How To Perform A Novobiocin Susceptibility Test

If you’re interested in bacteria, then you may have heard of the novobiocin susceptibility test. To put it simply, the test was designed decades ago in order to help differentiate certain types of bacteria.

How To Perform A Novobiocin Susceptibility Test

The bacteria in question was Staphylococcus saprophyticus, which is a Gram-positive bacteria, meaning that it stains a certain color in a Gram test.

The bacterium is notorious for causing urinary tract infections. However, there are various versions of it, and they vary in resistance to antibiotics like novobiocin.

Therefore, it was important to differentiate between the two, and this is where the test can help. But how can you perform a novobiocin susceptibility test? IOn our guide below, you’ll find out exactly yhow to carry out the test.


Our aim in carrying out a novobiocin susceptibility test is to determine the susceptibility pattern of bacterium to novobiocin, a type of antibiotic. 


The result of using novobiocin is that it interferes with unpackaging and repackaging of DNA.

When? It does it during the bacterial cell cycle and during the process of DNA replication. To do its interfering, the antibiotic binds to DNA gyrase (an enzyme) and blocks the activity of the adenosine triphosphatase. 

Whether there is susceptibility to the novobiocin antibiotic is decided upon by putting a paper disc that has been impregnated with novobiocin on an agar plate that has been seeded with the organism that is being investigated and tested on.

During the incubation period, when the organism is multiplying, the cells get exposed to the novobiocin as it’s diffusing into the agar. 

If the bacteria organism is susceptible to novobiocin, you will notice the appearance of zones of inhibition. These show where the antibiotic concentration has stopped the bacteria from growing further.

However, no inhibition zone will show whether the organism is resistant to novobiocin. This is why an agar plate is seeded a lot with the testing organism in order to make growth on the surface. 

After that seed period, a novobiocin disc is applied to the surface of the agar, and once the incubation has been done then the Kirby-Bauer method can be used to find out how sensitive the organism is to the novobiocin. 


Step 1

Impregnate the novobiocin disc with 5ug of novobiocin, right onto 6mm diameter paper discs. 

Step 2

Prepare a suspension of the “test isolate” in a tryptic soy broth in a test tube. The broth should be equal to something like a McFarland 0.5 standard. The isolate should be between 18 and 72 hours. 

Step 3

Then take a sterile swab and immerse it in the suspension, turning it against the sides of the test tube in order to get rid of excess. After that, take the swab and inoculate a blood agar plate. Do this by streaking your swab over the whole plate surface. 

Step 4

The agar surface now needs to dry, but you should only let it do so for about 15 minutes. Then apply the novobiocin disc. Next, take a sterile swab and swab over the entire plate and edges, going in different directions. This will prepare a whole range of growth across it. 

Step 5

Now get some flamed forceps that have been dipped in alcohol, and aseptically (free of pathogenic organisms) apply the novobiocin disc to the surface of the plates.

The best way to do this is to gently push the discs down. After that, you should aerobically incubate the plate. This should be done at anywhere between 35 and 37 degrees centigrade, and should last somewhere between 18 and 24 hours.

Step 6

Once that time has elapsed, measure the diameter of the inhibition zones. Don’t worry, they should be very visible. Measure them by using a metric ruler, or if you don’t have one, use some sliding calipers instead.


If you’ve followed the method of the experiment correctly, then you should come to some expected results. 

If the result is positive, and the organism is sensitive to the novobiocin antibiotic, then you will have measured an inhibition zone greater than 16 mm.

On the other hand, a negative result will see you measuring it to either 16 mm exactly, or a figure that is less than that. A negative result means that the organism is resistant to the novobiocin antibiotic. 

Benefits And Flaws Of The Test


There are a variety of benefits to performing this test and following the method accurately. For one thing, it is a great way to distinguish staphylococcus saprophyticus from other staphylococci that are coagulase-negative (bacteria that commonly live on a human’s skin).

On top of that, experiments such as these have allowed the CoNS (coagulase-negative staphylococci) to be divided into two categories for extra ease of classification.

One of the categories is susceptible to the novobiocin antibiotic, while the other has resistance.


However, there are also a handful of flaws in the experiment. For one thing, the novobiocin disc can give us inaccurate and misleading results if it is used on isolates that are not from urinary specimens, so it is best to be careful and accurate.

There are some other human isolates that are also resistant to novobiocin, besides the pens that we look at with this experiment.

Testing should really only be carried out on isolated colonies of: coagulase-negative Gram positive cocci that are aerobic and catalase-positive. 

On top of this, you should only perform molecular, mass spectrometry, biochemical, or immunological testing on colonies that are from pure cultures. 

Final Thoughts

If you want to find out how susceptible the certain bacteria are to the antibiotic named novobiocin, then the novobiocin susceptibility test is a great way to find out their levels – or if they’re resistant.

Follow our methodology carefully and study the results with precision to find out.

Jennifer Dawkins

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