Targets And Probes

The in situ analyses of hydrated biofilms may be carried out using a variety of probes targeted generally at polysaccharides, proteins, lipids, or nucleic acids. In addition, other probes such as dextrans, ficols, and polystyrene beads may be used to assess general properties such as charge, hydrophobicity, permeability, or the determination of diffusion coefficients. Probes are most frequently conjugated to fluors although colloidal reflective conjugates (gold, silver) may be used.27 Recently, quantum dots (QDs) have shown great promise as multiwavelength fluorescent labels. Colloidal QDs are semiconductor nanocrystals whose photoluminescence emission wavelength is proportional to the size of the crystal. Kloepfer et al.34 reported that cell surface molecules, such as glycoproteins, made excellent targets for QDs conjugated to wheat germ agglutinin. This new class of fluorescent labels may open opportunities for in situ detection of matrix chemistry. As indicated above, the option exists for probe independent examination of major biopolymers and other constituents in hydrated biofilm and floc material providing a basis for detailed examination of these structures and ground truthing of the fluorescent and reflection based probe dependent approaches.

6.3.1 Polysaccharides 6.3.1.1 General Probes

A range of stains with specificity for beta-D-glucan polysaccharides are used as general stains, these include calcofluor white and congo red. Ruthenium red has also been used as a light microscopy stain for detection of EPS. Probes for glycoaminoglycan such as Alcian blue may also be used as a general stain for "polysaccharides." Wetzel et al.35 demonstrated its use for determination of total EPS in microbial biofilms, in this case it was used indirectly and not for microscopy. Due to the complexity of the EPS the likelihood of finding a true total polysaccharide probe appears to be very limited.

6.3.1.2 Lectins

Lectin-like proteins have a long history of application in the biological sciences.36 Currently, lectins are regarded as proteins with a lectin-carbohydrate and a lectin-protein binding site and are characterized on the basis of their interactions with specific monosaccharides. Lectins are produced by many organisms including plants, vertebrates, protists, slime molds, and bacteria where they function as cell/surface-recognition molecules.37 Recognition of the specific site is controlled by stereochemistry, however, the carbohydrates also interact with lectins via hydrogen bonds, metal coordination, van der Waals, and hydrophobic interactions.38 (See also review articles and comprehensive books on lectins.39-42)

The difficulty of isolating a single polymer type from a complex biofilm matrix may be comparable to the situation at the cellular level.43 Neu et al.16 noted that if one considers the potential of carbohydrates to encode information in terms of sac-charides it is even larger than that of amino acids and nucleotides. The latter two compounds can only build 1 dimer whereas one type of monosaccharide can form 11 different disaccharides. Further, 4 monosaccharides, a common number in the repeating unit of polysaccharides, may form 35,560 different disaccharides.37 If each of the estimated number of bacterial species (4,800,000) secretes one protein and one polysaccharide this would be 9,600,000 EPS compounds; a very conservative estimate.44 As a consequence, there is a need to establish an in situ technique for the assessment of glycoconjugate distribution in floc systems. At present the most promising approach to achieve this is the application of fluorescent-lectin-binding-analysis (FLBA) in combination with CLSM. Labeled lectins have been successfully used in many microbial pure culture studies to probe for cell surface structures.45-48 Fluor conjugated lectins have also been used fairly extensively in complex environments including, marine habitats49 and freshwater systems.1,15,16,18,30,50,51

As noted by Neu and Lawrence9 lectins may represent a useful probe for in situ techniques to three-dimensionally examine the distribution of glycoconjugates in fully hydrated microbial systems. The many lectins available, offer a huge and diverse group of carbohydrate specific binding molecules waiting to be employed for an in situ approach.52 The above listed studies all suggest that lectins may be applied successfully to extract information regarding the nature of the EPS. Fluor-conjugated lectins effectively reveal the form, distribution, and arrangement of EPS in three dimensions. Figure 6.4 illustrates this phenomenon showing the distribution of EPS using Solanum tuberosum, Cicer arietinum, and Tetragonolobus purpureas lectins and confocal laser microscopy to examine a microcolony in a river biofilm, note the multiple layers of EPS identified by each lectin and their spatial distribution. Figure 6.5 illustrates the distribution of binding sites for lectins within a river floc from the Elbe River. As also shown in Figure 6.5, FLBA has been combined with fluorescent in situ hybridization (FISH; see review by Amann et al.)53 to allow localization and identification of bacteria associated with the binding of specific lectins.17 This visualization is extremely useful as a starting point for additional questions regarding the EPS. However, the major goals of quantification and chemical identification remain more elusive. Neu et al.16 evaluated lectin binding in complex habitats in detail. They showed that it was possible, through digital

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