Comparison of microscope techniques for the examination of biofilms

Biofilms developed on solid supports suspended within a continuous culture model water system. The system inoculum was composed of a diverse consortium of microorganisms obtained from a naturally occurring aquatic source. Biofilms were removed and either directly visualised or fixed and stained prior to examination. Several techniques for microscopy examination of biofilms in situ on the substrata supporting their growth have been used in this study. These have included transmission electron microscopy (TEM), scanning electron microscopy (SEM), environmental scanning electronmicroscopy (ESEM), episcopic differential interference contrast microscopy (DIC) with and without fluorescence, Hoffman modulation contrast microscopy (HMC), atomic force microscopy (AFM) and scanning confocal laser microscopy (SCLM). A comparison of the advantages of the individual techniques to visualise biofilms is discussed. Cross-sections prepared for TEM analysis gave useful information about the spatial relationships of microorganisms within the biofilm matrix, whilst SEM enabled the surface topology of the biofilms to be examined at a high magnification. The preparation required for TEM and SEM, may however, result in the inclusion of artefacts. ESEM and AFM allow direct visualisation of intact hydrated specimens at high magnification. AFM images may be rotated and manipulated to provide accurate measurement of individual microorganisms with relative ease. SCLM was used to investigate not only the presence and the viability of the biofilm consortium but also biofilm/substrata interactions. Light microscopy techniques, although unable to reproduce the high magnification of the methods described above, are still of importance in the examination of intact biofilms. HMC allows the in situ examination of biofilms, a clear image is produced without artefacts. DIC may be used to examine biofilms on opaque surfaces and if used in conjunction with fluorescent vital stains can be used to assess the viability of the microbial population.