ANTIBIOTIC SENSITIVITY TESTING | DISC DIFFUSION METHOD

Introduction:

Antibiotic sensitivity testing plays a pivotal role in clinical pathology laboratories, aiding in the assessment of clinical isolates’ susceptibility to antibiotics. This test can be conducted by placing small paper disks soaked in antibiotics on agar plates, with the resulting zone of bacterial growth inhibition serving as a measure of the antibiotic’s effectiveness against the specific organism. The methods used for this testing vary among countries. In the United Kingdom, a comparative approach is employed, where the susceptibility of the test organism is compared to that of a known, susceptible control strain. This comparison can occur on the same agar plate (Stoke’s method) or on separate plates (Kirby-Bauer method).

A prevalent global approach is the standardized test, involving the comparison of inhibition zone diameters against standard zones, as defined in a reference chart. Different regions have developed their own unique methods. For instance, Western Europe uses the ICS (International Collaborative Study) method, France follows the SFM (Societe Francaise de Microbiologie) method, Germany utilizes the DIN (Deutsches Institut fur Normung) method, and Scandinavian countries adopt the SIR (Swedish International Reference) method. In the United States, the Kirby-Bauer method, recommended by the National Committee for Clinical and Laboratory Standards (NCCLS).

Two primary method are used for conducting sensitivity tests:

• Disc Diffusion Method:

This routine method entails placing paper disks soaked in a specified quantity of antibiotic on agar plates containing pure cultures of the target organisms. The antibiotics diffuse into the surrounding medium, leading to the formation of visible clear zones, which are then measured and compared to control results.

• Agar or Broth Dilution Method:

This more specialized method exposes organisms to varying concentrations of antibiotics in agar or broth. It provides more detailed information, particularly regarding the minimum inhibitory concentration (MIC) of an antibiotic.

1. Disc Diffusion Method:

The disc diffusion method is a common and routine method employed in clinical laboratories. In this approach, small filter paper disks that have been soaked in a known quantity of antibiotic are strategically placed on agar plates that contain a pure culture of the target organisms.

The process involves the following steps:

Preparation:

Filter paper disks are impregnated with a precise amount of antibiotic.

1. Inoculation: These prepared disks are then carefully positioned onto agar plates that have been inoculated with a pure culture of the microorganisms under investigation.
2. Antibiotic Diffusion: Over time, the antibiotics begin to diffuse from the disks into the surrounding agar medium. As a result, the concentration of the antibiotic in the medium increases nears the disk.
3. Inhibition of Microbial Growth: In areas where the antibiotic concentration is sufficient, it prevents the growth and division of the microorganisms. As a result, a visible and clear zone devoid of microbial growth appears around the antibiotic-soaked disk.
4. Measurement and Comparison: The diameter of this clear zone is subsequently measured and compared to predetermined standards or controls. This comparison is crucial for assessing the sensitivity of the microorganisms to the antibiotic being tested.

The disc diffusion method provides a practical and widely-used method for determining the susceptibility of microorganisms to antibiotics in clinical settings.

Methods for Antibiotic Sensitivity Testing Using Disc Diffusion Method

Antibiotic sensitivity testing through the disc diffusion method offers two distinct methods:

1. Kirby-Bauer Method:
   • In the Kirby-Bauer method, antibiotic discs are applied to both the test strains and control strains separately on different plates.
   • The zones of inhibition surrounding the antibiotic discs on the test strains are then compared to those of the control strains.
2. Stokes Method:
   • In the Stokes method, test and control strains are applied on the same plate, arranged in such a way that the test strain is located on one side of the antibiotic disc, and the control strain is on the other side.
   • This method is preferred over the Kirby-Bauer method because it employs the same disc and the same medium for both the test and control strains.

Challenges and Considerations in Disk Diffusion Method:

1. Choice of Media: The selection of the appropriate agar medium is critical. Diagnostic agars should not be used for susceptibility tests. For example, the NCCLS method requires the use of Muller-Hinton agar, while the BSAC method uses IsoSensitest agar.
2. Inoculum Density: The density of the microbial inoculum must be carefully controlled. An excessively heavy inoculum can lead to smaller zone diameters, while a light inoculum can result in larger zones. A 0.5 McFarland standard is typically used to achieve semi-confluent growth.
3. Antibiotic Disc Quality: The antibiotic content of the discs is of utmost importance. Discs with an excessively high concentration, such as homemade ones, can lead to inaccurate susceptibility results. Proper storage conditions, especially for β-lactam antibiotics, are essential to maintain disc potency. Some β-lactam antibiotic discs are kept refrigerated and/or desiccated.
4. Use of Control Strains: Whether employing a comparative or standardized method, control strains should always be included in the testing process to ensure the quality and potency of the antibiotic discs.
5. CO2 Incubation Impact: Incubation in an atmosphere containing CO2 can alter the pH of the agar medium and may result in smaller inhibition zones, particularly when testing macrolides against Haemophilus influenzae.
6. Depth of medium: Plates should have a consistent level depth of 4 mm. Zones of inhibition increases as the depth of agar decreases.

Antibiotic sensitivity testing using the disc diffusion method demands meticulous attention to these factors to ensure accurate and reliable results.

Procedure for Antibiotic Sensitivity Testing Using Disc Diffusion Method

To conduct antibiotic sensitivity testing using the disc diffusion method, follow these steps:

1. Isolate Colonies:
   • Select a minimum of four to five well-isolated colonies of the same morphological type from an agar plate culture.
   • Use a wire loop to touch the top of each colony and transfer the growth into a sterilized tube containing 4 to 5 ml of an appropriate broth medium (e.g., BHI broth).
2. Incubation:

• • Incubate the broth culture for 2-8 hours at a temperature of 35-37°C.

