Antibiotic Sensitivity Test (Kirby Bauer Method)

Aim

To become acquainted with Kirby Bauer procedure for the evaluation of antimicrobial activity of chemotherapeutic agents.

Principle

The available chemotherapeutic agents vary in their scope of antimicrobial activity. Some have a limited spectrum of activity being effective against only one group of microorganism. Others exhibit broad spectrum of activity against a range of microorganisms. The drug susceptibilities of many pathogenic microorganisms are known, but it is sometimes necessary to test several agents to determine the drug of choice.

A standard filter paper disc agar diffusion procedure known as Kirby Bauer method is used to determine the drug susceptibility of microorganisms. This method allows the rapid determination of the efficiency of the drug by measuring the diameter of the zone of the inhibition that results from the diffusion of the agent into the medium surrounding the disc. The susceptibility of an organism to the drug is determined by the size of the zone. The size of the zone in turn is dependent on various factors such as

1) the number of discs used

2) the growth rate of the organism

3)  Degree of susceptibility of the organism

A measurement of the diameter of the zone of inhibition in millimeter is made and its size is compared to that expressed in a standardized chart. Based on this comparison the test organism is determined to be resistant, intermediate or susceptible to the antibiotic.

Determination of antibiotic effectiveness against specific pathogen is essential to the proper therapy. The spread of drug resistant pathogen is one of the most serious threat to successful treatment of microbial disease. In treatment and control of infectious diseases especially when caused by pathogens that are often drug resistant. Sensitivity testing is used to selective effective antimicrobial drugs. Testing can show which agents are most effective against a pathogen.

Materials Required

• Broth cultures of E.coli, Staphylococus, Bacillus and Pseudomonas species
• Mueller Hinton Agar plates
• Antibiotic Discs: streptomycin, chloramphenicol, penicillin, 
• Forceps
• Sterile cotton swabs
• Glass marking pencil

Procedure

1. Mueller Hinton Agar was prepared, sterilized and dispersed into sterilized petri plates.
2. The bottom of each plate was labelled with the name of the microorganism to be inoculated  
3. The plates were inoculated with the respective microorganism using aseptic techniques as follows:
4. a) A sterile cotton swab was dipped into a well-mixed test culture and excess inoculum was removed by pressing the saturated swab against the inner wall of the culture tube.
5.  b) Using the swab, entire agar surface was streaked horizontally, vertically and around the outer edge of the plate to ensure a heavy growth over the entire surface.

      5. All the culture plates were allowed to dry for about 5 minutes
     6.  Using a sterile forceps an antibiotic disc was placed on the the agar surface and it was pressed lightly on the agar surface with the forceps to keep it in place. 4-5 discs were placed on an MHA plate and the plates closed

      7. All the plates were incubated in inverted position for 24-48 hours at 37⁰C.

Observation

The plates were observed for zone of inhibition after 24 hours. The diameter of the inhibition zones were measured and tabulated for each organisms. Based on the measurement, the test organism was determined to be resistant, intermediate or susceptible to the antibiotics.

Result

Four different antibiotics were used for testing the susceptibility. E. coli was resistant to Streptomycin, Tetracycline, Bacitracin and susceptible to Chloramphenicol. Staphylococcus was susceptible to Streptomycin, Tetracycline, Bacitracin and Chloramphenicol. Bacillus was susceptible to Streptomycin, Tetracycline, Bacitracin and Chloramphenicol.  Pseudomonas was resistant to Bacitracin, moderately susceptible to Chloramphenicol, Tetracycline and susceptible to Streptomycin

Antibiogram of E. coli (left hand side)

Sl No	Name of Antibiotic	Strength	Diameter of zone of Inhibition	Remark
1	Streptomycin	10 mcg	10 mm	Resistant
2	Chloramphenicol	30 mcg	20 mm	Susceptible
3	Tetracycline	30 mcg	5 mm	Resistant
4	Bacitracin	10 units	No zone	Resistant

Antibiogram of Staphyloccoccus (left hand side)

Sl No	Name of Antibiotic	Strength	Diameter of zone of Inhibition	Remark
1	Streptomycin	10 mcg	25 mm	Susceptible
2	Chloramphenicol	30 mcg	35 mm	Susceptible
3	Tetracycline	30 mcg	17 mm	Susceptible
4	Bacitracin	10 units	17 mm	Susceptible

  Antibiogram of Bacillus (left hand side)

Sl No	Name of Antibiotic	Strength	Diameter of zone of Inhibition	Remark
1	Streptomycin	10 mcg	26 mm	Susceptible
2	Chloramphenicol	30 mcg	30 mm	Susceptible
3	Tetracycline	30 mcg	30 mm	Susceptible
4	Bacitracin	10 units	18 mm	Susceptible

 Antibiogram of Pseudomonas (left hand side)

Sl No	Name of Antibiotic	Strength	Diameter of zone of Inhibition	Remark
1	Streptomycin	10 mcg	20 mm	Susceptible
2	Chloramphenicol	30 mcg	16 mm	Moderately Sensitive
3	Tetracycline	30 mcg	13 mm	Moderately Sensitive
4	Bacitracin	10 units	No zone	Resistant