Generation of Greater Bacterial Biofilm Biomass using PCR-Plate Deep Well Microplate Devices

The goal of this procedure is to compare the standard peg lid biofilm device to a polypropylene deep well biofilm device through crystal violet staining and antimicrobial susceptibility testing. A standard peg lid biofilm device has 96 pegs and fits on top of a standard 96-well microtiter plate allowing for high throughput biofilm experiments. The peg lid provides a surface for biofilm growth and biofilms grown on the pegs can be further analyzed in downstream experiments.

However, the plates use a maximum volume of 200 microliters making it more challenging to study biofilm formation and biomass using experiments that demand greater quantities of biofilm. Here, we describe the deep well biofilm device for growing static biofilms. The deep well biofilm device utilizes a 96-well deep well microtiter plate fitted with a semi-skirted 96-well PCR plate as the peg lid.

This method allows for a maximum plate volume of 750 microliters, provides more peg surface area for biofilm growth, and is lower in cost as compared to the standard biofilm device. We will describe and compare the protocols for crystal violet staining of biofilms to determine biofilm biomass, as well as determination of the minimum biofilm eradication concentration, also known as the MBEC. Shown is a schematic overview of the experiment that will be described.

Streak desired bacterial strains from a cryopreserved stock directly onto a nutrient agar plate. Incubate agar plates overnight with optimal strain conditions. The following day, inoculate a single colony in five milliliters of growth media and grow cultures in an incubator overnight.

The biofilm plates will be filled as shown. Fill the outer wells with 750 microliters of sterile water and negative control wells with 750 microliters of sterile growth media. Fill the remaining wells with 675 microliters of sterile growth media.

Standardize overnight cultures to an optical density at 600 nanometers of 1.0. Create a tenfold dilution series until a 10 to the minus three dilution is reached. Inoculate media wells with the 10 to the minus three diluted culture for a final bacterial dilution of 10 to the minus four and final total in-well volume of 750 microliters.

Carefully insert the peg lid into the biofilm plate. Incubate plates at optimal strain conditions with maximum shaking of 160 RPM for 24 hours. Carefully remove the peg lid from the biofilm growth plate and rinse the biofilms in a new deep well plate filled with 800 microliters of sterile PBS per well.

Transfer the peg lids to a new deep well plate containing 800 microliters per well of 0.1%weight by volume crystal violet and allow to stain for five minutes. Rinse the lids again in a fresh deep well plate filled with PBS to remove excess stain. After allowing the lids to dry for 10 minutes in a biosafety cabinet, destain the pegs in a deep well plate filled with 800 microliters of 30%ascetic acid per well for five minutes.

Remove the peg lid and mix thoroughly to evenly distribute the stain. Transfer 200 microliters from the deep well biofilm device into a standard 96-well microtiter plate and read the optical density at 550 nanometers. The antimicrobial challenge plates will be prepared as shown.

Fill the outer wells with 750 microliters of sterile water and the positive and negative control wells with 750 microliters of growth media. Fill the remaining wells with 750 microliters of the desired antimicrobial dilution series. Carefully remove the peg lid from the device and rinse in a sterile deep well plate with 800 microliters per well of PBS.

Transfer the peg lid to the antimicrobial challenge plate and incubate for the desired exposure timeframe. Aseptically remove peg lids from the antimicrobial exposure plate and rinse in a sterile deep well PBS-filled plate. Remove the peg lid from the PBS and transfer to a new deep well plate containing 750 microliters of recovery media per well.

Sonicate the plate in a sonicating water bath for 30 minutes. Remove the peg lid and replace with a standard non-pegged flat top microtiter plate lid to avoid contamination. Incubate at optimal strain conditions overnight.

The following day, transfer 200 microliters from the deep well plate to a standard microtiter plate and read the optical density at 600 nanometers. Crystal violet staining is an effective and widely used method of approximating the biomass of bacterial biofilms. Crystal violet staining of the standard and deep well devices showed that both bacterial species grew significantly more biomass on the deep well device as compared to the standard device with Escherichia coli forming 2.1-fold more biomass compared to the standard biofilm device and Pseudomonas aeruginosa forming 4.1-fold more biomass.

These results were consistent with our hypothesis that the increased surface area of the deep well biofilm device would allow for increased biomass accumulation. Despite greater variability in technical replicate CV-stained values for both strains growing on the deep well device, no statistically significant differences for either species'biological replicates were noted using pair-wise two-way ANOVA or student's T-test with P values being greater than 0.05. This finding shows that the biofilm formation by deep well devices form reproducible biofilms similar to that of standard devices.

Our results after standardizing biofilm biomass for volume and peg surface area between the standard and deep well devices did indicate a slight but significant material preference between the two bacterial species tested. On the polypropylene deep well device, Pseudomonas aeruginosa demonstrated a 1.4-fold relative increase in biomass formation and Escherichia coli demonstrated a 1.52-fold relative decrease in biomass formation with the inverse effect observed on the polystyrene standard device. This difference in bacterial species preference for biofilm device peg material indicates that the results cannot be cross-compared between biofilm devices.

Antimicrobial challenge of 24-hour biofilms was tested using the quaternary ammonium compound benzalkonium chloride which is known to be an effective inhibitor of bacterial biofilm formation, but a less effective antimicrobial for biofilm eradication. Our results showed that BZK BMEC values obtained from the deep well biofilm device increased by an average of sixfold for Escherichia coli and eightfold for Pseudomonas aeruginosa when compared to growth on the standard biofilm device. The BZK MBEC results were more variable as expected since it is a less effective antimicrobial for biofilm eradication and resulted in a range for Escherichia coli MBEC values on both devices due to a couple outlier biofilm pegs that survived at higher concentrations.

We also compared the disinfectant bleach, which is known to be a very effective antimicrobial for bacterial biofilm eradication. Bleach demonstrated a slightly higher MBEC value when tested in the standard device as compared to the deep well device for both species, leading to a fourfold increase in Escherichia coli and a twofold increase in Pseudomonas aeruginosa respectively. However, these differences were within the range of reproducible error between devices and demonstrated much less variability than BZK, confirming bleach as a more effective antimicrobial for biofilm eradication.

A major limitation of each biofilm device would be the different plastic composition of the pegs and the resulting variability in adherence and biomass formation of different bacterial species to these surfaces. Additionally, chemical interactions and compatibilities between different antimicrobials and the plastic peg material must be taken into account before performing the described protocol. In conclusion, this protocol and the findings from our biomass comparisons on the deep well and standard biofilm devices show that both are capable of cultivating robust Escherichia coli and Pseudomonas aeruginosa biofilms with the deep well device providing significantly more biomass per peg surface area than the standard device.

The deep well biofilm device offers a viable, affordable alternative for static, high throughput biofilm cultivation and screening studies that require larger amounts of biofilm for downstream analysis. Due to the differences in plastic material compositions between each device, the CV-stained biofilm biomass and MBEC values cannot be directly compared between devices. However, if experiments are consistently conducted on the same device, results obtained between isolates and antimicrobials are comparable.

The deep well biofilm device is recommended for labs that would like to study biofilms in high throughput assays using low-cost materials.