4) Procedure

The following steps outline the detailed procedure for microbial screening of antimicrobial compounds:

1. Step 1: Selection of Microbial Strains
   1. Select a range of microbial strains that represent different pathogens, including Gram-positive and Gram-negative bacteria, fungi, and other relevant microorganisms (e.g., Mycobacterium tuberculosis, Candida species, Escherichia coli, Staphylococcus aureus).
   2. Ensure that the selected strains are well-characterized, including their susceptibility profiles, and that they are stored in appropriate conditions (e.g., frozen stocks or agar slants) for future use.
   3. Verify that the strains are free from contamination and meet the required quality standards for antimicrobial testing.
2. Step 2: Preparation of Microbial Cultures
   1. Inoculate the microbial strains into appropriate culture media (e.g., agar plates, liquid broth) and incubate under the required conditions (e.g., temperature, atmospheric conditions) to achieve log-phase growth.
   2. Check for purity and uniformity of the microbial cultures before testing, and discard any contaminated or non-uniform cultures.
   3. Prepare the required concentration of microbial inoculum, typically by adjusting the optical density (OD) of the culture to a specified value (e.g., 0.5 McFarland standard) to ensure consistent inoculum size for screening.
3. Step 3: Preparation of Antimicrobial Compounds
   1. Prepare a series of dilutions of the antimicrobial compounds to be tested. This can be done by dissolving the compounds in an appropriate solvent (e.g., DMSO, PBS) and diluting to the desired concentrations (e.g., micromolar to nanomolar range).
   2. Ensure that the solvent used for the compound does not interfere with microbial growth or assay results. Use appropriate solvent controls when necessary.
   3. For each compound, prepare the stock solution in sufficient volume to allow for multiple replicates and testing across different concentrations.
4. Step 4: Microbial Screening Assay
   1. Perform the antimicrobial susceptibility testing using the chosen assay method. Common methods include disk diffusion (Kirby-Bauer method), broth microdilution, or agar dilution techniques.
   2. For the disk diffusion method, place filter paper disks impregnated with the antimicrobial compound on an inoculated agar plate and incubate. Measure the zone of inhibition to determine the antimicrobial activity.
   3. For broth microdilution, inoculate the wells of a 96-well plate with the microbial culture and compound dilutions, then incubate and assess bacterial growth using optical density or visual inspection for turbidity.
   4. Record the minimum inhibitory concentration (MIC) for each compound, which is the lowest concentration of the antimicrobial agent that prevents visible growth of the microorganism.
5. Step 5: Controls and Quality Assurance
   1. Include positive and negative controls in each screening run to ensure the validity of the results. Positive controls should be known antimicrobial agents with established activity against the chosen microbial strains, while negative controls should include compounds with no expected antimicrobial activity.
   2. Ensure that the assay conditions (e.g., temperature, incubation time) are consistent and that all reagents, media, and equipment are appropriately calibrated and validated.
   3. Verify that the compound concentrations and inoculum sizes are accurate and that the data are reproducible across multiple replicates and assay runs.
6. Step 6: Data Analysis and Interpretation
   1. Analyze the data to identify the antimicrobial activity of the tested compounds. Record the zone of inhibition (for disk diffusion) or the MIC values (for broth microdilution) for each compound at different concentrations.
   2. Compare the results with the positive and negative controls to ensure the accuracy of the assay.
   3. Prioritize compounds with the lowest MIC values or largest zones of inhibition for further validation and potential lead optimization.
7. Step 7: Documentation and Reporting
   1. Document all screening results, including compound identification, concentration tested, strain used, assay method, and the observed antimicrobial activity (e.g., MIC or zone of inhibition).
   2. Prepare a Microbial Screening Report that summarizes the results, including an analysis of the antimicrobial activity of each compound, as well as recommendations for further development or testing.
   3. Ensure that all data is properly stored and available for future analysis, regulatory compliance, and intellectual property protection.

5) Abbreviations

• MIC: Minimum Inhibitory Concentration
• OD: Optical Density
• PCR: Polymerase Chain Reaction
• HIB: High Inoculum Bacteria

6) Documents

The following documents should be maintained throughout the microbial screening process:

1. Microbial Screening Protocol
2. Screening Data and Results Reports
3. Compound Preparation and Dilution Records
4. Assay Control and Validation Records

7) Reference

References to regulatory guidelines and scientific literature that support this SOP:

• FDA Guidance for Industry on Antimicrobial Drug Development
• Scientific literature on antimicrobial screening methods and best practices

8) SOP Version

Version 1.0: Initial version of the SOP.