3. Turbidity Adjustment:

• • Adjust the turbidity of the broth culture using a BaSO4 standard (0.5 unit). This adjustment is made through visual comparison, reading the tube against a white background with contrasting black lines.

4. Inoculum Preparation:

• • After adjusting the turbidity of the inoculum suspension, use a sterile cotton swab on an applicator to dip into the suspension within 15 minutes.
  • Rotate the swab several times while pressing firmly on the inside wall of the tube above the fluid level to remove excess inoculum.

5. Inoculation of Agar Plate:

• • Inoculate the dry surface of a Mueller-Hinton agar plate by streaking the swab over the entire agar surface.
  • Repeat streaking twice, rotating the plate approximately 60 degrees each time.

6. Application of Sensitivity Discs:

• • Place the appropriate sensitivity disks on the agar surface, ensuring they are 24 mm apart from center to center.

7. Incubation:

• • Invert the plates and place them in an incubator set at 35-37°C within 15 minutes after applying the discs.

8. Examination and Measurement:

• • After 16-18 hours of incubation, examine each plate and measure the diameters of the zones of inhibition, including the diameter of the discs.

9. Interpretation:

• • • Interpret the sizes of the inhibition zones by comparing them with zones of control strains or by referring to a reference table.

  

Control Strains:

• • With each batch of testing, include a control strain that is known to be sensitive to the antibiotics used. Control strains can be obtained from the National Collection of Type Cultures (NCTC).
  • Compare the zones of inhibition of test organisms with those of these control strains to verify the efficiency of the discs on a daily basis.
  • Common control strains include:
    • • • Staphylococcus aureus
        • Escherichia coli
        • Pseudomonas aeruginosa
        • Clostridium perfringens (anaerobic)

Media for Routine Susceptibility Testing:

• Choice of Mueller-Hinton Agar:

Mueller-Hinton agar is commonly used in routine susceptibility testing for several reasons: a. It demonstrates good batch-to-batch reproducibility. b. It contains low levels of sulphonamide, trimethoprim, and tetracycline inhibitors. c. It supports satisfactory growth for most pathogens. d. There is an extensive dataset available regarding susceptibility tests performed using this medium.

• Media with Thymidine or Thiamine:

Some media containing thymidine or thiamine can counteract the inhibitory effects of sulphonamides and trimethoprim. This may result in smaller or less distinct inhibition zones or even no zone at all. When Mueller-Hinton agar contains thymidine, it is essential to add thymidine phosphorylase or lysed horse blood to counteract the thymidine’s effects.

• Use of Blood Agar or Chocolate Agar:

For certain organisms that do not grow well on Mueller-Hinton agar, such as Streptococcus pyogenes or S. pneumoniae, blood agar or chocolate agar is employed for sensitivity testing.

Sensitivity Testing for Bacteria with Special Growth Requirements

The standard Kirby-Bauer and other diffusion tests are primarily designed for rapidly growing pathogens. However, testing these organisms with slower growth rates may lead to larger inhibition zones, potentially resulting in inaccurate susceptibility results. To address this issue, optimal growth conditions must be provided for the strains being tested. This can be achieved by:

• Lower Incubation Temperature: Adjusting the incubation temperature can be effective for certain bacteria. For example, Methicillin-resistant Staphylococcus aureus (MRSA) may appear sensitive to methicillin when incubated at 37°C, while they are resistant at 30°C (or with 5% NaCl added to the medium). This phenomenon is due to the non-homogeneity of the bacterial population. The resistant portion of the population grows optimally at 30°C and may not be detected at 37°C due to slower growth. Lowering the incubation temperature to 30°C can yield more accurate results for strains like MRSA and others with similar growth characteristics.

Strains that may exhibit improved growth at 30°C and benefit from sensitivity testing at this temperature include:

1. Methicillin-resistant staphylococci
2. Yersinia spp., Klebsiella ozaenae, certain non-fermenter Gram-negative rods
3. Pseudomonas putida, Ps. fluorescens, some strains of P. cepacia, Aeromonas spp., and some Moraxella spp.

Special Nutrient Requirements for Sensitivity Testing

1. Additional Supplements: Some strains of enterobacteriaceae, including E. coli, Citrobacter, Klebsiella, Proteus, and Salmonella spp., form dwarf colonies on routine media. These strains may require supplemental nutrients, such as CO2, thiamine, glutamic acid, or other compounds, to support larger colony growth and facilitate sensitivity testing.
2. Dwarf Colony Formation in Staphylococcus aureus: In certain cases, strains of Staphylococcus aureus may form dwarf colonies on routine media. To enhance their growth for sensitivity testing, supplementation with thiamine and menadione may be necessary.
3. Interference with Antibiotics: It’s important to note that some of the supplemented substances used to support growth may interfere with the activity of specific antibiotics. For example, CO2 can affect the interpretation of zone sizes for aminoglycosides, macrolides, and tetracyclines. Therefore, modifications to the zone size interpretation may be required.

Special Interpretation Tables:

When conducting sensitivity testing for slow-growing strains or strains with specific growth requirements, special interpretation tables become essential. This is particularly relevant for organisms like Haemophilus, Neisseria, Streptococcus pneumoniae, and anaerobes.