Purpose

This test is used for presumptive identification and differentiation of beta-hemolytic group A streptococci (Streptococcus pyogenes susceptible) from other beta-hemolytic streptococci. It is also used to distinguish staphylococci species (resistant) from micrococci (susceptible).

Principle

The antibiotic bacitracin inhibits the synthesis of bacterial cell walls. A disk (TaxoA) impregnated with a small amount of bacitracin (0.04 units) is placed on an agar plate, allowing the antibiotic to diffuse into the medium and inhibit the growth of susceptible organisms. After incubation, the inoculated plates are examined for zones of inhibition surrounding the disks.

Method

 1. Using an inoculating loop, streak two or three suspect colonies of a pure culture onto a blood agar plate.

 2. Using heated forceps, place a bacitracin disk in the first quadrant (area of heaviest growth). Gently tap the disk to ensure adequate contact with the agar surface.

 3. Incubate the plate for 18 to 24 hours at 35°-37°C in ambient air for staphylococci and in 5% to 10% carbon dioxide (CO2) for streptococci differentiation.

4. Look for a zone of inhibition around the disk.

Expected Results

Positive: Any zone of inhibition greater than 10 mm; susceptible (Figure 1, A).

 Negative: No zone of inhibition; resistant (Figure 1, B).

Limitations

 Performance depends on the integrity of the disk. Proper storage and expiration dates should be maintained.

Quality Control

Positive: Streptococcus pyogenes (ATCC19615)—susceptible Micrococcus luteus (ATCC10240)—susceptible

Negative: Streptococcus agalactiae (ATCC27956)—resistant Staphylococcus aureus (ATCC25923)—resistant

Fig1. Bacitracin (A disk) Susceptibility. Any zone of inhibition is positive (Streptococcus pyogenes); growth up to the disk is negative (Streptococcus agalactiae).