Assessment of a new antibiotic (Culture sensitivity testing)

Assessment of a new antibiotic activity can be done by culture sensitivity testing. Antibiotic
susceptibility testing is important for choosing the correct antibiotic for treatment.

Testing bacteria for antibiotic susceptibility:

 Cup plate or disk plate method (diffusion method)

 Broth dilution method.

Agar diffusion method:

 Suitable nutrient medium plates are prepared

 Bacterial culture isolated from the infection is spread on the surface of the medium. Let the
surface gets dried.
 Cups are made within the solid medium. (Approximately 6 cups for each plate of 5-8 mm in
diameter). Alternatively paper disks impregnated with antibiotic can be used.
 Standard concentrations of antibiotics are applied into the cups.
 The plates are left for diffusion of antibiotic at low temperature. Based on the type and
concentration each antibiotic diffuses at different rates.
 Incubate the plates for 18 to 24 hours. (35 oC to 37 oC).
 Record the zones of inhibition in mm.

Interpretation:

The width of the zone is compared to a standard table of predetermined zone widths representing
antibiotic concentrations in the agar that correlates with the concentration of antibiotic achievable in
the plasma of a patient using the manufacturer’s recommended dosage.

If the zone of inhibition is wider than the predetermined zone, the bacterial species is considered to be
susceptible to the antibiotic.

If the zone of inhibition is less than the predetermined zone, the bacterial species is considered to be
resistant to the antibiotic.

If the zone of inhibition is equal to the predetermined zone, the result is intermediate.

Broth dilution method (MIC):

Test tubes or wells containing increasing concentrations of each antibiotic to be tested are inoculated
with a fixed volume of nutrient broth containing standard concentration of bacterial culture. The tubes
are incubated for 18 to 24 hours approximately at 35oC to 37 oC and observed for turbidity. A clear
sample is a clear indication of inhibition of bacterial growth. The MIC is the lowest concentration of the
antibiotic that inhibits the growth of the antibiotic.
To determine whether the bacterial culture is susceptible, intermediate or resistance, the MIC is
compared with that of concentration of the antibiotic that can be achieved in the plasma of the patient
using the manufacturer’s recommended doses.

If the MIC is less than the range of the antibiotic within the plasma, the bacterial culture is susceptible.

If the MIC is more than the range of the antibiotic within the plasma, the bacterial culture is resistant.

If the MIC is equal to the range of the antibiotic within the plasma, the result is intermediate.