AFM imaging of microbial biofilms is a new application for AFM. Biofilms are now recognized as a critically important mechanism by which microorganisms colonize surfaces and protect themselves from their environment. We mostly think of bacteria as free-floating cells thanks to decades of simplistic illustrations in textbooks. Instead, this planktonic form is just a short phase of the life cycle that allows the bacteria to disperse and colonize new sites. A direct approach to accessing these capabilities is to grow microbes directly on coupons (~10×10 mm) that are easily handled in culture conditions and fit perfectly in the AFM for imaging.

In medicine, biofilms simultaneously are essential to human health and can cause a range of problems. Biofilms on epithelial surfaces such as the gut are an integral component and critical to our health. But when “normal” bacteria are introduced to the wrong place or “bad” bacteria get in (for example through surgical site infections, implants, or devices) biofilms have serious consequences: they are difficult to detect, treat, and can pick up drug resistance from other microbes. Surface treatments of medical devices to prevent biofilm formation has become an active field of research and technology developent.

Bacterial biofilm found in the water reservoir of a CPAP machine. The biofilm was mechanically disrupted, leaving pockets of extracellular matrix where bacterial cells were torn out. In the intact area, high-magnification scans show bacterial cells (surface ripples ~2 nm tall) emerging out of a dense network of fimbriae (~10 nm tall) (shaded topography image).

Visualization of the extracellular matrix consisting of a fimbriae network.

Most of us don’t realize it but microbial biofilms are everywhere! The slimy stuff in your drain, all your sinks, the drip pan of your coffee machine, the green stuff in your water pitcher, the seals around your washer, the puddle at your doorstep. Yep, all biofilms!

Check out this example of what happens when you leave a piece of mica in a rain puddle for 48 hours. Life happens, and the AFM gives us to the ability to image amazing details. From a puddle to microbes to macromolecular assemblies.

Light microscopy overview of a rain puddle biofilm on mica with the HR-AFM. The cantilever is positioned in the corner of the protist, seen in the AFM topography image. The large protist is surrounded by the many small, pill-shaped bacteria and a pollen (top left). Two flagella emanate from the top left corner of the protist next to the pollen.

AFM has two additional capabilities that are relevant to biofilm research, the ability to measure forces (stiffness and adhesion of the biofilm) and to disrupt specific areas and structures with nanolithography. The MolBio3D AFM and the HR-AFM are very affordable options to work on biofilms both dry and in liquid. Additional correlation with light microscopy can link structural observation with AFM to optical methods (fluorescent staining, live-dead assays) using the LS-AFM.