Harvesting and Disaggregation: An Overlooked Step in Biofilm Methods Research

The overall goal of these procedures is to harvest and disaggregate biofilms in the optimal way so as to increase reproducibility. Three techniques will be demonstrated in this video, each focusing on the harvesting and disaggregation from a different surface type. This video will demonstrate the proper techniques for one, vortexing and sonicating biofilm off of a hard non-porous surface such as a polycarbonate coupon from the CDC biofilm reactor.

Two, the scraping and homogenizing of the biofilm off of the hard non-porous borosilicate glass surface commonly found in the drip flow reactor. And three, the scraping, vortexing, and sonicating of biofilm from a porous silicone tubing surface commonly found in the catheter model. Hi, my name is Kelly Buckingham-Meyer here at the Standardized Biofilm Methods Lab.

In our lab, we break biofilm methods down into four components:growth, treatment, sampling, and analysis. In the peer-reviewed literature, details of how biofilms are sampled or harvested and disaggregated from a surface are typically sparse. This is the reason for this video.

Since our lab develops and tests standardized biofilm methods, we conducted an extensive literature review where the three most common biofilm harvesting and disaggregation methods were identified. Those methods are vortexing and sonicating biofilm from a polycarbonate coupon, scraping and homogenizing biofilm from a borosilicate glass coupon, and scraping, vortexing, and sonicating biofilm from silicone tubing. While methods written on paper are powerful, we hope that the video with the technical details presented by my coworker Lindsey Miller and I will be helpful.

To begin, grow mature Pseudomonas aeruginosa 15442 biofilm according to the ASTM Standard E2562, the Standard Test Method for Quantification of Pseudomonas aeruginosa Biofilm Grown with High Shear and Continuous Flow using the CDC Biofilm Reactor. At the end of the 48-hour growth period, prepare to treat and sample coupons according to the ASTM Standard E2871, the Standard Test Method for Determining Disinfectant Efficacy Against Biofilm Grown in the CDC Biofilm Reactor Using the Single Tube Method. Then aseptically insert autoclaved splash guards into sterile 50 milliliter conical tubes using flame-sterilized forceps.

Repeat for all tubes that will receive treatment. Tubes for control coupons do not need a splash guard. Aseptically remove a rod from the CDC biofilm reactor and rinse it in 30 milliliters of sterile dilution water.

This will remove the loosely attached cells from the biofilm. Hold the rod parallel to the bench top over the sample tubes with splash guards. Using a flame-sterilized Allen wrench, loosen the set screws to drop a biofilm-coated coupon into the tube.

Repeat for the desired number of coupons. Remove the splash guards and place the splash guards in a separate container for sterilization. Using a five milliliter serological pipette, slowly pipette four milliliters of treatment or control into the tubes so that the liquid flows down the inside of the wall of the tube.

Gently swirl the bottom of the tube so that any air bubbles under the coupon are displaced. Allow for 30 to 60 seconds between each addition. At the end of the specified contact time, pipette 36 milliliters of neutralizer into the tubes in the same order that the treatment or control was applied.

Vortex each tube on the highest setting for 30 plus or minus five seconds and ensure that a complete vortex is achieved. Place tubes in the tube rack suspended in the degassed sonicator such that the water level in the bath is equal to the water level in the tubes. Sonicate the samples at 45 kilohertz, 100%power, and normal function for 30 plus or minus five seconds.

Repeat the vortex and sonication cycles and end with a final vortex, for a total of five cycles. Finally, serially dilute the sample, plate the dilution on R2A agar and incubate the plates for 24 hours at 36 degrees Celsius. Count the colonies as appropriate to the plating method and record the data.

This next biofilm harvesting and disaggregation technique is useful for hard non-porous coupons that are the standard in the drip flow reactor. Grow a mature Pseudomonas aeruginosa 15442 biofilm according to the ASTM Standard E2647 the Standard Test Method for Quantification of Pseudomonas aeruginosa Biofilm Grown Using Drip Flow Biofilm Reactor with Low Shear and Continuous Flow. Set up the sampling station to include a sampling board, 95%ethanol in a beaker, an alcohol burner, hemostats, a coupon removal tool, beakers with sterile dilution water, and dilution tubes for rinsing the coupons.

Turn the pump off, remove the channel cover and use sterile coupon removal tool and hemostats to remove the coupon, being careful not to disturb the biofilm. Rinse the coupon by gently immersing with a fluid motion in 45 milliliters of sterile dilution water contained in a 50 milliliter centrifuge tube. Immediately reverse the motion to remove the coupon.

Place the coupon into a beaker containing 45 milliliters of sterile dilution water. Scrape the biofilm-covered coupon surface in a downward direction for approximately 15 seconds using a sterile spatula or scraper. Rinse the spatula or scraper by stirring it in the beaker.

Repeat the scraping and rinsing process three to four times ensuring full coverage of the coupon surface. Rinse the coupon by holding it at a 60 degree angle over the sterile beaker and pipetting one milliliter of sterile dilution water over the surface of the coupon. Repeat for a total of five rinses.

The final volume in the beaker is now 50 milliliters. Working in the biosafety cabinet, homogenize the scraped biofilm sample. Attach a sterile homogenizer probe to the homogenizer.

