Bacitracin Test: Principle, Procedure, Results

Last updated on June 25th, 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:

Quality Control

Perform sterility testing and performance testing blood agar plate and/or chocolate agar plate according to CLSI guidelines. Test the disk potency after each shipment or purchase of the bacitracin disk with the appropriate test organism.

Test organismBacitracin (10 IU) zone size
Haemophilus influenzae ATCC 10211No zone  
Staphylococcus epidermidis ATCC 1222812 mm

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 CO2.
  4. Look for a zone of inhibition around the disk.
Bacitracin (A disk) test for identifying Streptococcus pyogenes
Image 2:Bacitracin (A disk) test for identifying Streptococcus pyogenes

*Note: If used on direct sputum culture plates, use chocolate agar for bacitracin and blood agar plate for optochin. Addition of bacitracin disk (not Taxo A) to chocolate agar inhibits upper respiratory microbiota and improves detection of Haemophilus influenzae.


  1. Bacitracin sensitive: Any zone of inhibition around the disk. For example, Streptococcus pyogenes
  2. Bacitracin resistant: No zone of inhibition around the disk. Streptococcus agalactiae

Image sources:

  • Image 1:
  • Image 2: University of Florida
About Acharya Tankeshwar 474 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.