed swab over the entire agar surface of Mueller Hinton Agar (MHA), Blood Agar, 5 percent, or Tryptic Soy Agar (TSA) plate in two planes.

4. Use a sterile swab to create a lawn of growth over the entire plate, swabbing in three directions and around the edge of the plate.

5. Allow 15 minutes for the agar surface to dry before placing a disc. Using sterile forceps aseptically apply one novobiocin disc to the infected agar surface and lightly press down to ensure full contact with the medium.

6. Incubate plate aerobically at 35–37°C for 18–24 hours. Using sliding callipers or a metric ruler, measure the diameter of the zone of inhibition around the novobiocin disc in millimetres and note as susceptible or resistant.

Results

Keep an eye on the growth on the plate’s surface, especially around the antibiotic disc. The bacterium is susceptible to the antibiotic if there is a visible zone of inhibition surrounding the disc. The bacteria is resistant to the antibiotic if there is no zone.

Novobiocin Susceptibility Test

Positive: If the zone of inhibition is bigger than 16mm, the organism is susceptible to the antibiotic.

Negative: Novobiocin resistance is indicated by a zone of inhibition less than or equal to 16mm.

Advantages

1. Based on their novobiocin sensitivity test, coagulase-negative staphylococci (CoNS) have been split into two groups. Staphulococcus epidermidis, Staphulococcus haemolyticus, Staphulococcus hominis subsp. hominis, Staphulococcus capitis, Staphulococcus lugdunensis, Staphulococcus saccharolyticus, Staphulococcus warneri, and other species belong to the CoNS group that shows novobiocin susceptibility.

2. Staphylococcus cohnii, Staphylococcus xylosus and  Staphylococcus pulvereri belong to the novobiocin resistant group.

3. Novobiocin sensitivity test can be used to differentiate Staphylococcus saprophyticus from other CoNS in clinical specimens.

Limitations

1. When used on isolates other than those from urinary samples, the novobiocin sensitivity test is ineffective and can produce false findings.

2. Coagulase-negative staphylococci (CoNS) that aren’t Staphylococcus saprophyticus, like Staphylococcus cohnii, Staphylococcus xylosus, or Staphylococcus pulvereri, may also be resistant to novobiocin sensitivity test.

3. Only isolated colonies of Gram-positive cocci that are aerobic, catalase-positive, coagulase-negative, and catalase-positive in clusters will be examined.

4. For the conformation of organisms, biochemical and immunological studies are also advised.

Further Readings

Reference