Confocal Microscopy

CLSM, combined with in situ hybridization techniques, has been applied with considerable success to visualize the structure of soil microbial communities. The basic principle of CLSM is to first create an image that is composed only of emitted fluorescence signals from a single plane of focus. This is done using a pinhole aperture, which eliminates any signal that may be coming from portions of the field that are out of focus. A series of these optical sections is scanned at specific depths and then each section is "stacked" using imaging software, giving rise to either a two-dimensional image that includes all planes of focus in the specimen or a computer-generated three-dimensional image. This gives us unprecedented resolution in viewing environmental specimens, allowing for better differentiation of organisms from particulate matter as well as giving us an insight into the three-dimensional spatial relationships of microbial communities within their environment (Fig. 4.4).

FIGURE 4.4 Confocal laser scanning microscopy image of a nematode feeding on a bacterial biofilm (with permission from W. C. Ghiorse, Cornell University).

biosensors and marker gene technologies

Introduced marker genes, such as luxAB (luminescence), lacZ (,5-galactosidase), and xylE (catechol 2, 3-dioxygenase) are now being used more frequently in soil microbial ecology studies (Table 4.2). One such gene that has attracted a lot of attention in rhizosphere studies is gfp, which encodes the green fluorescent protein (GFP). Green fluorescent protein is a unique bioluminescent genetic marker, which can be used to identify, track, and count specific organisms into which the gene has been cloned that have been reintroduced into the environment (Chalfie et al., 1994). The gfp gene was discovered in and is derived from the bioluminescent jellyfish Aequorea victoria (Prasher et al., 1992). Once cloned into the organism of interest, GFP methods require no exogenous substrates, complex media, or expensive equipment to monitor and, hence, are favored over many fluorescence methods for environmental applications (Errampalli et al., 1999). GFP-marked cells can be identified using a standard fluorescence microscope fitted with excitation and emission filters of the appropriate wavelengths. One reason for such keen interest in GFP is that there is no background GFP activity in plants or the bacteria and fungi that interact with them, thereby making gfp an excellent target gene that can be introduced into selected bacterial or fungal strains and used to study plant-microbe interactions (Errampalli et al., 1999). Basically, gfp is transformed into either the chromosome or a plasmid in a bacterial strain, where it is subsequently replicated. Various gene constructs have been made, which differ in the type of promoters or terminators used, and some contain repressor genes such as lacI for control of gfp expression. Once key populations in a sample are known and isolates obtained, they can be subsequently marked with gfp or other genes producing detectable products in order to track them and assess their functions and interactions in soil and the rhizosphere. In addition to GFP, red-shifted and yellow-shifted variants have been

TABLE 4.2 Major marker methods used for bacterial detection (modified from Errampalli et at., 1999).

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