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Fluorescence microscopy for visualization of soil microorganisms -- a 
review

Biology and Fertility of Soils
Issue: Volume 39, Number 5
Date:  April 2004
Pages: 301 - 311
http://dx.doi.org/10.1007/s00374-004-0722-x


Ying Li (1), Warren A Dick (1, 2) and Olli H. Tuovinen (1, 3)

(1) Environmental Science Graduate Program, Ohio State Univ
(2) School of Natural Resources, Ohio State Univ
(3) Department of Microbiology, Ohio State Univ

Abstract


Direct microscopic observation of microorganisms is
an important tool in many microbial studies. Such observations
have been reported for Protozoa, fungi, inoculated bacteria, and
rhizosphere microorganisms but few studies have focused on
indigenous bacteria and their spatial relationship within various
microhabitats. Principles and applications of epifluorescence
microscopy and confocal laser scanning microscopy for visualization
of soil microorganisms in situ are reviewed. Both cationic and
anionic dyes (also commonly referred to as fluorochromes if they
are fluorescent) have been used based on their ability to bind to
specific cellular components of microbial cells. Common fluorochromes
used for imaging of microbial cells include acridine orange, ethidium
bromide, fluorescein isothiocyanate, 5-(4,6-dichlorotriazinyl)
aminofluorescein, 4prime,6-diamidino-2-phenylindole, europium chelate,
magnesium salt of 8-anilino-1-naphthalene sulfonic acid, and calcofluor
white M2R.

Combining fluorescence staining techniques with soil thin section
technology allows one to obtain images of microorganisms in situ.
Soil texture and the procedures used for resin embedding are important
factors affecting the quality of stained soil thin sections. Indeed, 
general
limitations of applying fluorescence microscopy to soil ecological studies
are the non-specific binding of dyes to the soil matrix and the
autofluorescence of some soil components. The development of
fluorescent in situ hybridization and confocal laser scanning microscopy
techniques provides new potential for microbial distribution studies.

Keywords:  Confocal laser scanning microscopy - Fluorescence microscopy
- Fluorochromes - In situ hybridization - Microbial stains


Introduction

Soil is a heterogeneous system where the behavior of
microorganisms is affected by their immediate microenvironment.
Soil aggregates are considered to be the basic
microhabitats of soil microorganisms (Hattori 1988).
Therefore, the microbial distribution is closely related to
the structure and composition of soil aggregates. There
are two approaches used to study microbial distribution in
soil aggregates: (1) fractionation based on location of
microorganisms in the outer or inner layers of soil
aggregates, and (2) direct observation of soil microorganisms
in situ by means of microscopic techniques.

In the fractionation approach, microbial analyses (e.g.,
isolation, enumeration, or DNA extraction) are conducted
on water-washed fractions (from outer to inner layers) of
soil aggregates (Hattori 1988). Traditionally, after microorganisms
are extracted from the soil, they are cultured
using different media for enrichment or for isolation.
Isolation is needed for phenotypic testing, whereas DNA
extraction and PCR amplification of target sequences are
used for the characterization of 16S rRNA-based phylogeny.
Molecular techniques of soil microbial ecology
bypass the need to culture microorganisms, but the
efficiency of DNA extraction from soil is believed to be
widely variable (Lloyd-Jones and Hunter 2001; Miller et
al. 1999; Tien et al. 1999). The elution of soil microorganisms
is never complete due to the rapid and strong
sorption of some microbial cells to clay particles and
other mineral surfaces and trapping within micropore
spaces. Not knowing what culture conditions to use also
causes many microorganisms to be undetected. As a
result, methods based on culture recoveries underestimate
the actual microbial population by at least 1 or 2 orders of
magnitude (Hartmann et al. 1997). Exact estimates cannot
be given because the recovery is likely to vary from one
soil system to another.

Direct microscopic observations of soil microorganisms
have been performed with fluorescence microscopy,
transmission electron microscopy, and scanning electron
microscopy. Transmission electron microscopy and scanning
electron microscopy can provide high resolution
images and are, therefore, useful in ultrastructural studies
of soil particles and microorganisms (Foster 1988). For
situations where micron-level resolution is adequate,
fluorescence microscopy is often used. Examples include
the enumeration of microorganisms or the determination
of the spatial distribution of soil microorganisms in their
microhabitats (Tippktter et al. 1986; Altemller and van
Vliet-Lanoe 1990; Postma and Altemller 1990; Pickup
1995; DeLeo et al. 1997; Fisk et al. 1999). Different
fluorescent dyes have also been used, in combination, to
differentiate metabolically active cells from inactive cells
in soil microbial populations (Bhupathiraju et al. 1999a,
1999b).

Confocal laser scanning microscopy provides a new
potential for soil microbial distribution studies. Confocal
laser scanning microscopy has gained popularity since the
1990s and was originally used primarily in the biomedical
and life sciences. With its many advantages, this technique
has attracted the attention of microbial ecology
researchers studying biofilms, compost processes, and
biofouling (Lawrence et al. 1991; Caldwell et al. 1992a;
Surman et al. 1996; Swope and Flickinger 1996;
Chalmers et al. 1997).

The purpose of this review is to summarize the
principles and applications of fluorescence microscopy as
applied to soil microbial studies. The emphasis of this
review is on stains and techniques for embedding soil thin
sections for imaging using fluorescence microscopy.

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