MBEC Assay Protocol Using the 96-Well Plate Sonicator UIP400MTP

The MBEC assay is used to determine the concentration of antimicrobial agents needed to eliminate biofilms formed on peg lids. To assess biofilm viability, the biofilms must first be detached from the peg lids of a 96-well plate. Sonication is the most reliable and efficient method for this detachment. The multi-well plate sonicator UIP400MTP is specifically designed to process assay plates with maximum efficiency and convenience, streamlining and accelerating high-throughput MBEC assays.

Sonication for Biofilm Detachment

Sonication is a crucial step in Minimum Biofilm Eradication Concentration (MBEC) assays, enabling the effective dislodging of biofilm cells from their surface attachment. Biofilms are inherently structured communities of microorganisms encased in an extracellular matrix, which makes them significantly more resistant to antimicrobial agents compared to planktonic cells. During MBEC assays, sonication uses ultrasonic waves to generate controlled cavitation, disrupting the biofilm matrix and releasing embedded cells into a suspension. This step ensures that the biofilm cells are evenly dispersed in the recovery medium, facilitating accurate viability assessments through plating, dilution, or spectrophotometric methods. Without proper biofilm detachment, residual matrix components could shield cells, leading to underestimation of antimicrobial efficacy. Therefore, sonication is indispensable for obtaining reliable and reproducible MBEC values, reflecting the true eradication potential of the tested agents. The multi-well plate sonicator UIP400MTP allows for the facile high-throughput sample preparation in assay plates.

Minimum Biofilm Eradication Concentration (MBEC) Assay Protocol

Step 1: Biofilm Formation

1. Prepare the bacterial suspension:
   Grow bacteria in appropriate media to log-phase growth.
   Dilute the bacterial culture to a defined optical density (e.g., OD600 ~0.1).
2. Inoculate the 96-well plate:
   Add the bacterial suspension (e.g., 150–200 µL) into each well of a standard 96-well microtiter plate.
3. Attach the peg lid:
   Place the peg lid onto the inoculated plate to allow biofilm formation on the peg surfaces.
4. Incubate the plate:
   Incubate the setup at an appropriate temperature (e.g., 37°C) for 24–48 hours without shaking to promote biofilm growth.

Step 2: Treatment with Antimicrobial Agents

• Prepare antimicrobial solutions:
  Prepare a range of antimicrobial concentrations in fresh media.
  Expose biofilms to antimicrobial agents:
  Remove the peg lid from the bacterial culture and rinse it in sterile saline or PBS to remove planktonic cells.
  Place the peg lid into a new 96-well plate containing the antimicrobial solutions.
• Incubate the plate:
  Incubate for a defined period (e.g., 24 hours) to allow antimicrobial exposure.

Step 3: Sonication with the 96-Well Plate Sonicator UIP400MTP

The sonication step is critical for detaching biofilms from the peg lids to assess viability. Follow these steps for the UIP400MTP sonicator:

1. Prepare the setup:
   Fill a fresh 96-well plate with recovery medium (e.g., neutralizing broth or fresh growth medium) in each well.
2. Transfer the peg lid:
   Remove the peg lid from the antimicrobial treatment plate.
   Rinse the peg lid in sterile saline or PBS to remove residual antimicrobial agents.
3. Position the plate and sonicator:
   Attach the peg lid to the recovery medium plate.
   Place the recovery medium plate onto the UIP400MTP sonicator platform, ensuring the plate is centered and stable.
4. Adjust sonication parameters:
   Set the sonication parameters at the UIP400MTP (Settings can be adjusted to the biofilm):
   Amplitude: 70–100%.
   Sonication time: 1–3 minutes (adjust based on biofilm robustness) at cycle mode.
5. Sonicate:
   Start the sonication process. The ultrasonic vibration will dislodge the biofilms from the peg surfaces into the recovery medium.
6. Monitor the process:
   Use the pluggable temperature sensor to monitor the sample temperature in the wells. The UIP400MTP can be connected to a lab chiller for cooling.
7. Post-sonication handling:
   Immediately transfer the recovery medium (now containing detached biofilms) into a fresh sterile plate for downstream analysis.

(A) Plate containing TSB with 2% glucose used for biofilm formation, cell recovery, and
determination of MIC and MBCB; (B) Lid with pins for formation of staphylococcal biofilms. determination of MIC and MBCB; (B) Lid with pins for formation of staphylococcal biofilms.
The biofilm cells formed on the pins were dislodged by sonication (Hielscher Ultrasound Technology) for 5 min in 96-well plates containing fresh culture medium for recovery of the cells.
(Picture and study: ©de Oliveira et al., 2016)

Step 4: Viability Assessment

Plate and culture detached biofilms:

1. Perform serial dilutions of the recovery medium and plate onto agar to enumerate colony-forming units (CFU).
   Alternatively, use a colorimetric or fluorescence-based viability assay.
2. Record results:
   Determine the MBEC as the lowest concentration of antimicrobial that eradicated detectable biofilm viability.

Design, Manufacturing and Consulting – Quality Made in Germany

Hielscher ultrasonicators are well-known for their highest quality and design standards. Robustness and easy operation allow the smooth integration of our ultrasonicators into industrial facilities. Rough conditions and demanding environments are easily handled by Hielscher sonicators.

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Read here how the UIP400MTP is used for biofilm detachment as a sample preparation step during the ASTM E2799 protocol for testing disinfectant efficacy against Pseudomonas aeruginosa biofilm using the MBEC assay.