About this test
What is the purpose of the test?
Bacitracin is an antibiotic interfering with the synthesis of peptidoglycan, a major component of bacterial cell walls. Different types of bacteria have different degrees of susceptibility to bacitracin. This test determines whether the bacterium is either sensitive (susceptible) to bacitracin or resistant to the drug. Knowledge about bacitracin susceptibility is valuable in identification of Gram positive cocci, some of which are susceptible and others of which are resistant. For different groups of Gram positive cocci, susceptibility may be to antibiotic concentrations of only 0.04 U (units) of bacitracin, while other identifications are aided by knowledge of susceptibility to 2.0 U of the antibiotic.
How is bacitracin susceptibility determined?
Susceptibility to bacitracin is determined by placing a bacitracin-impregnated paper disk on a nutrient agar plate seeded with the microbe under investigation. As the microbe multiplies during incubation to produce a lawn of confluent growth, cells are exposed to the antibiotic diffusing into the agar from the paper disk. If the bacteria are susceptible to bacitracin, there will be a visible zone of inhibition forming around the disk, representing an area where the antibiotic concentration has prevented bacterial growth. Should the microbe be resistant, the lawn of cells will form visible growth up to the margin of the disk. This test can be performed using disks impregnated with either 0.04 U or 2.0 U concentrations of bacitracin. In either instance, the mechanism of the test is the same.
What medium is used?
The medium used for growing the bacterial lawn is typically either blood agar or a nutrient rich, general-purpose medium like nutrient agar . In VirtualUnknown:Microbiology™, we use nutrient agar .
How is the test performed?
An inoculum from a pure culture is transferred aseptically to a sterile plate of nutrient agar, typically by use of cotton-tipped applicator (swab). In a clinical setting, the applicator often contains other fibers than cotton to prevent any toxic residue found in cotton after processing from interfering with the growth of delicate pathogens. The inoculum is spread over the plate to distribute the bacteria as evenly and thoroughly as possible over the entire agar surface. An antibiotic-impregnated disk containing bacitracin is then aseptically placed in the center of the agar surface. The inoculated plate is incubated at 35-37 C for 24 hours and the results are determined. Growth of the lawn up to the margin of the disk indicates the bacterium is resistant to the antibiotic. An obvious clear zone around the disk, termed the zone of inhibition , indicates the bacterium to be susceptible to the antibiotic.
What reagents are needed?
None. The disk is added before incubation. No additions are made following incubation.
To perform this test in VUMIE Online© Online Lab, complete the following steps:
Inoculation of Medium
1. Select the nutrient agar plate medium.
2. Complete the process of a tube-to-lawn aseptic transfer to inoculate the medium.
Addition of the Antibiotic Sensitivity Disk
3. Once the lawn has been created and BEFORE incubating the plate, add select a Bacitracin disk in the desired concentration and place it on the lawn.
4. Replace the cap on the inoculum tube and the plate lid.
Incubation of the Inoculated Medium
5. Place the inoculated tube into the 35-37 C incubator.
6. Press the New Day button to move forward 24 hours.
Determination of Test Results
7. Retrieve the incubated culture from the incubator.
8. Observe the growth on the surface of the plate, especially in the vicinity of the antibiotic disk. If there is a visible zone of inhibition surrounding the disk, an area where the lawn has not grown due to the antibiotic in the disk, the microbe is susceptible to the antibiotic. If no zone is present, the microbe is resistant to the antibiotic.