This procedure directly compares planktonic and biofilm resistance for a bacterial strain that can form a biofilm. First, establish a biofilm, replenish the media and apply serial dilution of the antimicrobial agent of choice. Then allow the cells to recover in fresh, medium.
Now assay for viability of the cells by plating on LB auger. Ultimately determining the minimal bactericidal concentration of planktonic and biofilm cells is used to show the increase in resistance that results from growth in a biofilm. Generally, individuals new to this method will struggle because they contaminate adjacent wells, or they fail to let the prongs of the multi-pronged device cool down enough so that they don't kill the bacteria.
Culture, the wild type and mutant strain of bacteria for 16 hours in a rich medium at 37 degrees Celsius, dilute the saturated overnight cultures into a fresh medium like M 63, minimal medium, supplemented with magnesium, sulfate and arginine. Then Eloqua. 100 microliters per well.
Allotting 24 wells for each strain, incubate for 24 hours at 37 degrees Celsius. Next, prepare 10 x stock solutions of a dilution series of antibiotic for seven wells and store on ice. Using a multi-channel pipette, remove the spent supernatant that contains planktonic cells.
Then add 90 microliters of M 63 to all of the wells and add 10 microliters of the appropriate antibiotic stock solution to the no antibiotic control. Well, for each replicate and strain, add 10 microliters of water. Incubate the micro titer plate for 24 hours at 37 degrees Celsius.
Using a multichannel pipette, remove the spent supernatant that contains planktonic cells. Add 115 microliters of M 63 to all wells and continue to grow the cultures for 24 hours. At 37 degrees Celsius.
Label one lb auger plate per one half 96. Well microtiter plate to sterilize the multi-pronged device, dip it in 100%ethanol and pass the prongs across the open flame of a bunsen burner. Let the prongs cool slightly.
Using the tips of the prongs. Transfer planktonic culture from each well of the microtiter plate to the surface of an LB auger plate. Place the LB auger plates at 37 degrees Celsius for 16 hours.
Then visually determine the cutoff for bacterial growth as the minimal bactericidal concentration of the antibiotic culture, the wild type and mutant strain of bacteria for 16 hours in a rich medium at 37 degrees Celsius, dilute the saturated overnight cultures one to 100 into fresh medium for antibiotic resistance assays to each well of a 96 well microtiter dish. Add 90 microliters of the bacterial dilution. Now to expose the planktonic cells to a concentration gradient of antibiotic.
Start with 10 x stock solutions of a dilution series of antibiotic like tobramycin mycin. Add 10 microliters of the antibiotic stalks as per experimental design. Then add 10 microliters of water to the no antibiotic control wells for each replicate and strain.
Incubate the microtiter plate for 24 hours at 37 degrees Celsius to assay for live cells. Start by labeling one LB auger plate for each half of a 96 Well microtiter plate. Sterilize the multi-pronged device by flaming with 100%ethanol.
Repeat once. Then using the multi-prong device, transfer about three microliters of planktonic culture from each well of the microtiter plate to the surface of an LB auger plate. Incubate the plates at 37 degrees Celsius for 16 hours.
Then determine the minimal bactericidal concentration of the antibiotic by identifying by eye the cutoff for bacterial growth. In this experiment, the two different minimal BACTERICIDAL concentration assays were used to compare the sensitivity of PA 14 wild type with a mutant strain that lacks the antibiotic resistance. Gene NDVB.
Here, the minimal bactericidal concentrations of the antibiotic tobramycin were measured for the biofilm. The MBCB for PA 14 is 100 micrograms per milliliter compared 12.5 micrograms per milliliter for the mutant. By contrast, the minimal bactericidal concentrations of tobramycin for the planktonic cells was eight micrograms per milliliter for both PA 14 wild type and mutant.
Once mastered, the NBCB can be done in around an hour, split up over five days.
