Identification of a reliable fixative solution to preserve complex architecture of bacterial biofilms for scanning electron microscopy evaluation

Authors: Dassanayake, Rohana; Falkenberg, Shollie; Stasko, Judith; Shircliff, Adrienne; Lippolis, John; Briggs, Robert

Submitted to: PLOS ONE
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/18/2020
Publication Date: 5/29/2020
Citation: Dassanayake, R.P., Falkenberg, S.M., Stasko, J.A., Shircliff, A.L., Lippolis, J.D., Briggs, R.E. 2020. Identification of a reliable fixative solution to preserve complex architecture of bacterial biofilms for scanning electron microscopy evaluation. PLoS One. 15(5):e0233973. https://doi.org/10.1371/journal.pone.0233973.
DOI: https://doi.org/10.1371/journal.pone.0233973

Interpretive Summary: Bacterial biofilms are organized communities of bacterial cells which grow by attaching to surfaces, each other, and are covered by a film-like substance called matrix. Bacteria form biofilms to protect from the host immune response and also due to the limitation of nutrients. Film-like substance is often damaged when biofilms are fixed before microscopic examination. Therefore, we compared several fixative solutions to identify which fixative could best preserve the architecture of bacterial biofilms. We identified one fixative that could preserve both film-like substance and bacterial cells in the biofilms to analyze the complex architecture of biofilms. This finding will help scientists to study the detailed structure of biofilms formed by other bacterial species.

Technical Abstract: Bacterial biofilms are organized sessile communities of bacteria enclosed in extracellular polymeric substances (EPS). To analyze organization of bacteria and EPS in high resolution and high magnification by scanning electron microscopy (SEM), it is important to preserve the complex architecture of biofilms. Therefore, fixation abilities of formalin, glutaraldehyde, and Methacarn (methanol/chloroform/acetic acid-6:3:1) fixatives were evaluated to identify which fixative would best preserve the complex structure of bacterial biofilms. Economically important Gram-negative Mannheimia haemolytica, the major pathogen associated with bovine respiratory disease complex, and Gram-positive Staphylococcus aureus, the major cause of chronic mastitis in cattle, bacteria were selected since both form biofilms on solid-liquid interface. For SEM analysis, round glass coverslips were placed into the wells of 24-well plates and diluted M. haemolytica or S. aureus cultures were added, and incubated at 37°C for 48-72 h under static growth conditions. Culture media were aspirated and biofilms were fixed with an individual fixative for 48 h. SEM examination revealed that all three fixatives were highly effective preserving the bacterial cell morphology, however only Methacarn fixative could consistently preserve the complex structure of biofilms. EPS layers were clearly visible on the top, in the middle, and in the bottom of the biofilms with Methacarn fixative. Biomass and three-dimensional structure of the biofilms were further confirmed spectrophotometrically following crystal violet staining and by confocal microscopy after viability staining, respectively. These findings suggest that Methacarn fixative is the best fixative to preserve the complex architecture of bacterial biofilms grown on glass coverslips for SEM evaluation.