Advances in Biofilm Experimental Techniques

All biofilm experimental systems should contain certain critical components: (1) the biofilm study reactor itself (where the surface accumulation of cells and biofilm will be studied) and (2) the environmental support system that controls the experimental conditions (e.g., temperature, flow velocity, nutrient concentrations, pH) of the bulk fluid. Under quiescent conditions, the rate of transport of dissolved nutrients and particulates (i.e., the bacteria) to the substratum may be the slowest in a series of rate processes. For example, particle transport may limit the rate of cell deposition in quiescent conditions. Thus, it is recommended that only experimental systems that create a gradient-less, well-mixed bulk fluid phase be employed. Otherwise, instead of determining the true kinetics of whatever biofilm process is of interest, the resultant data will erroneously reflect mass transfer limitations.

Invasive Biofilm Diagnosis. Detection of biofilms often requires direct sampling and removal of a finite quantity of biofilm from a reactor surface to be used in a number of destructive analytical procedures that may measure

(1) overall biofilm amount (cell mass or biofilm thickness),

(2) a component of the biofilm (biofilm carbon, cell number, biofilm nitrogen content), or (3) a biofilm cell component or cellular activity (ATP, cell protein, dehydrogenase activity, active DNA synthesis, species-specific 16S rRNA). Reactor designs that provide access for destructive sampling involve removable sections of substratum and are quite numerous. The reader is directed to a number of excellent reviews for further details (5,7). One drawback to destructive analyses is that the parameter determined represents an average over the entire sample area, which ignores any spatial variations in that parameter that may exist.

Almost any parameter determined by a molecular or chemical assay (ATP, DNA, total protein, biofilm polysaccharide content, 16S rRNA, cellular phospholipid signatures, specific enzyme levels, and oligonucleotide probes), cytochemical (acridine orange, DAPI stain, and Hoechst DNA stains) or immunofluorescent staining, or cellular activity (CTC stains, INT stains, and plate counting) techniques that has been applied to planktonic (freely suspended) cells can be used to quantify changes in biofilm on a substratum. Rodriguez et al. (64) reports on the potential of a fluorescent redox probe, 5-cyano-2,3-ditolyltetrazo-lium chloride (CTC), for direct observation of actively respiring bacteria. Oxidized CTC is colorless, but upon reduction by electron transport activity, the insoluble CTC-formazan fluoresces at 365 nm upon excitation. Respiring marine bacteria within thin (10-50 pm) biofilms attached to optically nontransparent polysulfone substrata were rapidly and easily enumerated. Respiring cell counts via CTC were also useful in directly assessing the efficacy of various biocidal control agents.

The advent of modern molecular biology has provided many useful diagnostic methods with which to analyze biofilm ecology. For example, Amann et al. (65) characterize the population structure of sulfidogenic biofilms, established in anaerobic bioreactors, by selective polymerase chain reaction (PCR) amplification and fluorescent microscopy. 16S rRNA common to the sulfate-reducing bacteria in the biofilm was selectively amplified by PCR and used to design both general and specific fluorescent hybridization probes. Biofilms, (5-10 pm thick) on glass cover slips immersed within laboratory anaerobic biofilm reactors, were fixed in formaldehyde and dehydrated prior to hybridization with the RNA probes. Hybridized biofilm samples were then viewed under epifluorescent microscopy.

Rogers and Keevil (66) report on the destructive sampling of a multispecies biofilm, intentionally inoculated with Legionella pneumophilia, followed by both immunogold and fluorescein immunolabeling for this species. Epi-scopic differential interference contrast microscopy was employed to simultaneously visualize the total biofilm community and the labeled Legionella species. This techniques was able to provide observations of variations in the biofilm community from one position to another (parallel to the substratum) but was unable to discern such variations with depth (perpendicular to the substratum).

McCarter et al. (67) report a very elegant series of genetic techniques to investigate bacterial responses to various stimuli, one being the adhesion to a surface. Using a transposon (mini-Mulux), the authors were able to incorporate recombinant reporter gene insertions adjacent to target gene promoter sites that encode for light production as a function of target gene expression. Light production is conveniently measured by exposure of X-ray or photographic film, visual examination, chemiluminescence, or photometry. Such techniques also obviate the need to disrupt biofilms prior to quantification. McCarter et al. employed a lux reporter gene adjacent to the chromosomal gene encoding for swarmer cell differentiation that Vibria parahaemolyticus experiences upon association with a solid surface. Resultant mutants thus emit light upon growth at a surface but not in suspension. Dagostino et al.

(68) also employed transposon mutagenesis to insert into an appropriate recipient bacteria a marker gene that lacks its own promoter. The premise is that if a suitable target gene is "on" at the surface, then expression of the marker will be observed only in the presence of the surface. Da-gostino et al. employed Escherichia coli C600 (pRK2013: mini-MuTetr lacZ) as donor and two marine bacteria as recipients. The authors were able to successfully isolate a transposon-generated mutant in which the lacZ gene was not expressed in either liquid or agar, but was expressed when grown on a polystyrene substratum.

Noninvasive Biofilm Diagnosis. The "holy grail" of biofilm research is a diagnostic method that creates as little disturbance to the biofilm and the reactor operation as possible. Regrettably, most parameters of interest in cell adhesion and biofilm accumulation require destructive sampling and analyses.

True noninvasive diagnostics of biofilm accumulation, amount, and biofilm-cell reactivity are limited to relatively few sophisticated techniques. The simplest of these techniques is on-line microscopy, which places the biofilm surface within the viewing field of a microscope. This technique requires a reactor dimension compatible with the microscope, and a flat reactor surface on which to focus. Microscopic observation of adherent cells can be quantified and preserved by use of video recorders or image analyzer systems that digitize observed images and save those images on a computer for future numerical interpretations.

