Bacitracin test : principle, procedure and expected results

Bacitracin Test: Introduction, Principle, Procedure and Expected Results

Use of bacitracin 10U for haemophilus screening

Introduction of bacitracin

Bacitracin is a peptide produced by Bacillus subtilis and was first isolated in 1945. It uses as an antibiotic and it disrupts Gram-positive bacteria by interfering with the cell wall and peptidoglycan synthesis. It is primarily used as a topical preparation because it can cause kidney damage when used internally. But here we discuss its diagnostic roles in microbiology. 0.04 U bacitracin uses for presumptive identification of Streptococcus pyogenes while 10 units for screening Haemophilus from sputum specimen during culture due to sputum having a variety of commensals those may interfere Haemophilus and due to being fastidious nature hard to recover.

Principle of Bacitracin

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. Haemophilus is resistant to bacitracin (10U) whereas most common bacteria are sensitive. It makes it easier to screen Haemophilus influenzae in sputum growing around the bacitracin disc.

Requirements for bacitracin test

Sputum specimen
bacitracin disc (10U)
Inoculating loop
Chocolate agar
Bunsen burner
Sterile forceps
CO₂incubator

Procedure of Bacitracin Test

Inoculate sputum into chocolate agar by inoculating loop.
After that, put the 10U bacitracin disc in the primary inoculum of chocolate agar.
Incubate chocolate agar plate at 37 °C in 5-10% CO₂ incubator for 24-48 hours.
After overnight incubation read the result.

Observations of Bacitracin Result

Observe the growth organism around the bacitracin disc. colony morphology of Haemophilus influenzae. The odor of the organisms.

Result interpretation of Bacitracin Test

For Haemophilus screening

Growth around the disc: Presence of Haemophilus species

No growth around the disc: Absence of Haemophilus species

Keynotes

Haemophilus species can be confirmed by using the following tests-

Colony characteristics

Colonies are Smooth, low convex, grayish, translucent, about 0.5-1.0 mm in size. The encapsulated strains usually produce larger and mucoid colonies and are about 1-3 mm in diameter.

Odor

Haemophilus species smell may be either bleach or seminal or dirty socks.

Oxidase test

As you know, Haemophilus species are oxidase test positive and therefore test should be positive.

Gram Stain findings

Gram-negative bacilli or gram-negative coccobacilli and sometimes filamentous may be observed.

Satellitism test for Haemophilus

Use of X, V and XV disks for Haemophilus species identification

Haemophilus species have varying requirements for X, V, and XV growth factors. Consequently, the significant differences in growth factor requirements of Haemophilus spp. allows for their differentiation. Differentiation is based on the presence or absence of growth around and/or between disks impregnated with factors X, V, and XV.

Note: Growth around XV disks but no growth at all around X and V disks alone while maybe in between X and V disks is normally suggestive for Haemophilus influenzae as shown below-

# Chocolate agar with bacitracin for screening Haemophilus ||Use of 10U bacitracin: 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. Haemophilus is resistant to bacitracin (10U) whereas most common bacteria are sensitive. It makes it easier to screen Haemophilus influenzae in sputum growing around the bacitracin disc as shown below-