Agriculture Reference
In-Depth Information
7.5.2 Fluorescence Microscopy
The number of bacteria in soil, their cell volumes, and the frequencies of dividing
cells can be determined by fluorescence microscopy and computerized image
analysis (Bloem et al.
1995
). Soil microbial biomass can be estimated by staining
with fluorescent dyes such as fluorescein isothiocyanate.
7.5.3 Fluorescence In Situ Hybridization
Fluorescence in situ hybridization (FISH) is a direct, cultivation-independent tech-
nique using rRNA-targeted oligonucleotide probes that is frequently used for the
identification of microorganisms in soils. While this technique allows selective
visualization of bacterial cells of different phylogenetic groups, it also has some
limitations, particularly regarding quantitative analysis of complex samples (Moter
and G
ยจ
bel
2000
; Peix et al.
2007
).
7.5.4 Stable Isotope Probing
Stable isotope probing (SIP) is a culture-independent technique that allows the
identification of microorganisms directly involved in specific metabolic processes.
In this method, labeled nucleic acids synthesized during assimilation of an isotopi-
cally enriched substrate are isolated and analyzed (Radajewski et al.
2002
). The
technique has been used to study forest soils. Genetic diversity is most commonly
studied by analyzing the diversity of genes encoding 16S rRNA (18S rRNA for
eukaryotes). These genes occur in all microorganisms and show species-dependent
variations in their base compositions. Three methods are commonly applied to
examine the diversity of 16S (and 18S) rDNA sequences in total DNA extracted
from soil microbial communities: denaturing gradient gel electrophoresis (DGGE),
temperature gradient gel electrophoresis (TGGE), and terminal restriction fragment
length polymorphisms (T-RFLP).
7.5.5 RNA Measurement
The composition of soil microbial communities can be estimated by reverse
transcriptase polymerase chain reaction (RT-PCR) followed by gel electrophoresis
of the amplified cDNA fragments (Duineveld et al
2001
). The analysis of specific
mRNAs reflects the expression of the corresponding gene in soil. Such measure-
ments can also be done by quantitative real-time RT-PCR, which allows the
