Lab 11/11

Overview of Experiment

  • The experiment involves testing the antibiotic sensitivity of four different bacteria using the Kirby-Bauer method.

Key Concepts and Background

  • Kirby-Bauer Test: A standard method used clinically to assess antibiotic sensitivity of bacterial isolates from patients.

  • Importance of Isolation: The test can only be performed effectively if the suspected pathogen is in pure culture. Mixed cultures render the results invalid.

Preparation Steps

  • Culturing Bacteria: The bacteria must be isolated in pure cultures before starting the test.

    • Bacteria to be tested:

    • E. Coli (abbreviated as E)

    • Enterococcus (group D strep, abbreviated as ante)

    • Staphylococcus aureus (abbreviated as S)

    • Salmonella (abbreviated as Sac)

  • Growth Medium:

    • Mueller Hinton Agar: A complex growth medium used universally in clinical labs for drug sensitivity tests.

    • It contains tailored ingredients designed for antibiotic testing.

Procedure - Performing the Test

  1. Materials Needed:

    • Mueller Hinton Agar Plate: One plate per person.

    • Sterile Swab: For transferring bacteria to the agar plate.

  2. Getting the Bacteria:

    • Each lab bench will have a test tube containing one of the four bacteria.

    • It is important to mix the test tubes before use.

  3. Swabbing the Plate:

    • Dip a sterile swab into the bacterial culture, swirl to collect bacteria, then swab the agar surface completely.

    • Swab the plate in a perpendicular manner for even coverage:

      • First swab over the surface completely.

      • Rotate the plate 90 degrees and swab again.

    • Important to swab the edge of the plate after covering the surface for best results.

  4. Adding Antibiotic Discs:

    • Antibiotic Discs: Four different antibiotics to be tested: Penicillin, Polymyxin B, Tetracycline, Vancomycin.

    • Forceps Care: Sterilize the forceps with alcohol before picking up each disc.

    • Placement of Discs: Position discs on the plate without crowding; a gentle tap ensures flat contact with the agar surface.

    • Document usage of antibiotics as necessary but not on the plate itself.

  5. Incubation:

    • Invert plates and incubate at 37 degrees Celsius for optimal bacteria growth.

Understanding Drug Diffusion and Minimal Inhibitory Concentration (MIC)

  • Diffusion of Antibiotics:

    • Upon contact with the agar, antibiotics diffuse laterally and down into the agar creating a concentration gradient.

    • Highest concentration at the disc, decreasing outward.

  • Minimal Inhibitory Concentration (MIC):

    • Defined as the lowest concentration of antibiotic that inhibits bacterial growth.

    • MIC is a critical factor in determining the effectiveness of an antibiotic against a specific bacterium.

Analyzing Results

  • Zone of Inhibition:

    • The area around the antibiotic disc where bacterial growth is inhibited is measured.

    • If the bacteria grow up to the disc, it indicates resistance to that antibiotic.

Safety and Precautions

  • Aseptic Technique:

    • Ensure that gloves are worn at all times to maintain a sterile environment and protect against Biosafety Level 2 organisms (Staphylococcus aureus and Salmonella).

  • Proper Disposal: Discard used swabs in the designated biohazard bag.

Documentation and Further Analysis

  • Label plates appropriately: name, date, and type of bacteria tested.

  • Ensure to record observations for further analysis. Potential questions to consider:

    • Did there is any growth on the plate?

    • Did the appearance change, and what could that indicate?

  • Understand that observations contribute to identifying the bacteria and their antibiotic resistance profiles.

Concluding Remarks

  • The experiment requires careful execution and attention to detail during the swabbing and disk placement. Understanding the results will lead to insights into bacterial resistance patterns and effective treatment options.