For about 14 years, confocal scanning laser microscopy (CSLM) has been a tool in the biological sciences. CSLM uses confocal apertures, or pinholes, to create a thin (0.4 im) depth of field, which eliminates out-of-focus light emitted from the laser-excited fluorophores. Laser light sources provide the intense, highly coherent, collimated light necessary to penetrate thick specimens. The laser light is used to excite fluorophores—either those intrinsic (e.g., chlorophyll) to the sample, or selected chemical or immunological stains intentionally applied to the sample. Resultant fluorescence that passes through the aperture is detected by a photomultiplier, and a digital image is collected. Using a computer-manipulated stage, optical sections can be collected from a specimen in three dimensions. This allows, for the first time, the ability to digitally locate cells on a substratum or within a biofilm, noninvasively (69). Numerous fluorochromes are available that can be used to localize and measure intracellular and extracellular conditions in three dimensions within living biofilms.

Camper et al. (70) detail image analysis software that can be used to collect images of bacterial adhesion patterns from the CSLM, digitize those images, then superimpose those images onto digitized images of the substratum itself, collected from any one of a number of other analytical instruments (e.g., X-ray photoelectron spectroscopy, atomic force microscopy, time-of-flight secondary ion mass spectroscopy); all images are collected at the same location on the substratum. Thus, one can now correlate the adhesion of a single cell to the local topographical or chemical properties of the substratum directly underneath the cell.

Lawrence et al. (71) illustrates the ability to optically section fully hydrated biofilms, both horizontally and sag-

itally, producing optical sections of undisturbed biofilms with a spatial thickness of 2 im. Any fluorescent probe (cellular DNA stain; immunofluorescent stain; pH-, ion-, and redox-sensitive stains; viability redox stains) or any combination of probes can be simultaneously recorded by the CLSM with great clarity and little background interference, due to the confocal exclusion of any fluorescence originating from excited fluorochrome above and below the focal point (Fig. 6). CLSM is a critical tool in population analysis of biofilms consisting of mixed strains, mixed species, and mixed cell lines and will allow detailed examination of the relationships between biofilm structure, adaptation, reactivity, and response to external stress (72).

Another noninvasive technique that can be used to collect information on the molecular chemistry of cell adhesion at the substratum-fluid interface is Fourier transform infrared spectroscopy (FT-IR) coupled with attenuated total reflectance (ATR) waveguides integrated within flow cells. Atoms and groups of atoms within a molecule vibrate with a characteristic frequency and will adsorb light at those frequencies. Light that contains infrared frequencies can be focused on a molecule, and the amount of light adsorbed is then measured as a function of frequency. Specific IR adsorptions can thus be assigned to particular bonds, and alterations in these bonds due to changes in local environment can be assessed from the resultant spectral details. Recent increases in the capability of FT-IRs and the focusing of the IR wave within specific crystal wave guides (germanium or zinc selenide crystals) allows one to establish a standing IR evanescent wave at the surface of the crystal. Fourier transform signal processing and multiple scanning allow aqueous samples to be processed where the IR spectra of molecules directly adjacent to the waveguide can be collected. In biological systems, the IR absorbance of water must be subtracted via computer manipulations to provide the IR spectra of molecules accumulating at the crystal surface. The effective depth of penetration for the evanescent IR wave is a function of crystal material and the wavenumber but ranges around 0.3-0.7 im, thus providing spectra that reflect those chemical species directly adjacent to the surface.

Figure 6. Confocal laser microscope optical section of a biofilm.

ATR/FT-IR has been used to study the effects of protein-conditioning films on alginate adsorption to the germanium surface (73). Alginate adsorbed in greater quantities to protein-coated ATR crystals than to uncoated ones. For the development of biofilms on ATR crystals, the observed spectrum is the integral of all biomolecules on the surface and over the entire surface. Bremer and Geesey (74) report the chemical changes as detected by FT-IR that occur upon inoculating with a microbial culture, at a germanium ATR crystal situated within a flow cell. Spectral intensities representing various bonds within proteins (amide I at 1,645/ cm; amide II at 1,550/cm) and polysaccharide (C-O stretches at 1,058/cm) are seen to accumulate with time, indicating the feasibility of the ATR/FT-IR system to detect biofilm formation. Because the IR wave can only penetrate less than 1 im into the biofilm, reported increases in ab-sorbance intensities most likely represent increases in cell surface coverage of the crystal and not increases in biofilm thickness. A plot of attached cells per area versus the increases in area under the amide I peak was generated to estimate a lower limit of detection by FT-IR of ~5 X 105 cells/cm2. In a medical application, ATR/FT-IR has been used to follow the penetration of an antibiotic (100 ig/mL ciprofloxacin) into the IR wave region underneath a P aeruginosa biofilm (75).

Several reports exist of the use of invasive but nondestructive analysis of solute concentration profiles, by microsensor chemical probes, as a function of spatial dimension within the developing biofilm. Microsensors (tip size < 15 im) exist that can detect various dissolved solutes including glucose, oxygen, pH, sulfide, and ammonia. DeBeer et al. (76) report using microoxygen sensors (tip size = 15 im) to estimate oxygen profiles, local oxygen uptake rates, and oxygen diffusion coefficients in a mixed-culture biofilm community cultivated within a laboratory biofilm reactor. Schramm et al. (77) determined microprofiles of oxygen and nitrate in nitrifying biofilms from a trickling filter and correlated these solute profiles with bacterial population profiles for Nitrosomonas and Nitro-bacter spp., as determined using fluorescence in situ hybridization of cells fixed with 16S rRNA oligonucleotide probes.

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