Bacitracin Test: Principle, Procedure, expected results

Identification chart for Gram Positive cocci Source:

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.

    Identification chart for Gram Positive cocci Source:
    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:

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

Image sources:

  • Image 1:
  • Image 2: University of Florida


About Acharya Tankeshwar 458 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.


  1. Dear Mr. Acharya;
    Congrants for such an useful blog.
    Recently i faced a difficult situation. In a diagnostic laboratory exam, i was asked to perform Bacitracin test to diagnose an unknown Microorganism. After one night incubation i saw an inhibition zone about 12 mm around the bacitracin disk. According to the brochure of the disk i used, inhibition zone more than 15 mm is interpretd as sensetive;So i wrote “Resistant” as the answer.
    Unfortunately i failed the exam and i was said that “any zone” around the disk should be interpreted sensitive.
    Now i am confused which one is correct; any zone or zone more than 15 mm.
    I am intensively looking forward to hearing your help.

    Yours sincerely, Fatemeh Azizmohamadi

    P.S: The bacitracin disk i used is produced in Rosco Diagnostica company

    • Dear Fatemeh Azizmohamadi
      Sorry to hear that you failed in that particular examination, believe me, that’s not the end. I am hopeful next time you will strike high and big and get success. Regarding Bacitracin sensitivity test, like i have mentioned in the blog post, we (in our diagnostic laboratory) do not measure the inhibition zone size. Any inhibition around the edge of bacitracin disk is reported as sensitive. This is in accordance with available practices and literature too.

  2. Is that possible to get charts for other gm pos, gm neg, variable Bacterial identification…
    Here is very good chart of coccie identification.

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