Bacitracin Sensitivity Test: Introduction, Principle, Test Procedure and Result Interpretaion and Keynotes
Introduction of Bacitracin Sensitivity Test
Bacitracin Sensitivity Test is applied to differentiate genera like Micrococcus and Stomatococcus from Staphylococcus when combined with other procedures such as the modified oxidase test. It is useful to test for presumptive identification of Group A streptococci ( Streptococcus pyogenes)
Principle of Bacitracin Sensitivity Test
Bacitracin is a polypeptide antibiotic derived from Bacillus subtilis that functions to block cell wall formation by interfering with the dephosphorylation of the lipid compound that carries peptidoglycans to the growing microbial cell wall. Bacitracin (0.04 units) inhibits the growth of Micrococcus and Stomatococcus and Streptococcus pyogenes while having no effect on Staphylococcus which is resistant.
Test Requirements for Bacitracin Sensitivity Test
- O.04 U bacitracin disk
- Test organisms
- McFarland Densitometer
- Glass Test tubes
- Blood agar/Mueller Hinton Agar supplemented with 5%
Sheep blood (preferred) - Swab stick
- Bunsen burner
- Inoculating wire
- Incubator
- Disk applicator/ forceps
- Control Strains (Positive control-Streptococcus pyogenes and Negative control-Staphylococcus aureus)
Test Procedure of Bacitracin Sensitivity Test
- Blood agar/Mueller Hinton Agar supplemented with 5%
Sheep blood is used in these antibiotic susceptibility tests due to being fastidious nature of organisms. - Emulsify heavy colonies in sterile saline matching the turbidity that standard (0.5 McFarland).
- Place a sterile cotton swab in the bacterial suspension and remove the excess fluid by pressing and rotating the cotton against the inside of the tube above the fluid level.
- The swab is streaked in three directions over the surface of the antimicrobial sensitivity testing agar (Blood agar/MHAsupplemented with 5%
Sheep blood to obtain uniform growth. - Allow the plates to dry for 10-15 minutes.
- Using sterile forceps or a suitable disk dispenser, place paper disks impregnated with a fixed concentration of an antibiotic, on the surface agar plates having distance disc to disc 25 mm while plate border to disk 15 mm.
- Incubate the plates at 37°C for 24 hours.
- Following overnight incubation, measure the diameter of the zone of inhibition in millimeters (mm) around each disk.
Result and Interpretation of Bacitracin Sensitivity Test
Sensitive/Susceptible: zones of inhibition greater than or equal to (≥) 10mm
Resistant: zones of inhibition less than (<) 10 mm
Susceptible: Micrococcus and Stomatococcus
Resistant: Staphylococcus species
Keynotes on Bacitracin Sensitivity Test
- 0.04 U bacitracin uses for presumptive identification of Streptococcus pyogenes .
- 10 units bacitracin is used for screening Haemophilus from sputum specimen during culture due to sputum having a variety of commensals that may interfere with Haemophilus and due to being fastidious nature hard to recover.