Rapid Report pubs.acs.org/biochemistry
Defining Molecular Details of the Chemistry of Biofilm Formation by Raman Microspectroscopy Paul R. Carey,*,† Blake R. Gibson,† Jordan F. Gibson,† Michael E. Greenberg,† Hossein Heidari-Torkabadi,‡ Marianne Pusztai-Carey,† Sean T. Weaver,† and Grant R. Whitmer† †
Department of Biochemistry and ‡Department of Pharmacology, Case Western Reserve University, Cleveland, Ohio 44106, United States S Supporting Information *
compounds that retard or eliminate biofilm growth. Several groups have suggested Raman spectroscopy as a possible candidate for analyzing bacterial biofilms. Different forms of Raman spectroscopy have been used, such as UV resonance Raman,5 confocal Raman,6,7 and Raman studies of planktonic cells and biofilm.8 However, these earlier studies did not reveal detailed molecular insights into the conversion of planktonic cells to biofilm. The studies presented here provide such details with the added advantage that planktonic cells are trapped in their growth phase by being frozen within 1 min, thus minimizing metabolic changes. Recently, we showed that freeze-dried bacteria yield highquality Raman spectra that are a source of detailed molecular information.9−11 Here we show that these data can be combined with the Raman spectra of films to yield unique insights into the chemical changes accompanying biofilm formation. The data on freeze-dried planktonic cells are obtained with a Raman microscope.9 Cells, at midgrowth phase, are washed to remove growth medium, flash-frozen, and freeze-dried. They are then mounted within small indentations on aluminum foil. For biofilm formation, the cells are grown in a shaker at 37 °C, rapidly washed, and suspended in an isotonic NaCl solution at 22 °C. Thus, they are stressed by a combination of cold shock and nutrient deprivation. After standing in the Al indentations for 48 h in an atmosphere of saturated water vapor, the cells are dried in an oven at 50 °C. The resultant film is washed with an isotonic NaCl solution to remove particles that are not part of the extracellular matrix. The dried film is then examined under the Raman microscope. A more detailed protocol is described in the Supporting Information. The method of drying the biofilm was selected on the basis of standard published protocols.12 Comparison of the spectra of the freeze-dried planktonic cells with those from the corresponding biofilm gives detailed information about the chemical changes accompanying biofilm formation. As a control, methyl violet staining experiments were undertaken12 to test for the presence of biofilm. The results are given in Figure 1 and show that both E. coli and S. epidermidis are forming biofilm. Further confirmation is given by photographs of the biofilm shown in Figure S1.
ABSTRACT: Two protocols that allow for the comparison of Raman spectra of planktonic cells and biofilm formed from these cells in their growth phase have been developed. Planktonic cells are washed and flash-frozen in
