Bacitracin Test: Principle, Procedure, expected results

Last updated on May 12th, 2021

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 with the peptidoglycan synthesis of bacteria. The presumptive identification of group A streptococci (GAS) is usually done by testing for sensitivity to bacitracin.


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 Streptococciis 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 it’s the only beta-hemolytic streptococci that gives a positive PYR reaction.

Biochemical test for identification of GPC
Identification chart for Gram Positive cocci Source:

Other Uses

Bacitracin test is a very sensitive assay to separate coagulase-negative staphylococci (resistant) strains with pigment or sticky colony morphology from significant anatomic sites from Rothia mucilaginosa (susceptible) and Micrococcus (susceptible).

Staphylococci should show no zone of inhibition around the bacitracin 0.04-U disk on blood agar plate.

Bacitracin differentiation may also be useful for staphylococci strains since the great majority of Micrococcus and Rothia organisms are susceptible to penicillin and most coagulase-negative staphylococci are not.

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 the disk.

Expected results of Bacitracin test:

Bacitracin (A disk) test for identifying Streptococcus pyogenes
Image 2:Bacitracin (A disk) test for identifying Streptococcus pyogenes
  1. Sensitive: Any zone of inhibition around the disk
  2. Resistant: No zone of inhibition

Quality control

  1. Streptococcus pyogenes: zone of inhibition
  2. Streptococcus agalactiae: no zone of inhibition

Image sources:

  • Image 1:
  • Image 2: University of Florida
About Acharya Tankeshwar 468 Articles
Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. I am working as an Asst. Professor and Microbiologist at Department of Microbiology and Immunology, Patan Academy of Health Sciences, Nepal. If you want me to write about any posts that you found confusing/difficult, please mention in the comments below.