Lab 5

Overview of Antibiotic Sensitivity Testing

Antibiotic resistance has emerged as a significant concern in clinical and hospital settings over the past few years. To address this, specific assays are used to determine how sensitive bacterial strains are to various treatments. The primary method explored here is the Kirby-Bauer Method, also formally known as the Standardized Disc Susceptibility Test.

Experimental Objectives and Materials

The objective of the antibiotic sensitivity experiment is to determine the threshold of susceptibility across different bacterial strains. This is achieved by creating a bacterial lawn and observing the efficacy of various antibiotics delivered via small, high-quality paper or cardboard discs.

Key materials required for the assay include:

• Agar Plates: Non-selective agar is used, specifically LB agar or Mueller-Hinton agar. These provide a nutrient-rich environment for the bacteria to grow without inhibiting any specific strains.

• Bacterial Cultures: Cultures should be grown in liquid media prior to the experiment.

• L Spreader: A disposable tool used to spread the bacterial culture evenly across the surface of the agar. These are disposed of in biohazardous waste after a single use.

• Antibiotic Discs: Small round cardboard materials impregnated with specific dosages of antibiotics. Each disc features a letter abbreviation for the drug and a number indicating the dosage amount.

• Sterile Forceps: Used to handle and place the antibiotic discs to maintain aseptic conditions.

• Pipette and Tips: For precise measurement and distribution of the bacterial culture, typically using sterile, autoclaved tips.

• Ruler: To measure the results in millimeters ($mm$).

Procedure for Bacterial Lawn Preparation

To ensure accurate results, the bacteria must be spread to achieve confluent growth, where the entire surface of the plate is covered in a uniform layer, often referred to as a "lawn."

1. Labeling: Mark the plate with the experimental number (e.g., PV05) and the current date.

2. Volume Adjustment: Set the pipette to approximately $150\,\mu L$ (microliters) of the culture by twisting the head of the pipette until the numbers reflect the desired volume.

3. Inoculation: Using a sterile tip, take the bacterial sample and apply it to the agar surface in dropwise steps around the plate to ensure equal distribution.

4. Spreading: Use a sterile L spreader to guide the culture around the plate. Most spreaders have a flat surface and a curved surface; the flat surface (without a line) should be used. Keep the lid as close to the plate as possible to avoid outside contamination.

5. Technique: Move the L spreader gently. It is normal to feel some resistance as the liquid culture is absorbed into the agar. If necessary, rotate the plate to ensure even distribution across the entire surface area.

6. Disposal: Once finished, dispose of the spreader in the appropriate biohazardous waste container.

Antibiotic Disc Application and Incubation

1. Placement: Using sterile forceps, remove a disc from the dispenser and place it on the agar. Discs should be placed roughly a finger's width away from the edge of the plate to allow space for a full zone of clearing.

2. Spacing: Arrange multiple discs (up to eight) with equal spacing to prevent overlapping results.

3. Securing: Gently tap the top of each disc with the forceps to ensure it is seated securely on the agar surface. Do not apply firm pressure, as this can break the agar surface.

4. Incubation: Invert the plate to prevent condensation from dripping onto the culture. Incubate the plate at $37\,^{\circ}C$ (degrees Celsius) overnight for active growth.

Measuring and Interpreting Zones of Inhibition

The next day, plates are examined for "zones of inhibition," which are the clear circular areas around the discs where bacterial growth has been inhibited or killed.

• Measurement: Use a ruler to measure the diameter of each zone in millimeters ($mm$). Measure directly across the center of the disc from one edge of the clearing to the other.

• Overlapping Zones: If two zones of inhibition overlap, the full diameter cannot be measured accurately. In this case, measure the radius (from the center of the disc to the outer edge of the clearing) in an area with clear bacterial growth and double that value ($Radius \times 2 = Diameter$).

• Interpretation Categories: Bacteria are classified into three categories based on the measurement:

  • Susceptible (S): The antibiotic is effective at killing or inhibiting the bacteria.

  • Resistant (R): The bacteria grow right up to the disc, or the clearing is smaller than a specific threshold, indicating the antibiotic is ineffective.

  • Intermediate (I): The bacteria show some sensitivity, but not enough to be classified as fully susceptible.

Case Studies and Interpretive Examples

Interpretations rely on reference charts specific to the bacterial species being tested (e.g., Staphylococcus or E. coli).

• Erythromycin (E):

  • Measurement: $31\,mm$.

  • Interpretation: According to the Staphylococcus chart, this falls into the Susceptible category, meaning it would be an effective clinical treatment.

• Gentamicin (G):

  • Measurement: $9\,mm$.

  • Interpretation: This falls into the Resistant category for Staphylococcus, meaning it is contraindicated for clinical use against this strain.

• Oxacillin (O):

  • Measurement: $0\,mm$ (no aura/clearing visible).

  • Interpretation: The strain is highly Resistant.

• Penicillin (P):

  • Thresholds for Penicillin are often very specific: Susceptibility requires a diameter $> 29\,mm$, while Resistance is defined as $< 28\,mm$.

  • In a specific lab observation, a penicillin zone measured at $36\,mm$, placing it in the Susceptible category.

  • In another scenario, despite a small zone of diminished growth, a measurement well below the threshold confirmed the strain was Resistant.

Data Recording and Analysis

Results should be meticulously recorded in a laboratory notebook, including:

1. The name of each antibiotic based on the disc abbreviation.

2. The measured diameter of the zone of inhibition in millimeters ($mm$).

3. The interpretation (Resistant, Intermediate, or Susceptible) derived from the reference chart.