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Page 4; Vol. 2 No. 1
A major reason for the Sabbatical at Cardiff University was to learn new nonculture techniques. In addition, collaboration with other colleagues in Cardiff, Manchester, and New Castle expanded opportunities to learn additional techniques. These have expanded our means of analyzing Biofilms including imaging techniques using 3-D Confocal with animation and SEM.
List of new Tests:
1. PCR/DGGE using selected primers able to amplify and segregate 16 RNA from a diverse microbial-rich population. This is appropriate for Biofilms, where 50% of the isolates are probably nonculturable, and a significant majority of the biofilm isolates may be anaerobes amplifying the difficulty in traditional recovery. As we move away from the “1-bug/1-disease” theme of Robert Koch, protocols are necessary that distinguish multispecies and microbial diversity.
2. 3-D imaging using Confocal Laser Scanning Microscopy (CLSM), the structure of Biofilms amplifies their uniqueness and defines much of their pathogenicity, “Structure Equals Function.” 3-D glasses, software and Poloxamer provide a detailed analysis of Biofilm growth. This particularly highlights the various staging (I-IV) that defines the life cycle of a Biofilm and outlines potential targets recognizing Stage I and Stage IV are most likely for anti-infective Biofilm therapy.
3. Co-aggregation and Synergy. Utilizing crystal violet analysis of biofilms grown in 96 pegs and resultant Optical Density, we measured the impact of environment (pH 5.5 vs. 7.0 and time) and strains on co-aggregation and synergy.
4. SEM/EA (Scanning Electron Microscopy combined with Elemental Analysis). In defining the structure of Biofilms, it is also important to correlate the source and elements that may be part of that environment. Hence, while in Cardiff and later at WVU, School of Engineering and NIOSH, utilized the JOEL microscopes with gold shadowing and x-ray microanalysis which allowed for detection of elements within the undisturbed structure of the Biofilm.
5. Poloxamer F-127, a reverse gel. With Dr. Paul Robinson at Purdue University, we became aware of the unique characteristics of reverse gels, which due to its structure enhanced and triggered Biofilm formation from organisms initially grown planktonically. Subsequently, with Dr. Steven Percival in Manchester, England, we expanded our use of poloxamer, employing F-127 to standardize potential of biofilm susceptibility testing, amplifying the reverse gel/Biofilm inducement.