The, overall, goal of this assay platform is to provide a meaningful screening work flow combining three, relevant, end points, viability, biomass, and biofilm matrix, for identification and characterization of new, anti-biofilm compounds. This platform can help answers key questions in the field of antibiofilm agent discovery, by allowing an early distinction of short versus long term chemotherapeutic effects in a quick, explaining compatible manner. The main advantage of this developed platform is that it combines different assays to provide a more complete picture of the anti-biofilm effects of chemical libraries, especially of natural compounds.
To begin the experiment, prepare untreated, control samples with 200 microliters of 10 to the sixth CFU per milliliter bacterial culture per well of a sterile, 96 microwell plate. Include media controls without bacterial culture. Next, on the same 96 well plates, prepare samples with four microliters of 50x antibiotic stock solution and 196 microliters of the bacteria culture per well, then, incubate the cultures.
Continue with resazurin staining by using a multichannel pipette to transfer the entire planktonic solution, carefully, without touching the biofilms or creating air bubbles, to a separate, clean, 96 well plate. Again, using a multichannel pipette, wash the biofilms, once, with sterile PBS by adding 200 microliters per well and remove the solution carefully. Add 200 microliters of 20 micromolar resazurin solution, per well, of the biofilm plate and incubate the biofilm, in darkness, at 200 rpm for 20 minutes, until the untreated biofilm controls are evenly pink.
Then, measure the fluorescence with a plate reader. Remove the resazurin stain carefully from the wells. Fix the biofilms with 200 microliters of methanol, for 15 minutes.
After fixation, remove the methanol and let the plate air dry for 10 minutes. Next, use a multichannel pipette to carefully add 190 microliters of 0.02%crystal violet solution to the biofilm. Avoid touching the sides of the wells, with the stain, while pipetting, and prevent the formation of air bubbles, and do not press the pipette to complete blowout.
Then, incubate the plate for five minutes at room temperature. With a multichannel pipette, remove the stain carefully. Wash the biofilm with 200 microliters of deionized water.
Let the wells dry for five minutes at room temperature, and dissolve the remaining stain in 33%acetic acid. Incubate the plate for one hour at room temperature. After incubation, read the absorbance at 595 nanometers.
Measure the turbidity at 595 nanometers of the prepared, planktonic solution plate. This can be done during the incubation of the crystal violet stained plate. Next, stain the planktonic bacteria, with resazurin, by adding 10 microliters of the resazurin stock, per well.
Pipette the solution to mix it well. Incubate the plate, in darkness, at room temperature, 200 rpm, for approximately five minutes, until the untreated controls are evenly pink. Then, measure the fluorescence.
Obtain one of the parallel sample plates for this staining, and use a multichannel pipette to remove the planktonic solution from the wells. Wash the wells, once, with steril PBS carefully, without touching the biofilms. Add 200 microliters of wheat germ agglutinin, or WGA solution, per well, to be stained.
Then, incubate the plate. After incubation, wash the cells with PBS, three times, to remove the unbound stain. Let the plate dry for 15 minutes at room temperature.
Visualize the samples with a fluorescence microscope and an FITC filter. Dissolve the bound stain in 200 microliters of 33%acetic acid per well. Seal the wells with strip caps and sonicate them using a water bath sonicator.
After sonication, incubate the plate, for one hour, at 37 degrees Celsius. After one hour, repeat sonication while keeping the wells sealed between the sonication steps. Measure the fluorescence with a plate reader.
Again, take one of the parallel sample plates for this staining, and using a multichannel pipette, remove the planktonic solution from the wells. Then, wash the wells once with sterile PBS. Add six microliters of the staining solution per well, and incubate the plate in darkness for 15 minutes.
Before microscopy, remove the excess liquid, manually, using a multichannel pipette. Capture the images using a fluorescence microscope with a FITC filter, for green fluorescence, to visualize viable cells and a TRITC filter, for red fluorescence, to visualize dead cells. Use one of the parallel sample plates for this staining, and remove the planktonic solution, from the wells, and wash the wells once with sterile PBS, using a multichannel pipette.
Next, add 200 microliters of the staining solution, per well, and incubate them, for 15 minutes, in darkness. Finally, measure the fluorescence with a plate reader. Two screenings were run to demonstrate the performance of the platform as a percent of the untreated biofilm control.
The screening of a cinchona alkaloid derivative library identified one, active compound. The screening of a library of abietane-type diterpenoids and derivatives, identified five active compounds. Penicillin G and Ciprofloxacin both significantly decrease the viability, biomass, and biofilm matrix PNAG content prior to biofilm formation.
However, this was not the case when post-exposure was used. The most prominent result was the increase, of the biofilm matrix, to over 200%when preformed biofilms were treated with high concentrations of Penicillin G.This fluorescent image confirms that Penicillin G kills about 50%of the preformed, Staph aureus, biofilm, bacterial population. The average of the green to red fluorescence ratios for the untreated biofilm wells indicate a predominance of live cells.
While Penicillin treated wells have about 56%live cells. The cells remaining alive, after Penicillin treatment, produced a significantly higher amount of extracellular polymeric substance, or EPS, when compared with untreated biofilms. While attempting this procedure, it's important to remember to pay attention to the manual handling of the pipette to avoid contamination of bacteria and stain between wells.
Following this platform, antibiofilm compounds effective against Staphylococcus aureus can be quickly identified and characterised. If other bacterial species are used, other optimization of the staining protocols will be needed, but the rational of the platform will remain the same.
