The continuous culture systems chemostat and turbidostat could also be used to culture the biofilms. The type of environment they are grown in, effects their ultimate morphological, clustering and adhesion character.
For Visualization, traditionally electron microscopy was the method of choice to examine microbial biofilms under high resolution. Unfortunately, sample preparation for electron microscopy results in dehydrated samples. Consequently, this approach provided a deceivingly simplistic view of biofilms, since the biofilm collapsed when water was removed. (Davey and Ootle, 2000) Phase microscopy was also an alternative to visualization of living biofilms. Biofilms are 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 electron microscopy (ESEM), episcopic differential interference contrast microscopy (DIC) with and without fluorescence, Hoffman modulation contrast microscopy (HMC), atomic force microscopy (AFM). ...
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. (Surman et al, 1996) Viability of the microbial biofilm populations can also be determined by LIVE/DEAD BacLight bacterial viability staining kit (Hentzer 2001)
The application of confocal scanning laser microscopes (CSLM) to biofilm research radically altered our perception of biofilm structure and function (Laurence 1991). Before the use of CSLM, electron microscopy was the method of choice to examine microbial biofilms under high resolution. Unfortunately, sample preparation for electron microscopy results in dehydrated
samples. Consequently, this approach provided a deceivingly simplistic view of biofilms, since the biofilm collapsed when water was removed. On the other hand, CSLM, which allows the visualization of fully hydrated samples, has revealed the elaborate three-dimensional structure of biofilms (Costerton 1995). CSLM is used very effectively to monitor Microbial Biofilms. It is used to investigate not only the presence and the viability of the biofilm consortium but also biofilm/substrata interactions. CSLM has been used very effectively to monitor biofilm development in flow cells. Flow cells are small continuous-flow systems with a viewing port that allows direct observation of the biofilm without disrupting the community. These systems are often once-flow, meaning that fresh medium