Bacitracin Test: Principle, Procedure, Results

Bacitracin is a bactericidal drug that is useful in treating superficial skin infections but is too toxic for systemic use. Bacitracin is a polypeptide antibiotic produced by Bacillus subtilis.

Bacitracin interferes with the peptidoglycan synthesis of bacteria. The presumptive identification of group A streptococci (GAS) is usually made 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 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 35°C 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

Acharya Tankeshwar

Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. As an asst. professor, I am teaching microbiology and immunology to medical and nursing students at PAHS, Nepal. I have been working as a microbiologist at Patan hospital for more than 10 years.

6 thoughts on “Bacitracin Test: Principle, Procedure, Results

  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

    1. 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.

  3. do we get zone around bacitracin around alpha haemolytic colonies also, we many a time observed this

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