ASTM E2799-22 Protocol for Antimicrobial Evaluation

The Hielscher UIP400MTP is a powerful and efficient multi-well plate sonicator designed to streamline the sonication step in the ASTM E2799-22 protocol. By delivering uniform ultrasonic energy across the entire microtiter plate, the UIP400MTP ensures consistent and reliable detachment of biofilms from peg lids without damaging the cells or compromising sample integrity. Its high-throughput capability and user-friendly operation significantly reduce processing time while enhancing reproducibility, making it an ideal solution for laboratories conducting biofilm viability assays and antimicrobial efficacy testing.

ASTM E2799

ASTM E2799 is a standardized test method designed to evaluate the efficacy of antimicrobial agents against biofilms, specifically focusing on Pseudomonas aeruginosa. Biofilms are complex microbial communities that adhere to surfaces and exhibit heightened resistance to antimicrobial treatments, making them a significant challenge in healthcare, industrial, and environmental contexts. This method uses the Minimum Biofilm Eradication Concentration (MBEC) assay to provide a reliable and reproducible framework for testing disinfectants under realistic biofilm conditions.

How Does Sonication contribute to ASTM E2799?

Sonication plays a critical role in the ASTM E2799 protocol by facilitating the detachment of biofilms from test surfaces, ensuring an accurate assessment of microbial viability after treatment. By using controlled ultrasonic energy, the protocol achieves efficient and consistent biofilm disruption without damaging the cells, enabling precise and reproducible results. This makes ASTM E2799 an essential tool for advancing biofilm research and developing effective antimicrobial strategies.

How to Run the MBEC Assay in accordance with ASTM E2799-22

The ASTM E2799-22 protocol outlines the steps for evaluating the efficacy of antimicrobial agents against biofilms. When using the Hielscher UIP400MTP microtiter plate sonicator, the protocol can be adapted to incorporate this advanced sonication method for biofilm detachment. Below is a description of the steps tailored to using the microplate sonicator UIP400MTP:

1. Preparation of Biofilms
   Prepare a sterile 96-well microtiter plate with detachable peg lids.
   Inoculate the wells with a standardized bacterial suspension in a suitable growth medium.
   Place the peg lid onto the plate, ensuring the pegs are fully immersed in the bacterial suspension.
   Incubate the plate under controlled conditions (e.g., temperature and time) to allow biofilm formation on the pegs.
2. Treatment with Antimicrobial Agents
   After biofilm formation, replace the medium with fresh medium containing the test antimicrobial agents at different concentrations.
   Reattach the peg lid and incubate under the conditions specified in the test design.
3. Preparation for Biofilm Detachment
   After treatment, rinse the peg lid by immersing it in a sterile rinse solution to remove residual antimicrobial agents and loosely adhered planktonic cells.
4. Biofilm Detachment Using the UIP400MTP Sonicator
   Prepare a fresh 96-well microtiter plate containing a recovery medium (e.g., neutralizing broth).
   Detach the biofilms by placing the peg lid into the wells of the recovery medium plate.
   Insert the plate into the UIP400MTP sonicator, ensuring the lid and plate are properly aligned.
   Set the sonicator to 70%-80% amplitude and sonicate for 3-4 minutes at cycle mode, to ensure consistent biofilm detachment without cell lysis.
5. Assessment of Biofilm Viability
   After sonication, process the recovery medium containing detached biofilm cells using standard microbiological or analytical techniques, such as:
   • Colony-forming unit (CFU) counts: Perform serial dilutions and plate on agar for viable cell enumeration.
   • Optical density (OD) measurements:Use spectrophotometric analysis to assess cell density.
   • Fluorescence-based viability assays:Quantify live and dead cells using fluorescent dyes.
6. Data Analysis
   Compare the viability data of treated and untreated biofilms to determine the efficacy of the antimicrobial agents.
   Calculate the minimum biofilm eradication concentration (MBEC) based on the concentration required to eliminate viable biofilm cells.
7. Quality Control
   Ensure consistent sonication conditions by regularly calibrating the UIP400MTP and verifying that the biofilm detachment process does not result in significant cell lysis or destruction.
   Include appropriate controls, such as untreated biofilms and medium-only blanks, to validate the results.

The biofilm cells formed on the pins were dislodged by sonication (Hielscher Ultrasound Technology, Teltow, Germany, UIP250MTP) for 5 min in 96-well plates containing fresh culture medium for recovery of the cells.
(Picture and study: ©de Oliveira et al., 2016)

By integrating the UIP400MTP into the ASTM E2799-22 workflow, the process of biofilm detachment is optimized for consistency, efficiency, and high-throughput applications.