Bacitracin Susceptibility Test: Principle, Procedure, and Results

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Bacitracin Susceptibility Test Introduction

Many types of biochemical and antimicrobial tests are being performed in a laboratory in order to detect the specific characteristic of a micro-organism especially a pathogen and its reactions to the certain enzymes.

One such biochemical test is Bacitracin susceptibility test. Bacitracin susceptibility test is one of the antimicrobial tests which are used for identifying a group A streptococcus from the other Beta-hemolytic species of Streptococci.

What is Bacitracin Susceptibility Test?

Bacitracin susceptibility test is considered as an antimicrobial disk tests used foe identifying the Group A Streptococci from the other species of Beta-hemolytic Streptococci.

Antimicrobial susceptibility tests are usually performed in order to differentiate a particular species from another specific genus.

Here, antimicrobial tests are used in order to determine the susceptibility of a bacterial species to a specific antibiotic.


Bacitracin is one of the bactericidal drugs which is used to treat the superficial skin infections. But these are rarely injected for systemic uses.

Bacitracin is obtained from the Bacillus subtilis which is one of the polypeptide antibiotics.

Bacitracin often interferes with the peptidoglycan and help in producing the bacteria and also in inhibiting their growth.

Bacitracin blocks the bactoprenol from transporting the sugars such as NAM and NAG sugars across the cell membranes, which further inhibits the production of peptidoglycan.

The growth of Group A Beta-hemolytic Streptococcus is inhibited by the Bacitracin. Where as the other species of beta-hemolytic streptococci are not inhibiting.

This helps us to differentiate the two different group of organisms. However, Bacitracin disks are mainly used against the Streptococcus pyrogens which helps in inhibiting the growth of an organism.

Bacitracin results in forming the zone of inhibition of about 12mm against the S. pyrogens, hence it helps us to determine the positive control of the organism while testing.

Bacitracin Susceptibility Test Objective

 The main aim of the Bacitracin susceptibility test is to differentiate the Group A Beta-hemolytic Streptococci form the other species of Beta-hemolytic Streptococci.

 To detect the pattern of antibiotic susceptibility in various organisms against Bacitracin.

Bacitracin Susceptibility Test Principle

Generally, the growth of Group A Beta-hemolytic streptococci on the blood agar is inhibited by using 0.04 units of Bacitracin disks.

Whereas the other similar species like Micrococci and Streptococci are also inhibited but this 0.04-unit disc, whereas the other Coagulate-negative Staphylococci are resistant to this.

Bacitracin susceptibility test discs are simply a filter paper disc, which are impregnated with 0.04 units of Bacitracin.

These impregnated discs are then placed on an agar, which allows the antimicrobial to diffuse along with the medium thus inhibiting the growth of the organisms.

This test result is evaluated after incubating based on the zone of inhibition that are formed around the discs.

If the growth is seen at the edges of the disk, the it is deemed as resistant, in other case if the zone is present in a circular zone around the stick, then it represents the inhibition and susceptibility of the organisms.

Usually, Bacitracin disks are time saving experiments along with minimum labor and materials, if they are used in the form of screening test before serological grouping.

It is also said that Group A Streptococci are more sensitive to Bacitracin than the Beta-Hemolytic strains of the other groups.

Hence it is advisable to perform Bacitracin susceptibility test through antimicrobial disks for a rapid diagnosis, especially for Group A Streptococci.

Micro Organisms Tested

 This test is generally used for Penicillin-susceptible test or for stick colonies of the Gram-positive cocci, which are found in groups and catalyzes the positive and negative coagulates.

 Here lemon-Yellow colored colonies are not detected as they are assumed as Micrococcus.

Bacitracin Susceptibility Test Reagents

Media Used:

 Blood agar or

 Muller Hinton Agar

Supplies Used:

 Bacitracin 0.04-unit discs

 Sterile forceps

 Swab

 Inoculation broth.

Bacitracin Susceptibility Test Procedure

Usually, two different kinds of methods are used for Bacitracin Susceptibility test based on the kind of culture media they are used.

The test can be performed either by using pure culture of an organism or directly via clinical samples. The methods of Bacitracin susceptibility tests are listed below.

1. Hebert’s Method Using Blood Agar Plates

Initially 0.1 McFarland suspension of the organism is performed using a over night culture of the organism.

Different sections of blood agar plate are inoculated which results in forming a lawn culture.

Here each section is inoculated in one specific direction and the area of inoculation should be at a separation of 10mm between each of the discs that were placed.

These are then left to dry for about 10 minutes and after drying they are placed on the agar using sterile forceps.

Then the disc is tapped using the sterile stick and adherence is ensured.

The plates are then incubated at a temperature of about 35 to 37ºC for about 24 hours.

After a period of incubation, the zone of incubation is observed thus results are measured.

Further the results are confirmed using serological testing.

2. Muller-Hinton Agar Method

This method is also used to observe the susceptibility of the fast-growing organisms. Here 0.5 Mc Far land suspension of the organisms is prepared using an over night culture of the organism.

The MHA plates disks are inoculated using a suspension with sterile swabs to form a bacterial lawn on the agar.

Then the medium is allowed to dry. After drying the antibiotic disks are placed on the agar placed using the sterile forceps and the discs are placed by maintaining a distance of about 10mm.

Then further the discs are tapped using sterile sticks.

Then the inverted plates are incubated at a temperature of about 35 to 37ºC for about 24 hours.

After incubation the zone of inhibition is observed and measured.

Bacitracin Susceptibility Test Results
Zone of InhibitionMediaResult
Zone of inhibition is 6mm or less than thatBlood agar or Muller Hinton AgarResistant
Zone of inhibition is greater than 10mmBlood agar or Muller Hinton AgarSusceptible
Zone of inhibition is between 6mm to 10mmBlood agar or Muller Hinton AgarIt indicates probable susceptibility so the tests should be repeated
Bacitracin Susceptibility Test Citations


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