Eub338

16S rRNA

Most Bacteria

Amann et al., 1990

UNIV 1389b

18S rRNA

Most Eucarya

Zheng et al., 1996

UNIV 1389c

16S rRNA

Archaea

Zheng et al., 1996

BET42a

23S rRNA

,3-Proteobacteria

Manz et al., 1992

Beta-AO233

16S rRNA

Ammonium oxidizers

Stephen et al., 1998

Some probes are "universal" in that they target all known organisms in the Domain Bacteria or the Domain Archaea. Other probes are used to target different divisions within the Domain Bacteria or genes with specific functions that are unique to a specific class of bacteria.

Some probes are "universal" in that they target all known organisms in the Domain Bacteria or the Domain Archaea. Other probes are used to target different divisions within the Domain Bacteria or genes with specific functions that are unique to a specific class of bacteria.

The FISH technique employs an oligonucleotide probe conjugated with a fluorescent molecule (or fluorochrome). The probe is designed to bind to complementary sequences in the rRNA of the 16S subunit of the ribosomes within bacterial cells. Because metabolically active cells contain a large number of ribosomes, the concentration of fluorescently labeled probe is relatively high inside the cells, causing them to fluoresce under UV light. The final result is high binding specificity and typically low background fluorescence. For simultaneous counting of subpopulations in a given sample, probes can be designed that bind to specific sequences of rRNA that are found only in a particular group of organisms (i.e., Archaea, Bacteria, or subdivisions of the Bacteria; see Table 4.1) and used in conjunction with one another. FISH can also be combined with microautoradiography to determine specific substrate uptake profiles for individual cells within complex microbial communities (STARFISH, substrate-tracking autoradiographic fluorescence in situ hybridization; Ouverney and Fuhrman, 1999; Lee et al., 1999). Because these methods label only metabolically active cells, the samples can be labeled simultaneously with dyes that bind to nucleic acids, such as 4',6-diamidino-2-phenylindole (DAPI) or acridine orange, to facilitate a total cell count using fluorescence microscopy (Li et al., 2004). FISH is useful especially when used in conjunction with confocal laser scanning microscopy (CLSM; see below) as it allows three-dimensional visualization of the relative positions of diverse populations, even within complex communities such as biofilms and the surfaces of soil aggregates (Binnerup et al., 2001). The key advantage of FISH is the ability to visualize and identify organisms on a microscale in their natural environment. Such techniques have enormous potential for studying micro-bial interactions with plants and the ecology of target microbial populations in soil; however, the binding of fluorescent dyes to organic matter resulting in nonspecific fluorescence is a common problem in soils with high organic matter contents, such as peats, or other particles with high surface charge, such as black carbon. Image analysis software is readily available and may be "trained" to detect only those aspects of an image that meet specified criteria.

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