The overall goal of this procedure is to grow bacterial biofilms under low or moderate sheer force using either a drip flow biofilm reactor or a rotating disc reactor. Coupons in the biofilm react as are first inoculated with bacteria and incubated at 37 degrees Celsius to allow the bacterial cells to adhere to the growth surfaces. Once the cells have attached to the coupons, a flow of growth media is started producing a sheer force that operates during biofilm development.
Mature biofilms can then be harvested for analysis observation of the biofilms. Using scanning electron microscopy can be performed to determine the ultra structure of the biofilms produced. I'm Blaze Balls.
The main advantage of this technique of existing methods like flow cells is that high biomass production and multiple identical biofilms are readily achieved. Demonstrating this procedure will be Rachel Stevenson and Kelly Schwartz, a technician and graduate student from my laboratory. The drip flow biofilm reactor consists of four chambers with input ports for media entry and outflow ports for waste exit.
Inside each chamber is a removable coupon upon which the biofilm grows. To assemble the drip flow biofilm reactor, place the coupons in each parallel chamber and secure the chamber lids. Autoclave the assembled reactor as well as the in fluent nutrient tubing to sterilize the system before use.
Also autoclave the biofilm growth medium to inoculate the sterilized drip flow reactor. Place the apparatus on a flat surface and clamp the effluent lines. Next, fill each chamber with 10 milliliters of sterile triptych soy broth based growth medium, and add 100 microliters of staphylococcus aureus.
Overnight culture grown in the same medium to each chamber separately. Place the inoculated reactor in a 37 degrees Celsius incubator for 18 hours to allow the cells to adhere to the coupon surface. After the incubation, UNC clamp the effluent tubing and place the reactor on an incline wooden block cutter to 10 degree angle.Aseptically.
Connect the fluent nutrient tubing to a bottle containing continuous flow nutrient broth. Feed the tubing line through the pump and prime the tubing by running the pump at full speed. Once the fluent tubing is primed, stop the pump and attach 22 gauge one inch needles to the end of each tube.
Wipe the chamber inlet stopper with an ethanol wipe and aseptically. Insert the needles through the inlet stopper. Turn on the pump and allow medium to drip over the coupons.
At a flow rate of roughly 125 microliters per minute, the media should flow downward from the inlet stopper port to the effluent port. Operate the reactor in continuous flow for three days. Occasionally checking the reactor for proper drainage.
Drainage is checked by visual inspection of the chambers to ensure media is not pooling in the chambers. If media is pooling, pinching, the outflow tubing will usually promote proper drainage to harvest the biofilms, stop the pump and remove the needles from the reactor. Then place the reactor on a flat surface.
After three days of continuous flow, staphylococcus aureus biofilms appear as yellow biomass spread along the length of the inoculated coupons. A scanning electron micrograph of the biofilm reveals staphylococcus aureus covering the coupon in a surface associated biofilm community. In order to quantify the biomass obtained, use sterile forceps to as septically, remove the coupons, harvest the bacteria using a cell scraper, then transfer them to a conical tube containing five milliliters of sterile PBS.
Using a tissue, homogenizer disaggregate the bacterial clumps until a uniform suspension is obtained. Colony forming units are quantitated by plating serial dilutions onto triptych sogar plates incubating for 24 hours at 37 degrees Celsius and counting colonies. The rotating disc biofilm reactor is comprised of a chemos stat through which growth medium is pumped.
A spinning disc with a star bar and 18 coupons designed to fit into the spinning disc. To assemble the reactor first, load the spinning disc coupons into the slots of the spinning disc. Next place the loaded disc inside a one liter glass beaker with an overflow port and cap the reactor with a number 15 rubber stopper with one hole for media flow and one hole for aeration.
Autoclave, the assembled reactor and the inlet tubing to sterilize the system. To inoculate the reactor, fill the beaker with 250 milliliters of sterile culture medium and add 500 microliters of an overnight culture of staphylococcus aureus grown in triptych soy broth. Place the reactor on a stir plate set at 250 rotations per minute and incubate it overnight at 37 degrees Celsius to allow the cells to adhere to the coupons.
Following the overnight incubation, connect the inlet tubing to the port on the medium reservoir and to the peristaltic pump. Turn on the pump and set the flow to 0.25 milliliters per minute. Allow the reactor to run for 24 hours at 37 degrees Celsius to harvest the resulting biofilms.
Stop the reactor and aseptically. Remove the disc without touching the coupons. Using sterile forceps, remove the coupons and carefully dip each one in sterile PBS to remove loosely attached bacteria for antimicrobial compound testing.
Aseptically, place the coupons into individual wells of a 96 well plate containing compounds of interest. Incubate the plate for four hours at 37 degrees Celsius. Then transfer the coupons into 1.5 milliliter micro centrifuge tubes containing one times PBS and disperse the biofilms using a homogenizer.
Finally, plate serial dilutions of the cells on nutrient agar medium to determine the number of viable colony forming units per sample as described earlier. This graph shows the number of colony forming units in biofilms prepared using the spinning disc reactor and exposed to hydrogen peroxide, the ability to produce up to 18 identical biofilms. Using the spinning disc reactor makes this technique ideal for antimicrobial compound testing.
After watching this video, you should have a good understanding of how to run drip flow and rotating disc reactor biofilms. These biofilm reactors offer alternatives to flow cell and microtiter plate biofilms, and are ideal when a high amount of biofilm biomass is needed or antimicrobial testing on established biofilms is desired.