Place the probe tip in the liquid and turn the homogenizer on and ramp up to 20, 500 RPM. Homogenize the sample for 30 seconds. Turn down the RPM and switch the homogenizer off.

Sanitize the probe between biofilm samples by homogenizing in nine milliliter sterile dilution blank at 20, 500 RPM for 30 seconds as described above. Homogenize a nine milliliter tube of 70%ethanol for 30 seconds and detach the probe and let it stand in the ethanol tube for one minute. Homogenize two additional dilution blanks.

Also, a sterile disposable homogenizer probe may be used for each sample. Serially dilute the homogenized sample, plate the dilutions on R2A using the appropriate plating method, and incubate the plates for 24 hours at 36 degrees Celsius. Count the colonies and record the data.

This final biofilm harvesting and disaggregation technique is useful for biofilm grown in porous tubing like the silicone tubing used in the catheter model. To begin this procedure, grow a mature E.coli 53498 biofilm in silicone catheter tubing. Prepare the sampling materials:rinsed tubes, a sterile centrifuge tube, an empty sterile Petri dish, flame-sterilized stainless steel hemostats, scissors, timer, and a ruler.

With the pump paused, use 70%ethanol to clean the outside of the tubing. Measure two centimeters from the end, avoiding the area attached to the connector, and mark tubing to determine cutting locations. With flame-sterilized scissors, cut the tubing on the two centimeter mark.

Rinse the tubing segment to remove planktonic cells. With flame-sterilized forceps, gently immerse tubing segment into 20 milliliters of sterile dilution water, then immediately remove. Place the segment into 10 milliliters of neutralizer.

With flame-sterilized forceps, hold the tubing segment and scrape with a sterile wooden applicator stick until all inner areas of the tubing have been scraped. Occasionally rinse the stick in the 10 milliliters of neutralizer and place the segment back into the sample tube. The scraped tubing segment is now the 10 to the zero or the zero dilution.

Vortex each tube on the highest setting for 30 plus or minus five seconds. Place the tubes in the tube rack suspended in the sonicator bath, such that the water level in the bath is equal to the liquid level in the tubes. Sonicate at 45 kilohertz, 100%power, and normal function for 30 plus or minus five seconds.

Repeat this vortex and sonication cycle, then end with the final vortex. Serially dilute the samples in buffered water. Plate on tryptic soy agar using the appropriate plating method and then incubate the tubes at 36 plus or minus two degrees Celsius for 24 hours.

Count the colonies as appropriate to the plating method used and record the data to calculate the arithmetic mean. These four images taken by Danielle Or and Blaine Fritz all help to illustrate the importance of this harvesting and disaggregation procedure. All four of these coupons were stained with crystal violet to help visualize the amount of Pseudomonas aeruginosa biofilm present on the coupons before and after the vortex and sonication process.

Coupon A is completely covered in biofilm, while coupons B, C, and D all have substantially less biofilm on their surface. Coupons B, C, and D were all subjected to the vortex and sonication process described previously. These two images taken by Lindsey Miller on a confocal microscope at 12.5X magnification depicts a Pseudomonas aeruginosa biofilm that was treated with the back light live/dead stain.

The image on the left was sonicated at 45 kilohertz and 10%power, while the image on the right was sonicated at 45 kilohertz and 100%power. Clearly, the coupon on the left has much more biofilm remaining on its surface compared to the coupon on the right. This final figure displays how the manipulation of different sonication parameters affects the amount of Pseudomonas aeruginosa biofilm removed from the coupon surface.

All of these coupons were sonicated at 45 kilohertz, 100%power, and on normal setting for 30 plus or minus five seconds. These images were taken at 12.5X magnification by Lindsey Miller. Coupons in row one were sonicated in dilution water, while the coupons in row two were sonicated in DE neutralizing broth.

It appears that there is less biofilm remaining on the coupons that were sonicated in DE broth which contains a surfactant compared to the dilution water. Coupons A, B, E, and F were all sonicated in a 10 milliliter volume, while coupons C, D, G and H were sonicated in 40 milliliters of solution. There is clearly less biofilm on the coupons which were sonicated in the smaller volumes.

Coupons A, C, E, and G were sonicated in the bath with three tubes at once, while coupons B, D, F, and H were sonicated in a bath with 12 tubes at once. It appears that the samples sonicated with fewer tubes exhibited superior biofilm harvesting. Ultimately, minimizing the volume in the tubes, reducing the number of tubes sonicated at once, and use of DE neutralizing broth all seem to contribute to enhanced biofilm harvesting.

There's no perfect method to harvest and disaggregate the biofilm, but some approaches are better for different surfaces and/or applications. The techniques demonstrated in this video are the three most common methods according to a literature review. These three techniques can provide a starting point for researchers.

It is important to take the time to validate the chosen harvesting and disaggregation method. All equipment is slightly different, so even if the method has been standardized, it is still prudent to confirm the process for the equipment used. Research has demonstrated that improper choices may lead to biased test results.

After watching this video, this information will hopefully help researchers make more informed decisions on which method to use and provide guidance on improving reproducibility for the three surface types and complimentary harvesting and disaggregation techniques.