PNNL

In this image on the cover of the January issue of Analytical & Bioanalytical Chemistry, PNNL scientists analyzed a digital image of a stained biofilm-covered sample to obtain red, green, and blue spectra. Their method quickly quantifies the biofilm intensity and brings scientists closer to finding ways to prevent biofilms from forming. Permission to reproduce given through http://creativecommons.org/licenses/by/4.0/. Enlarge Image.

Results: A simple and fast method to quantify biofilm growth over large areas shows promise for preventing the slimy coatings that plague surfaces exposed to wet environments. Biofilms, which form in seconds, are a many-layered problem. They are difficult to study, especially in marine environments. They also lead to biofouling, a bane to ships, sensors, heat exchangers, desalination equipment, medical implants, and more. Meanwhile, current standards for assessing anti-fouling coatings require up to two years of data collection.

Seeking to better understand how biofilms form and their effects on surfaces to aid development of coatings that prevent their formation, scientists at Pacific Northwest National Laboratory developed a method to quantify early stages of biofouling activity that requires only a digital photograph of a stained surface to be evaluated using a new image analysis algorithm for quantification. The scientists then can compare the results to determine how much biofilm has accumulated on a surface. The entire process takes minutes. Their research is featured on the cover of Analytical & Bioanalytical Chemistry.

Why It Matters: Once immersed in liquid, a surface is immediately bombarded with biomolecules and collects a film. In marine environments, bacteria and other microorganisms, such as diatoms and algae, colonize the film. Eventually, larvae of mussels and barnacles join the surface. On ships, biofouling dramatically increases drag, which decreases fuel efficiency-even thin films have a huge impact.

The concern with biofouling extends to many areas. Sensors deployed in the environment for national security are only effective as long as they remain clean. Heat exchanger efficiency drops dramatically from biofouling. One of the highest costs associated with desalination for clean water is preventing biofouling of membranes, while films on medical implants consistently cause infection. The PNNL team's work brings scientists closer to finding ways to prevent biofouling and its effects.

This figure illustrates the optical image analysis process. Staining samples enhances the visible contrast of biofouling. Digital image analysis then separates the data into color channels for accurate assessment of biofouling intensity. Enlarge Image.

Methods: The team developed a versatile method to make the heterogeneous structure of biofilms more visible and easier to quantify. They put 1-in.2 fiberglass sample coupons in culture with bacteria for six days. To enhance visibility of the biofilm that formed on the surface, they then applied their own broad-spectrum mixture of biomolecular stains to the samples. After taking digital photos, they applied an image analysis algorithm developed by Dr. Curtis Larimer, a postdoctoral fellow at PNNL, to quantify the overall amount of visible biofilm growth.

The algorithm objectively quantifies biofouling in the red, green, and blue spectra captured by the camera. Existing research methods rely on qualitative visual observations and the researcher's personal judgment when making the assessment.

"Our method removes human error and will make it easier to compare studies in different conditions or locations," said Larimer, lead author of the journal cover article.

The new method builds on standards that involve putting an anti-fouling coating on a sample and submerging it in water for up to two years. One of the team's goals was to dramatically reduce the exposure time needed so new materials could be evaluated more quickly. In the early stages of fouling, the film is thin and nearly transparent, and biomolecular staining made it easier to visualize. Another goal was to improve the outcomes of image analysis. Older image analysis algorithms merely calculated how much of the surface was covered with biofilm.

"It became clear to us that all samples reached 100% coverage early on," Larimer said. "So, measuring the area didn't give the full picture. By seeing the whole image, not just counting pixels, we can measure the area and intensity of fouling, and we are better equipped to collect data from a large set of replicates."

To prove their method can be applied across a spectrum of environments, they also used it successfully at PNNL's Marine Sciences Laboratory in Sequim, Wash. This time, samples were submerged for six to 10 weeks.

What's Next? The PNNL team currently is using the method in several studies of anti-fouling coatings targeted for use on marine energy platforms and industrial applications. They plan to publish a paper demonstrating the method's use and application in the marine environment. They also have a provisional patent application filed for the biomolecular stain. The team hopes to show that the image analysis method can be used in the field by capturing images on a cell phone.

Acknowledgments

Sponsor: The work was supported by the Chemical Imaging Initiative, a PNNL Laboratory Directed Research and Development program; the U.S. Department of Energy Office of Energy Efficiency and Renewable Energy; and the Intelligence Community Postdoctoral Research Fellowship Program.

Research Team: Curtis Larimer, George Bonheyo, Shane Addleman, Eric Winder, and Robert Jeters, PNNL; and Ian Nettleship, University of Pittsburgh

Reference: Larimer C, E Winder, R Jeters, M Prowant, I Nettleship, RS Addleman, and GT Bonheyo. 2015. "A Method for Rapid Quantitative Assessment of Biofilms with Biomolecular Staining and Image Analysis." Analytical and Bioanalytical Chemistry 408(3):999-1008. DOI: 10.