Antibiotic Sensitivity

 

Disc Diffusion Method

Aim:  To test the pathogens for antibiotic sensitivity by disc diffusion method.

Theory:

Once the causative organism of a specific disease in a patient has been isolated, it is up to the attending physician to administer a chemotherapeutic agent that will inhibit or kill the pathogen without causing serious harm to the individual. The method must be relatively simple to use, be very reliable, and yield results in as short a time as possible. The Kirby – Bauer method of sensitivity testing is such a method.

The Kirby-Bauer method is not restricted to antibiotics. It may also be used to measure the sensitivity of any microorganism to a variety of antimicrobial agents such as sulfonamides and synthetic chemotherapeutics. Antibiotics are chemotherapeutic agents of low molecular weight produced by microorganisms that inhibit or kill other microorganisms. Drugs, on the other hand, are antimicrobic agents that are man – made.

Principles:

        One method that is used to determine antibiotic susceptibility is the sensitivity disk method of Kirby – Bauer (named after W. Kirby and A. W. Bauer in 1966). The Kirby-Bauer assay is a standardized assay used to determine the susceptibility of bacteria to various antibiotics. The assay uses a filter paper disk impregnated with an antibiotic that is placed on agar. The antibiotic diffuses from the disk into the agar. The concentration of the antibiotic decreases as it diffuses away from the disk. The solubility of the antibiotic and its molecular size will determine the size of the area of infiltration around the disk. If an organism is placed on the agar it will not grow in the area around the disk if it is susceptible to the antibiotic. This area of no growth around the disk is known as a “zone of inhibition”. At the point around disk where bacterial growth begins the antibiotic as reached its “minimum inhibitory concentration”. Further away from the disk the antibiotic concentration is too low to inhibit the grow of the bacteria.

        Antibiotic susceptibility patterns are called antibiograms. Antibiograms can be determined by comparing the zone diameter obtained with the known zone diameter size for susceptibility. For example, a zone of a certain size indicates susceptibility, zones of a smaller diameter or no zone at all show that the bacterium is resistant to the antibiotic. Frequently one will see colonies within the zone of inhibition when the strain is antibiotic resistant.

        This method is also useful for determining the minimal inhibitory concentration (MIC) which is determined by measuring the diameter of growth inhibition (clear) zone surrounding the antibiotic disc.

Many factors are involved in sensitivity disk testing and must be carefully controlled. These include size of the inoculum, distribution of the inoculum, incubation period, depth of the agar, diffusion rate of the antibiotic, concentration of antibiotic in the disk, and growth rate of the bacterium. If these factors are carefully controlled, this type of testing is highly satisfactory for determining the degree of susceptibility of a bacterium to a certain antibiotic.

Requirements:

1.      Nutrient Agar

a.      Peptone                             1.0 g

b.      Meat or Beef Extract              0.5 g

c.      Nacl                                 0.3 g

d.      Agar                                  2.5 g

e.      Distilled water                     1,00.0 ml

2.      Nutrient broth culture of Bacteria

3.      Sterile filter paper disc

4.      Antibiotic solution or Antibiotic disc

5.      Spreader

6.      Alcohol

Procedure:

1.      On nutrient agar plates, add 0.1 ml of bacterial culture in aseptic condition.

2.      With the help of disinfected spreader, spread the bacterial culture on the nutrient agar.

3.      Pick up a sterile filter paper disc by the outer edge using flamed sterile forceps and dip the opposite edge of the disc in the antibiotic dilutions.

4.      Place the disc near the edge of the agar surface of the inoculated plate.

5.      Press gently with sterile forceps to ensure firm contact with agar surface.

6.      Keep these plate in refrigerator for 5 minutes to diffuse the antibiotic solution.

7.      Incubate all plates at 37 °C for 24 to 48 hours in an inverted position.

 

Observation:

1.      Examine all the plates for the zone for inhibition surrounding the discs.

2.      Measure diameter of zone of inhibition in millimeters.

Bacterial Culture	Diameter of Zone Inhibition in millimeter
	Penicillin	Chloramphenicol
Staphylococcus aureus
Bacillus subtilis

Results: