Bacitracin Test: Principle, Procedure, expected results

Bacitracin is a bactericidal drug useful in the treatment of superficial skin infections but too toxic for systemic use. Bacitracin is a polypeptide antibiotic produced by Bacillus subtilis. This drug interferes in the peptidoglycan synthesis of bacteria. The presumptive identification of group A streptococci (GAS) is usually done by testing for sensitivity to bacitracin.

Principle: Bacitracin test is used to determine the effect of a small amount of bacitracin (0.04 IU or 0.05 IU not higher) on an organism. Streptococcus pyogenes (Group A Streptococci is inhibited by the small amount of bacitracin in the disk; other beta-hemolytic streptococci usually are not.  Some laboratories do not recommend the use of 0.04 U bacitracin disk as Lancefield groups C and G streptococci may occasionally  also show susceptibility to bacitracin. PYR reaction can confirm the isolate as S. pyogenes as its the only beta-hemolytic streptococci that gives positive PYR reaction.

Procedure of Bacitracin test

  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 35oC in ambient air.
  4. Look for zone of inhibition around disk.

    Image 1: Identification chart for Gram Positive cocci

Expected results of Bacitracin test:

  1. Sensitive: Any zone of inhibition around the disk
  2. Resistant: No zone of inhibition

Quality control organisms used in Bacitracin test:

Image 2:Bacitracin (A disk) test for identifying Streptococcus pyogenes
  1. Positive: Streptococcus pyogenes
  2. Negative: Streptococcus agalactiae

Image sources: