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Applied and Environmental Microbiology, April 1999, p. 1753-1761, Vol. 65, No. 4
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Counting and Size Classification of Active Soil
Bacteria by Fluorescence In Situ Hybridization with an rRNA
Oligonucleotide Probe
Henrik
Christensen,1,*
Michael
Hansen,2 and
Jan
Sørensen2
Department of Veterinary Microbiology, Royal
Veterinary and Agricultural University, 1870 Frederiksberg
C,1 and Section of Genetics and
Microbiology, Department of Ecology, Royal Veterinary and
Agricultural University, 1871 Frederiksberg C,2
Denmark
Received 26 October 1998/Accepted 4 January 1999
 |
ABSTRACT |
A fluorescence in situ hybridization (FISH) technique based on
binding of a rhodamine-labelled oligonucleotide probe to 16S rRNA was
used to estimate the numbers of ribosome-rich bacteria in soil samples.
Such bacteria, which have high cellular rRNA contents, were assumed to
be active (and growing) in the soil. Hybridization to an rRNA probe,
EUB338, for the domain Bacteria was performed with a soil
slurry, and this was followed by collection of the bacteria by membrane
filtration (pore size, 0.2 µm). A nonsense probe, NONEUB338 (which
has a nucleotide sequence complementary to the nucleotide sequence of
probe EUB338), was used as a control for nonspecific staining. Counting
and size classification into groups of small, medium, and large
bacteria were performed by fluorescence microscopy. To compensate for a
difference in the relative staining intensities of the probes and for
binding by the rhodamine part of the probe, control experiments in
which excess unlabelled probe was added were performed. This resulted in lower counts with EUB338 but not with NONEUB338, indicating that
nonspecific staining was due to binding of rhodamine to the bacteria. A
value of 4.8 × 108 active bacteria per g of dry soil
was obtained for bulk soil incubated for 2 days with 0.3% glucose. In
comparison, a value of 3.8 × 108 active bacteria per
g of dry soil was obtained for soil which had been air dried and
subsequently rewetted. In both soils, the majority (68 to 77%) of
actively growing bacteria were members of the smallest size class (cell
width, 0.25 to 0.5 µm), but the active (and growing) bacteria still
represented only approximately 5% of the total bacterial population
determined by DAPI (4',6-diamidino-2-phenylindole) staining. The FISH
technique in which slurry hybridization is used holds great promise for
use with phylogenetic probes and for automatic counting of soil bacteria.
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INTRODUCTION |
For a long time plate counting has
been inadequate for estimating the active populations of bacteria in
soils (8, 40), and numerous methods have been tested as
alternatives. Recently, the frequency of dividing cells (9)
and counts of
2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium chloride-reducing cells (35), 5-cyano-2,3-ditolyl tetrazolium chloride
(CTC)-reducing cells (48), and
[3H]thymidine- or
[14C]leucine-incorporating cells (3, 15) have
been used to determine active bacterial populations. The limitations of
these assays include inaccurate conversion factors in the
frequency-of-dividing-cell, thymidine, and leucine methods (3, 9,
13, 17), as well as difficulties in using the CTC and thymidine
methods with certain soils (15, 48).
The fluorescence in situ hybridization (FISH) method developed recently
has great potential for improving determinations of active populations
of soil bacteria (2, 14, 20, 42, 49). Oligonucleotide probes
are designed based on signature nucleotide positions in the bacterial
16S rRNA and may be used to target either a narrow group of organisms
or a broad group of organisms. High levels of rRNA (equivalent to high
ribosome numbers), which result in high detection signals with the FISH
technique, are observed in active populations of bacteria in which
protein synthesis is occurring either in nondividing or dividing cells
(12, 30, 37). In contrast, starved bacteria are known to
contain low levels of rRNA (18) but might be detected by
FISH under some circumstances (44). Similarly, 16S rRNA
levels decrease below the detection level in stressed bacteria
(44), and moribund bacteria are not expected to contain
detectable levels of rRNA.
The majority of soil bacteria are known to be less than 0.5 µm in
diameter (6, 7, 8, 17, 27), and it has been suggested that
this population of small bacteria consists mainly of gram-positive
organisms (32). These bacteria do not grow well under
laboratory conditions (7), and their growth rate, as
determined by the thymidine technique, may be lower than the growth
rate of larger bacteria (4). Still, the polulation dynamics and functional role of the small bacteria are unclear (45). With the FISH technique it may be possible to directly detect the
active bacterial populations in the soil and to determine the size
class distribution and the responses of the bacteria to different soil conditions.
FISH protocols for bacterial cells that are extracted from soil
(20) and bacterial cells that occur in smears
(49) are difficult to perform because bacteria associated
with soil particles are excluded and because the background
fluorescence signals from the soil smears are high,
respectively. To determine bacterial numbers in soil without an
extraction step, a slurry hybridization protocol followed by membrane
filtration and fluorescence microscopy was developed (14).
By this procedure, it is possible to include the important fraction of
adsorbed bacteria reported to exhibit a high level of activity
(5) and to exclude the high background signal from minute
clay and organic soil particles by collecting bacterium-sized
particles with a filter. It should be noted that the slurry
hybridization-membrane filtration method was originally developed
with laboratory-grown bacteria added to soil. Hence, when
indigenous soil bacteria are counted, some limitations due to
nonspecific staining may be encountered. The aims of this
investigation were, therefore, to determine the populations of active
bacteria belonging to different size groups in soil and to modify
the slurry hybridization protocol to compensate for nonspecific
staining in soil samples.
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MATERIALS AND METHODS |
Soil sampling and treatments.
Bulk soil samples were
collected from a sandy loam soil (63% sand, 17% silt, 17% clay,
1.1% C, pH [CaCl2] 5.5 [26]) at an experimental farm (Højbakkegård, Tåstrup, Denmark). This freshly collected soil was used during the developmental study to modify the
slurry hybridization protocol. In additional experiments, glucose was
added or a drying-rewetting cycle was included as described below
before the bacterial populations were counted. All of the soil samples
used for hybridization (5 to 10 g) were fixed in 100 ml of an
ice-cold (pH 7.0-buffered) paraformaldehyde solution (1).
After fixation overnight at 4°C, each sample was homogenized three
times for 1 min in a Waring blender operated at the maximum speed. The
cup containing blended soil was cooled on ice for 3 min between
homogenization steps. The blended sample was stored at 
20°C after
140 ml of filter-sterilized distilled water, 10 ml of 1 M Tris-HCl
buffer (pH 7.5), and 250 ml of 96% ethanol were added. The dry weight
of the soil sample was calculated on the basis of the soil moisture content.
Total counts of soil bacteria as determined by DAPI
staining.
DAPI (4',6-diamidino-2-phenylindole) staining was used
to determine the total numbers of soil bacteria. Samples corresponding to 0.1 mg (dry weight) were stained in 10 ml of filter-sterilized distilled water containing 1 µg of DAPIs per ml for 10 min (14, 36). Bacteria were collected by filtration through a
0.2-µm-pore-size black polycarbonate filter (diameter, 25 mm;
Millipore). The filters were mounted in paraffin oil, and counts were
determined with a fluorescence microscope as described below for the
filter specimens obtained from in situ slurry hybridization experiments.
In situ slurry hybridization with rhodamine-labelled
oligonucleotide probes.
To target the active soil bacteria, 16S
rRNA probe EUB338 (for the domain Bacteria) having the
nucleotide sequence 5'-TGAGGATGCCCTCCGTCG-3' (43)
was used. A different probe, NONEUB338, having the nucleotide sequence
complementary to the nucleotide sequence of EUB338, was used to
determine the nonspecific binding of a probe to the soil bacteria. The
nucleotide probes were labelled with rhodamine (tetramethyl rhodamine
isothiocyanate) (Sigma Chemical Co., St. Louis, Mo.) at a 1:1 ratio by
using an amino linker (Applied Biosystems, Foster City, Calif.), and
the rhodamine-labelled probes were purified by high-performance liquid
chromatography. The staining intensities of different batches of the
probes varied slightly because of slight differences in the ratio of
rhodamine to oligonucleotide (data not shown).
Sample volumes equivalent to 0.5 mg (dry weight) of soil were taken
directly from the blended samples while they were being stirred
magnetically. A dehydration process in which a sample was centrifuged
(10,000 × g, 5 min) and then resuspended in 80 and
100% ethanol was used (14). Soil pellets were then vacuum dried to evaporate the ethanol and resuspended in 200 µl of standard hybridization buffer (0.9 M NaCl, 20 mM Tris [pH 7.0]) containing 0.1% Nonidet P-40 detergent (Sigma). The samples were centrifuged again, and each pellet was resuspended in 200 µl of hybridization buffer containing 15% formamide and incubated at 37°C for 30 min. The effects of formamide concentrations ranging from 0 to 30% on the
cell counts were determined during the developmental study.
Then rhodamine-labelled probe EUB338 or NONEUB338 was added to each
slurry at a concentration of 12 ng µl
1. This
concentration was found to result in optimal staining, and higher probe
concentrations did not result in higher cell numbers (data not shown).
The standard protocol developed also included adding unlabelled probes
EUB338 and NONEUB338 at a concentration of 36 ng µl1
together with rhodamine-labelled probes EUB338 and NONEUB338, respectively. The effects of concentrations of unlabelled probe ranging
from 0 to 36 ng µl1 on the cells counts were determined
during the developmental study.
Hybridization took place at 37°C for 18 h by using continuous
rotation in a hybridization oven. In comparison, a shorter
hybridization
time (3 h) resulted in lower counts of indigenous
bacteria (the
counts were reduced 50% or more) (data not shown). After
hybridization
each slurry was transferred to 10 ml of hybridization
buffer containing
15% formamide and incubated at 37°C for 10 min.
The significance
of this washing procedure was determined during the
developmental
study by using wash times ranging from 2 to 60
min.
After hybridization, the soil bacteria were collected by filtration
onto 0.2-µm-pore-size black polycarbonate filters (diameter,
25 mm;
Millipore) in a filtration manifold with stainless steel
chimneys which
were prewarmed to the hybridization temperature.
The filters were
rinsed with 20 ml of filter-sterilized distilled
water at room
temperature (25°C) and stored at 4°C until fluorescence
microscopy
counting was performed (within 1 week). For longer
periods of storage,
the filters were frozen at
20°C. Prior to
microscopy, the filters
were mounted on glass slides in liquid
paraffin
oil.
Fluorescence microscopy counting and determining the size classes
of active soil bacteria.
To count the bacteria in different size
classes, a Zeiss Axioplan 2 fluorescence microscope equipped with a
Plan-Neofluar objective (magnification, ×100; numerical aperture, 1.3)
and a 100-W type HBO high-pressure mercury lamp was used. Zeiss no. 2 and 15 filters were used to visualize bacteria stained with DAPI and
rhodamine, respectively. Each bacterium was assigned to one of the
following three size classes as defined by spheres of the G12 New
Porton graticule (27, 34): small, medium, and large,
representing cell widths of 0.25 to 0.5, 0.5 to 0.7, and 0.7 to 1.0 µm, respectively. This classification was chosen since bacteria
belonging to the same species predominantly change in cell length
rather than in cell width in response to variable growth conditions
(24).
For soil samples hybridized with a rhodamine-labelled probe,
autofluorescence may sometimes give erroneous results (
14,
28). Therefore, a bacterial cell was counted only if
autofluorescence
was absent, which was ascertained by placing a Zeiss
no. 2 filter
(normally used for DAPI staining with UV light)
immediately behind
the rhodamine filter without moving the microscopic
field. Autofluorescing
cells were never detected with the Zeiss no. 15 filter when samples
were prepared for microscopy without the rhodamine
probe.
During the developmental study, at least 200 fields of 50 by 50 square
units of the G12 New Porton graticule were inspected
for each filter.
In subsequent studies of treated soils, at least
four replicates of
hybridization experiments were used, and at
least 100 cells were
counted for each sample. At low cell densities
a total of 600 microscopic fields were inspected, which corresponded
to 1% of the
sample filter. Statistical differences between counts
obtained with
different treatments were analyzed by a
d test
(
10).
The
d test was used because the variance
was not the same for
all of the data sets compared. Comparable levels
of variance are
required to perform the
t test. The
d test follows a Fisher-Behrens
distribution, as described
by Campbell (
10).
For selected samples confocal laser scanning microscopy (CLSM) was used
to confirm the distinction of active bacteria from
nonactive bacteria
or nonbacterial soil particles. The type of
confocal microscope used
(model TCS4d; Leica Laser Technik, Heidelberg,
Germany) and the
associated equipment have been described previously
by Hansen et al.
(
23). In this study, we used soil samples pretreated
with
glucose to stimulate bacterial activity. The filters were
first scanned
at random by using the optical part of the confocal
microscope, and
active bacteria were separated from nonactive
bacteria and nonbacterial
soil particles by using the following
criteria: light intensity,
morphology, and color. Particles were
confirmed to be active bacteria
by examining pictures taken by
CLSM. Each recording consisted of
a stack of images with a vertical
distance of 0.3 µm. The stack was
subsequently combined into one
image by maximum intensity projection
performed by the software
of the model TCS4d
microscope.
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RESULTS |
Optimization of hybridization conditions.
To count
indigenous, active soil bacteria, the protocol for in situ slurry
hybridization (14) had to be optimized, notably because
nonspecific staining was expected to be significant. Addition of
formamide to the hybridization buffer is known to decrease the melting
point of the DNA double helix (11), and the optimal formamide concentration for hybridization at 37°C was determined to
obtain a high-stringency protocol. Figure
1 shows the effects of different
formamide concentrations on the hybridization signal for both specific
probe EUB338 and nonspecific probe NONEUB338. The total population of
cells generally gave the highest counts in the presence of 15%
formamide for both probe EUB338 and probe NONEUB338, and this standard
concentration (15% formamide in hybridization buffer) was used for all
subsequent experiments. All three size classes of bacteria exhibited
the same pattern as the total bacterial population (data not shown).
Hence, significantly higher numbers were obtained for the small size
class of bacteria with the EUB338 probe when 15% formamide was used
than when other levels of formamide were used, and for the medium size
class significantly higher numbers were obtained in the presence of 5, 10, 15, and 20% formamide than in the presence of 0 and 30%
formamide. When the NONEUB338 probe was used, the numbers obtained in
the presence of 15% formamide were significantly higher than the
numbers obtained in the presence of 0 and 10% formamide for the small
size class and significantly higher than the numbers obtained in the
absence of formamide for the medium size class.
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FIG. 1.
Total numbers of cells (sums of values for all size
groups) targeted with specific probe EUB338 (A) or nonspecific probe
NONEUB338 (B) at different formamide concentrations. The number of
cells obtained with 15% formamide was defined as 100%. The bars
indicate standard deviations (n = 3).
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The optimal stringency wash time was determined at a fixed formamide
concentration of 15% and a hybridization temperature
of 37°C. Figure
2 shows that the highest counts generally
were
obtained after 10 min of washing. Hence, for the small size class
of bacteria, significantly higher numbers were obtained after
wash
times of 2 and 10 min than after washing for 60 min. For
the medium
size class, the numbers obtained after 2, 10, and 30
min of washing
were significantly higher than the numbers obtained
after 5 and 60 min.
Finally, for the large size class (and for
the total of all size
classes), the numbers obtained after 10
min of washing were
significantly higher than the numbers obtained
after all other wash
times. The lower values obtained after 2
and 5 min were probably due to
masking of the bacteria by excess
stain when the wash time was too
short. Hence, the final standard
protocol used for in situ slurry
hybridization of indigenous active,
soil bacteria included a formamide
concentration of 15% in the
buffer and a reaction time of 18 h at
37°C, followed by 10 min
of washing.
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FIG. 2.
Numbers of cells in size groups (0.25 to 0.5, 0.5 to
0.7, and 0.7 to 1.0 µm) targeted with specific probe EUB338 after
different stringency wash times. The number of cells obtained with a
wash time of 10 min was defined as 100%. The bars indicate standard
deviations (n = 3).
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Finally, probe penetration into gram-positive soil bacteria may be
difficult and was investigated for
Streptomyces sp. and
Frankia sp. by using lysozyme treatment (
21,
22).
To examine
this, we dehydrated soil samples with ethanol and then
incubated
them in a 1-, 2-, or 3-mg ml
1 lysozyme
solution. After this, the dehydration procedure was
repeated, and the
preparations were washed with 1% toluene in
96% ethanol. The lysozyme
treatment had no effect on the numbers
of cells targeted by the EUB338
probe (data not shown). This is
in agreement with results reported by
Roller et al. (
39) and
Zarda et al. (
49), who
found that lysozyme did not increase
the cell counts of gram-negative
bacteria. Treatment with lysozyme
was therefore omitted in the final
standard protocol for in situ
slurry
hybridization.
Correction model for nonspecific binding of probe.
We propose
a model for calculating the number of active bacteria in soil (see
below) based on the assumption that sufficient unlabelled probe (DNA)
should outcompete labelled DNA at both specific and nonspecific DNA
binding sites. Figure 3 shows that the
cell counts for all three size classes obtained with the EUB338 probe
decreased significantly when different amounts of the corresponding unlabelled probe were added. However, no effect of titration with unlabelled probe was observed for the smaller bacteria when they were
hybridized with the NONEUB338 probe, indicating that all nonspecific
staining in the small cells was due to binding of the rhodamine
component of the probe. For medium and large cells, however, the counts
obtained with the NONEUB338 probe also decreased as the concentration
of unlabelled probe increased. This shows that for larger cells,
nonspecific staining caused by DNA binding becomes more important than
rhodamine. This is probably due to the lower surface-to-volume ratio of
the larger bacteria if it is assumed that rhodamine binding is
associated with the cell wall.
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FIG. 3.
Numbers of cells in size groups (0.25 to 0.5, 0.5 to 0.7, and 0.7 to 1.0 µm) targeted with specific probe EUB338
( ) or nonspecific probe NONEUB338 ( ) in the presence of
different concentrations of the corresponding unlabelled probes. The
number of cells obtained without unlabelled probes was defined as
100%. The bars indicate standard deviations (n = 3).
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The amount of unlabelled probe EUB338 which was sufficient to titrate
out binding by DNA of labelled probe EUB338 was defined
on basis of the
bacteria in the smallest size class because these
bacteria were
numerically dominant. A constant low number of counts
was reached at 12 ng µl
1 (statistically significant), and maximal
titration was observed
with 36 ng of unlabelled probe per µl for the
small size class
(Fig.
3). For the medium and large size classes the
counts were
still decreasing and significantly lower in the presence of
36
ng of unlabelled probe µl
1 than in the presence of
0, 12, and 24 ng of unlabelled probe
µl
1; however, at
this concentration the counts were only 13 and 9%,
respectively, of
the counts obtained without unlabelled probe.
The numbers of cells in
the medium and large size classes represented
7 and 6%, respectively,
of the total counts in samples hybridized
with labelled EUB338 probe
(data not shown). Addition of 36 ng
of unlabelled probe
µl
1, therefore, reduced the counts to less than 1% of
the total counts
for the medium and large size classes, and 36 ng of
unlabelled
probe µl
1 was found to be sufficient to
titrate out binding due to the
DNA part of the
probe.
The model in Table
1 suggests that the
numbers of active bacteria determined by in situ slurry hybridization
in soil samples
can be calculated based on the following four
components: (i)
homologous binding of the DNA component of the probe to
target
rRNA; (ii) heterologous binding of the DNA component of the
probe
to target rRNA; (iii) binding of the DNA component of the probe
to nontarget rRNA; and (iv) binding of the rhodamine component
of the
probe to cell surfaces. In our protocol, the total number
of cells
determined with labelled, specific probe EUB338 (N
sp)
and labelled, nonspecific probe NONEUB338 (N
non) includes
the
numbers due to both DNA and rhodamine binding. However, similar
samples hybridized with labelled probe plus an excess amount of
the
corresponding unlabelled probe provide counts (N
sp,unl and
N
non,unl, respectively) resulting from rhodamine binding
alone,
because the unlabelled probe outcompetes the targets of the
labelled
probe. To determine the number of cells related to DNA binding
alone, the counts related to rhodamine binding are subtracted
from the
total counts to obtain N
sp N
sp,unl and
N
non N
non,unl,
respectively. Finally, the
corrected number of active bacteria
related to homologous hybridization
of the probe to the target
rRNA is obtained from (N
sp N
sp,unl)
(N
non N
non,unl), as
indicated in Table
1. While N
sp
and N
non depend on the ratio
of rhodamine to DNA in both
probe EUB338 and probe NONEUB338,
the calculated number of active cells
depends only on the labelling
of EUB338, because components from
nonspecific DNA and rhodamine
binding cancel each other out.
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TABLE 1.
Model for calculating numbers of actively growing
bacteria in soil, as determined by a slurry in situ hybridization
protocol performed with rhodamine-labelled oligonucleotide probes
targeting rRNA and controls containing excess unlabelled probe
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Detection limit.
To determine the number of cells targeted
by fluorescence-labelled oligonucleotide probes directed against
rRNA, the minimum growth rate which can be determined by
fluorescence microscopy has to be considered. We investigated the
relationship between growth rate and in situ hybridization signal in
soil samples which were amended with formalin-killed bacteria obtained
from pure cultures of Arthrobacter globiformis DSM
20124T, Pseudomonas putida MM 3 (16),
Pseudomonas fluorescens DF 17 (41), and
Corynebacterium sp. strain H 19 (13). Bacteria
could be detected easily by the EUB338 probe when the generation times were 5 h or less before the bacteria were added to the soil. In contrast, slowly growing bacteria (generation times, 1 to 2 days or
more) could not be detected by the probe (data not shown). The range of
generation times between 5 h and 1 day was not investigated.
Bacterial cells were counted with the conventional fluorescence
microscope based on staining intensity, cell shape, and color.
To test
our identification of the active bacteria, selected soil
samples were
investigated by CLSM by using the improved spatial
resolution and
narrow emission spectrum of this instrument to
give high-quality
images. Active bacteria, nonactive bacteria,
or nonbacterial soil
particles were first identified and assigned
to size classes by eye by
using conventional fluorescence microscopy.
Without moving the
microscopic field, we then recorded the images
by CLSM. As Fig.
4 shows, the particles identified as
active bacterial
cells by eye had sharper boundaries when they were
analyzed by
CLSM than the nonbacterial soil particles had, whose shapes
were
blurred. Furthermore, the light intensity was greater for the
active bacteria than for the nonactive bacteria.
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FIG. 4.
CLSM images of active bacteria (broad arrows), nonactive
bacteria (thin arrows), and nonbacterial soil particles (dotted arrows)
preselected randomly by conventional microscopy following in situ
slurry hybridization (EUB338 probe). Active and nonactive bacteria
representing different size classes (0.25 to 0.5 µm [A through D]
and 0.5 to 0.7 µm [E and F]) are shown. The size bars in panels E
and F apply to all images.
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Case study of pretreated and untreated soils.
The final
standard hybridization protocol, including the correction for
nonspecific binding, was tested with soil samples representing the
following conditions: (i) soil which had been air dried for 2 days and
then amended with a glucose solution (final concentration, 3 mg of
glucose per g [dry weight] of soil) by rewetting the preparation to
field capacity 2 days before hybridization; (ii) soil which had been
air dried for 2 days and then rewetted to field capacity 2 days before
hybridization; and (iii) soil which had been fixed directly after
collection from the field and had a water content of 9.8% on a dry
weight basis. The experiment with glucose-treated soil was repeated
four times, and the other two experiments were repeated twice. In all
repetitions, hybridizations were performed with at least four
replicates. The results given below are the means for all experiments
for each soil treatment.
Figure
5 shows that the
numbers of specifically stained cells (N
sp) decreased in
the following order: glucose-amended soil,
air-dried and rewetted soil,
untreated soil. After the addition
of unlabelled probe, the counts
(N
sp,unl) were reduced by 35 and
38% in the
glucose-treated and dried and rewetted soils, respectively,
but only by
6% in the untreated soil. As Fig.
5 also shows, the
counts obtained
with the nonspecific NONEUB338 probe rarely decreased
when excess
unlabelled NONEUB338 probe was added; therefore, the
nonspecific counts
were interpreted as being mostly due to rhodamine
binding. In most
cases, the nonspecific staining was thus completely
compensated for by
adding unlabelled probe, so that the numbers
of active cells in these
soils could be calculated from the counts
obtained with the EUB338
probe after subtraction of the counts
obtained with the EUB338 probe
plus unlabelled probe.
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FIG. 5.
Numbers of active soil bacteria determined by
fluorescence in situ slurry hybridization by using rhodamine-labelled
specific (Nsp) and nonspecific (Nnon)
oligonucleotide probe EUB338. Unlabelled probes were included in order
to determine the numbers of cells exhibiting nonspecific binding to the
rhodamine part of probe (Nsp,unl, Nnon,unl).
The numbers of active bacteria were calculated by compensating for both
types of nonspecific labelling ([Nsp Nsp,unl] [Nnon Nnon,unl]).
The data represent the results of four experiments performed with
glucose-treated soil (A), two experiments performed with air-dried and
rewetted soil (B), and two experiments performed with untreated soil
(C). Each experiment included four or five independent hybridizations,
and the standard deviations thus represent 16, 8, and 9 trials, for the
glucose-treated soil, air-dried and rewetted soil, and untreated soil,
respectively. The cells were assigned to three size classes, as
indicated.
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We anticipated that the data obtained for the untreated soil collected
under relatively dry conditions would represent the
natural
low-activity soil conditions under which many small bacteria,
but
relatively few active bacteria, would be detectable by the
in situ
slurry hybridization protocol. The results indicate that
no active
bacteria could be detected in the dry control soil (Fig.
5C),
reflecting the detection limit of the method. However, rewetting
air-dried soil clearly resulted in higher numbers of active bacteria
(3.8 × 10
8 cells g of soil
1), with the
greatest increase occurring in the abundant small-size
group of
bacteria. Finally, glucose amendment also resulted in
a significant
increase in the number of active bacteria (4.8 ×
10
8
cells g of soil
1), and the small size group was again the
predominant
group.
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DISCUSSION |
The present study shows that the majority of the active soil
bacteria, as determined by FISH, are the smallest bacteria (i.e., bacteria with diameters of less than 0.5 µm). The level of activity of these bacteria has not been investigated in detail (45), but the results correspond well with the finding that radiolabelled thymidine and leucine were actively incorporated into such bacteria extracted from soil (4, 5). Addition of a labile carbon source, such as glucose, to soil or rewetting of air-dried soil, resulting in a release of nutrients (38), are both
well-known treatments which increase bacterial activity dramatically
(8, 9, 13, 47). The increased counts determined after
rewetting alone or rewetting plus amendment with glucose are considered evidence that the hybridization protocol and the model for calculating the numbers of active soil bacteria performed correctly.
The rRNA content has been correlated directly with the growth rate for
fast-growing bacteria (19, 29), but this relationship may
not be valid during slow growth or starvation (12, 30, 44).
The relationship between growth rate and sensitivity of the FISH
protocol was investigated for laboratory-grown bacteria added to soil.
