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 <1 min to reduce the time for metabolic changes during processing, prior to freeze-drying. Biofilm is formed by standing cells in 50 μL indentations in aluminum foil in an atmosphere of saturated water vapor for 24-48 h. The results for Escherichia coli type K12 cells, which do not readily form biofilm, are compared to those for Staphylococcus epidermidis cells, which prolifically synthesize biofilm. For E. coli, the Raman spectra of the planktonic and biofilm samples are similar with the exception that the spectral signature of RNA, present in planktonic cells, could not be detected in biofilm. For S. epidermidis, major changes occur upon biofilm formation. In addition to the absence of the RNA features, new bands occur near 950 cm-1 and between 1350 and 1420 cm-1 that are associated with an increase in carbohydrate content. Unlike the case in E. coli biofilm, the intensity of G base ring modes is reduced in but A and T base ring signatures become more prominent. For S. epidermis in the biofilm's amide III region, there is evidence of an increase in the level of β-sheet structure accompanied by a decrease in α-helical content. The presence of biofilm is confirmed by microscope-aided photography and, separately, by staining with methyl violet.