Bacitracin Susceptibility Test- Principle, Procedure, Results, Uses

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What is Bacitracin Susceptibility Test?

Bacitracin susceptibility test is one of the antimicrobial disk tests for the identification of group A Streptococci from other β-hemolytic Streptococci.

  • Antimicrobial susceptibility tests are mostly performed for the differentiation of different species of a particular genus.
  • Antimicrobial disks are used to determine the susceptibility of bacteria to particular antibiotics.
  • Bacitracin is a bactericidal drug that is used in the treatment of superficial skin infections and is rarely injected for systemic use.
  • Bacitracin is obtained from Bacillus subtilis as a polypeptide antibiotic. It interferes with the peptidoglycan production in bacteria, which then inhibits the growth.
  • It blocks bactoprenol from transporting NAM and NAG sugars across the cell membrane, thereby inhibiting the further synthesis of peptidoglycan.
  • The growth of Group A β-hemolytic Streptococci is inhibited by Bacitracin whereas other β-hemolytic Streptococci are not. This helps in the differentiation between the two groups of organisms.
  • Bacitracin disks are mainly used against Streptococcus pyogenes as it inhibits the growth of the organism.
  • Bacitracin results in about 12mm of the zone of inhibition against S. pyogenes, which is then used as positive control while testing against other groups of organisms.

Objectives of Bacitracin Susceptibility Test

  • To differentiate Group A beta-hemolytic Streptococci (primarily S. pyogenes) from other beta-hemolytic Streptococci.
  • To observe the pattern of antibiotic susceptibility of different organisms against Bacitracin.

Principle of Bacitracin Susceptibility Test

  • The growth of Group A beta-hemolytic Streptococci on blood agar is inhibited by 0.04 units Bacitracin disc.
  • Micrococci and streptococci are also inhibited by 0.04 units disc, while all coagulase-negative staphylococci are resistant.
  • Bacitracin susceptibility test discs are filter paper discs impregnated with 0.04 units of Bacitracin. The impregnated disks are then placed on the agar where allows the antimicrobial to diffuse through the medium and inhibit the growth of the organism.
  • The test result is evaluated after incubation on the basis of the zone of inhibition observed around the disks.
  • If the growth of the organism is observed up to the edge of the disk, it is deemed resistant, whereas the presence of a circular zone around the disk represents inhibition and susceptibility.
  • Bacitracin discs can save considerable time, labor, and materials if used as a screening test before serological grouping.
  • It has been observed that Group A Streptococci were more sensitive to Bacitracin than beta-hemolytic strains of other groups.
  • Hence Bacitracin susceptibility test via antimicrobial disks is suggested as a rapid diagnostic agent for Group A Streptococci.

Microorganism tested

  • Bacitracin susceptibility test is used for penicillin-susceptible or “sticky” colonies of Gram-positive cocci in clusters that are catalase-positive and coagulase-negative from invasive-site specimens to separate Staphylococci from Micrococci.
  • Lemon yellow-colored colonies are not tested as those are presumed to be Micrococcus.

Media, reagents, and supplies used


  • Blood agar or
  • Mueller Hinton Agar


  • Bacitracin 0.04U disks
  • Swabs
  • Sterile forceps
  • Broth for inoculation

Procedure of Bacitracin Susceptibility Test

Two different procedures are available for the Bacitracin Susceptibility test using two different nutrient media. The testing can be done with both pure cultures of an organism obtained via subculture or directly from the clinical samples. Procedures for two different methods of Bacitracin susceptibility test is given below:

Hebert’s method using Blood Agar plates

  1. 0.1 McFarland suspension of the organism is prepared from the overnight culture of the organism.
  2. Different sections of the blood agar plate should be inoculated, resulting in lawn culture. Each of the sections should be inoculated in one particular direction, and the area of inoculation should allow a separation of 10 mm between each disk to be placed on the plate.
  3. After about 10 minutes for the drying, the disks are placed on the agar with sterile forceps. The disks are then tapped with a sterile stick to ensure adherence.
  4. The plates are then incubated at 35-37°C for 24 hours.
  5. After incubation, the zone of inhibition is observed and measured. Further confirmation can be obtained by serological testing.

Mueller Hinton Agar method

  1. Mueller Hinton agar can also be used to observe the susceptibility of fast-growing organisms.
  2. 0.5 McFarland suspension of the organism is prepared from the overnight culture of the organism.
  3. The MHA plates are then inoculated with the suspension with sterile swabs to form bacterial lawns on the agar.
  4. After about 10 minutes of drying, the antibiotic disks are placed on the agar plates with sterile forceps, allowing a distance of about 10 mm between the disks.
  5. The disks are tapped with a sterile stick to ensure adherence of the disk to the plates.
  6. The inverted plates are then incubated for 24 hours at 35-37°C.
  7. The zone of inhibition is observed and measured. Further confirmation can be obtained by serological testing.

Result Interpretation of Bacitracin Susceptibility Test

Bacitracin Susceptibility Test- Principle, Procedure and Result Interpretation

Image Source: Bailey and Scott’s Diagnostic Microbiology. Elsevier.

Zone of inhibition Media Result
6 mm or less Blood Agar or Mueller Hinton Agar Resistant
Greater than 10 mm Susceptible
Between 6 mm and 10 mm Tests are to be repeated as it indicates probable susceptibility.

Uses of Bacitracin Susceptibility Test

  • Bacitracin susceptibility test is primarily used for the differentiation of Group A beta-hemolytic Streptococci from other beta-hemolytic Streptococci.
  • It can also be used to observe the pattern of antibiotics susceptibility of different organisms against Bacitracin.
  • Bacitracin differentiation may also be useful for penicillin-susceptible strains since the great majority of Micrococcus, and Rothia organisms are susceptible to penicillin, and most coagulase-negative staphylococci are not.

Limitations of Bacitracin Susceptibility Test

  • Old blood agar plates that have dried should not be used for susceptibility testing as the diffusion of antibiotics on such agar is reduced, which might result in false-negative results.
  • Different zone of inhibition sizes might be observed with different concentrations of Bacitracin; thus, differential disks (0.04 U) should be used instead of sensitivity disks (10 U).
  • Light inoculum might result in a false zone of inhibition, and thus, sufficient inoculum resulting in confluent growth should be used.
  • For further confirmation, serological testing like latex agglutination tests should be performed to obtain accurate confirmation.
  • Staphylococci show no zone of inhibition around the bacitracin 0.04-U disk on BAP. Zone of inhibition of sizes greater than7 mm but less than the 10-mm breakpoint may be obtained for Micrococcus if incubation is not a full 24 h or MH agar is used.

References and Sources

  • Biochemical Tests for the Identification of Aerobic Bacteria. (2016). Clinical Microbiology Procedures Handbook,– 
  • Bacitracin Susceptibility Test Discs. DD015. HiMedia Laboratories.
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About Author

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Anupama Sapkota

Anupama Sapkota has a bachelor’s degree (B.Sc.) in Microbiology from St. Xavier's College, Kathmandu, Nepal. She is particularly interested in studies regarding antibiotic resistance with a focus on drug discovery.

1 thought on “Bacitracin Susceptibility Test- Principle, Procedure, Results, Uses”

  1. Great post. Wondering why I can’t see a photo of the agar plate after I click on the link shown in the “Search Results” section?


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