When thinking about bacteria, we often imagine bacterial cells floating in liquid medium. But, in reality, this planktonic lifestyle is only a very small episode in the bacterial life cycle. To be able to survive environmental stress like nutrient shortages and attacks launched by cleaning brushes, antibiotics or disinfectants, they attach to surfaces and build colonies that are reinforced with a self-produced extracellular matrix (ECM). The ECM acts as glue to keep the colony together so that the single bacteria can support each other but also act as a protective barrier. The conglomerate of bacteria in their self-produced extracellular matrix is called biofilm. Researchers from the Centre for the Advancement of Integrated Medical and Engineering Sciences (AIMES) at Karolinska Institutet in Sweden succeeded to take a unique look at Salmonella bacteria growing biofilm on semi-solid agar substrate. To be able to do that, the researchers used Salmonella that were previously tagged with green fluorescent protein (GFP) and added EbbaBiolight 680 to the semi-solid agar substrate on which the bacterial colonies were growing. They presented their findings in a study by Choong et al., which was recently published in the scientific journal Biofilms. EbbaBiolight are small, non-toxic fluorescent tracer molecules (optotracers) that don't disturb bacterial growth or biofilm formation. Also, EbbaBiolight fluorescence is switched-on only when the optotracer is bound to its target, which in the case of EbbaBiolight 680 has been identified as the ECM protein curli. Thus, EbbaBiolight 680 labels the ECM of gram-negative, curli producing bacteria like Salmonella or E. coli and allows imaging with extremely low background fluorescence. To be able to monitor many cultures simultaneously in an automated fashion, the researchers devised a semi-high throughput approach in which they used 6-well plates containing agar that was previously supplemented with EbbaBiolight 680 and placed a small amount of bacteria right in the middle of each dish to start-off the colony growth and biofilm formation.
The properties of the EbbaBiolight molecule allowed the researchers to monitor ECM formation in great detail and together with GFP-expressing bacteria revealed specific regions of active bacterial growth and the spatiotemporal co-localisation of biofilm components that expanded in a well-structured manner, projecting radial channel patterns (see video). Using GFP and EbbaBiolight 680 as fluorescent markers for bacteria and ECM does not only support fluorescence imaging but also enables fluorescence spectroscopy which facilitates data collection and subsequent analysis of the growth pattern in different regions of the bacterial biofilm. In summary, the semi-high throughput methodology using EbbaBiolight 680 provides a dynamic real-time analysis of biofilm formation on solid surfaces and might advance biofilm research by allowing the researchers to take biofilms and their structure into consideration when studying bacterial infections.