The results demonstrated that cells with generation times of 5 h
of less could be easily detected, and the data thus supported the
conclusion that the in situ slurry hybridization technique detects only
the most active bacteria in soil samples. When flow cytometry was used
to quantify light emission by a fluorescein-labelled EUB338 probe, a
lower limit of detection was set at generation times of 4 to 7 h
for cultures of Escherichia coli and Burkholderia cepacia (46). However, we cannot eliminate the
possibility that starved and dormant bacteria may also be detected as
active bacteria when their rRNA levels are still relatively high. This
has been observed for Salmonella typhimurium
(44), and a high level of protein turnover probably
accompanied by elevated rRNA levels has been found in starved marine
bacteria (31).
In soil samples nonspecific staining may result from binding of either
the oligonucleotide (DNA) component or the fluorochrome component of
the probe (1, 46). When the FISH technique is used with
environmental samples, nonspecifically stained cells are therefore
often observed and must be corrected for by subtracting counts obtained
by hybridization with a nonspecific probe (28, 33). With
soil samples hybridized with labelled probe EUB338 it was not possible
to correct for this by directly subtracting counts obtained with the
NONEUB338 probe because higher counts were sometimes obtained with the
NONEUB338 probe than with the EUB338 probe (Fig. 5). This finding was
related to slight differences between the ratios of rhodamine to DNA
components in different batches of the probes. To solve this problem,
we added different amounts of unlabelled (rhodamine-free) probes to the
soil samples in order to titrate out the nonspecific binding of the
labelled probe. The contribution of nonspecific binding of DNA was
compensated for by subtracting counts obtained with the nonspecific
probe from counts obtained with the specific probe after correction for
rhodamine binding.
The numbers of active bacteria found in the glucose-treated and the
dried and rewetted soil samples represented approximately 5% of the
total bacterial counts (5 × 109 to 9 × 109 cells g [dry weight] of soil1)
determined by DAPI staining. This estimate corresponds well with the
percentage of active cells determined by the CTC reduction assay
performed with bulk soil from the same barley field (48). This is apparently in strong contrast to other reported data, which
showed that 41% of the total DAPI counts could be detected with EUB338
probe hybridization by using soil smears (49). However, controls including nonspecific probe NONEUB338 were not included in the
previous study, and it is likely that the numbers of bacteria in the
samples were overestimated.
The great differences between replicates shows the importance of
including at least four replicates with the four combinations of probes
in order to obtain consistent results for the different size groups of
soil bacteria. The high number of replicates makes the method
labor-intensitive and expensive in terms of materials. Additional
improvements in the protocol for in situ slurry hybridization should include testing alternative fluorochrome labels for
the oligonucleotide probes in order to obtain minimal binding to soil particles and more uniform labelling. To reduce the labor
involved, automatic counting procedures based on CLSM, as demonstrated
by Bloem et al. (9), could be incorporated into the
FISH technique. Despite the obvious limitations at present, including
the relatively poor detection limit, our protocol for in situ
hybridization to rRNA to determine the number of active soil bacteria
is useful and could be improved. The sensitivity for specific groups of bacteria might be increased by performing repeated
homogenization-centrifugation extraction for soil bacteria
(32). This approach, in combination with soil perturbations,
might be used to determine the functions of major soil bacterial
groups, such as the Acidobacterium and Verrucomicrobia divisions (25), which have been
poorly characterized or not characterized by cultivation methods but
are known from 16S rRNA gene sequence comparisons.
| |
ACKNOWLEDGMENTS |
Palle Hobolth is acknowledged for synthesizing the
oligonucleotide probes. We thank Lars Kongsbak Poulsen and Svend J. Binnerup for their support and advice in developing the in situ
hybridization method.
This work was financed by the Danish Center for Microbial Ecology and
by the Danish Biotechnology Research Programme 1996-1999 (grant 9502015).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Veterinary Microbiology, Royal Veterinary and Agricultural University, Stigbøjlen 4, 1870 Frederiksberg C, Denmark. Phone: 4535282783. Fax:
4535282757. E-mail: kvlhc{at}unidhp.uni-c.dk.
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Abstract
Introduction
