Question

Consumer Product Experiments

Your job is to write an experiment to test a type of antimicrobial product or to test for levels of contamination in commonly used products. Either way, you will need to formulate a hypothesis that you present in an introductory paragraph.

• Introduction (with hypothesis)
• Materials (list of all materials required [most groups need 4-8 petri dishes])
  • divide materials into 3 categories: Lab supplies, Bring from home, ask to be purchased
• Methods (step by step directions)

Answer 1

Introduction:

Hypothesis- Characterizing antimicrobial agent by Interceptive breakpoint in susceptibility testing, to test the effectiveness and type of antimicrobial agent.

• Testing the susceptibility of an agent has an immense impact on characterizing an antimicrobial agent and determining its effectiveness, in terms of “resistant’ or “sensitive” is respect to a specific microbial strain.
• FDA and WHO have recognized that many pathogenic strains have emerged as resistant strains, causing devastation in terms of human and animal health.
• Antimicrobial susceptibility testing (AST), has major importance in clinical microbiology, for determining the property of a therapeutic regime.
• The results of such tests require proper interpretation and understanding. Hence, “interceptive breakpoints” are necessary to standardize each method used for such testing.
• Certain criteria for breakpoint setting are:

1. Needs to be set before the antimicrobial agent is considered as therapeutic agent or used clinically.

2. Requires prior understanding of pharmacokinetics and pharmacodynamics, and the other basic information of the agent to be tested,

3. Breakpoints should be again reviewed after a certain period of clinical application also.

4. Breakpoints should be revised after a certain time, and revised breakpoints established, if required.

• Several organizations have published, such standardization methods. The standard protocol of AST and interceptive breakpoints are considered, is developed by Clinical Laboratory and Standard Institute (CLSI), formally called National Committee for by Clinical Laboratory Standards (NCCLS) or European Committee on antimicrobial susceptibility testing (EUCAST).

Materials:

Petri-plates, antimicrobial agents (samples to be tested), disks or strips (for incorporating the agent) . microbial culture, media( Muller-Hinton Agar).

Method:

Disk diffusion testing:

• The antimicrobial agent is incorporated (in different concentrations), into disks, tablets or strips (of specific dimensions).
• The agents then placed on solid medium of standardized, documented formulations. Usually Muller-Hinton Agar.
• The standardized medium amount: 0.5 mm (+/- 4. Aapproximately 25 mL in a 90 mm circular plate, 31 mL in a 100 mm circular plate, 71 mL in a 150 mm circular plate, 40 mL in a 100 mm square plate. The plates should be dry, with no droplets of water.
• Microbial colonies are used in form of suspension. Turbidity determined (Mc Farland standard: 0.5, approximately 1x 10 ⁸ cfu/ml). Usually 15 minutes suspension preferred.
• The plates are inoculated by swab/ streak technique.
• Disks containing the agent are usually placed after 15 minutes of inoculation. Normally 6-8 disks per plate. Control is maintained, with disk without agent.
• Incubation of plates as per specific time. The plates with confluent lawn growth are considered.
• The zone of inhibition is measured and compared as per AST documents.

Sensitive/ resistant:

• Breakpoints may be used to determine the susceptibility of the antimicrobial agent, to characterize the microbe or its components (toxin, capsule etc.) as either sensitive or resistant towards the antimicrobial agent.
• Susceptible or sensitive: Have been defined as, growth of the microbial strain is inhibited by an antimicrobial agent concentration, in the range found for the wild type strains.
• Resistant: Have been defined as, growth of the microbial strain is inhibited by an antimicrobial agent concentration, higher the range, observed for the wild type strains.

EUCAST have further described susceptibility as: Determined by applying breakpoint in a defined phenotypic system.

• Clinically resistant: Designated as R. Described by a level of antimicrobial susceptibility, resulting in a higher than expected chances of therapeutic failure.
• Clinically Sensitive: Designated as R. Described by a level of antimicrobial susceptibility, resulting in the desired or improved therapeutic outcome.
• Clinically Intermediate: Described by a level of antimicrobial susceptibility, resulting in an intermediate therapeutic outcome