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Luijten, M. L., Roelofsen, W., Langenhoff, A. A., Schraa, G., and Stams, A. J. (2004).
Hydrogen threshold concentrations in pure cultures of halorespiring bacteria and at a site
polluted with chlorinated ethenes. Environ. Microbiol. 6, 646650.
McInnes, D. M., and Kampbell, D. (2000). The bubble stripping method for determining
dissolved hydrogen (H2) in well water. Field Anal. Chem. Technol. 4, 283296.
Sanford, R. A., Cole, J. R., Loffler, F. E., and Tiedje, J. M. (1996). Characterization of
Desulfitobacterium chlororespirans sp. nov., which grows by coupling the oxidation of
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Microbiol. 62, 38003808.
Sanford, R. A., Cole, J. R., and Tiedje, J. M. (2002). Characterization and description of
Anaeromyxobacter dehalogenans gen. nov., sp. nov., an arylhalorespiring facultative
anaerobic myxobacterium. Appl. Environ. Microbiol. 68, 893900.
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Sung, Y., Ritalahti, K. M., Sanford, R. A., Urbance, J. W., Flynn, S. J., Tiedje, J. M., and
Loffler, F. E. (2003). Characterization of two tetrachloroethenereducing, acetate
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[14] Advances in Microscopy: Microautoradiography of

Single Cells
By JEPPE LUND NIELSEN and PER HALKJR NIELSEN
Abstract

Microautoradiography (MAR) is an efficient method to obtain reliable

information about the ecophysiology of microorganisms at the single
cell level in mixed communities. Data obtained by the traditional MAR
method can now be improved significantly when MAR is combined with
fluorescence in situ hybridization (FISH) with oligonucleotide probes for
the identification of target organisms. This chapter discusses how to use
METHODS IN ENZYMOLOGY, VOL. 397
Copyright 2005, Elsevier Inc. All rights reserved.

0076-6879/05 $35.00
DOI: 10.1016/S0076-6879(05)97014-6

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MARFISH in various ecosystems with emphasis on the type of information to be obtained, incubation conditions, and detailed protocols for
the MAR technique. It also discusses new MAR applications and the type
of information that can be obtained, e.g., the use of dual labeling to
investigate the simultaneous uptake of several substrates by individual
prokaryotes.
Introduction

The microbial consortia in natural or engineered systems typically

consist of many groups of prokaryotes, many of which are still unidentified.
Our understanding of their role and function in the environment is even
inferior. We have only a very limited understanding of a few distinct
groups, very often those with easily recognizable morphologies or
specialized activities. A traditional way of obtaining information on the
roles of microbes in the environment is to isolate and cultivate the microorganisms using more or less selective media. However, only a small
proportion of the microorganisms may be cultivated, and if they are, their
physiology and activity as investigated under culture conditions may
not necessarily reflect the activities and functions in the environment.
The application of molecular techniques has revealed the identity of many
uncultured organisms in complex environmental systems, which cannot be
detected by direct cultivation approaches. Therefore, cultureindependent
techniques are compulsory in order to investigate the physiology and
ecology of these uncultivated microorganisms.
A number of new staining methods and molecular methods have been
applied to provide information about the activity of prokaryotes directly in
complex microbial systems, and microautoradiography is probably the
most efficient and most versatile method to date. MAR has been used in
microbial ecology for many years (Brock and Brock, 1966, 1968), and since
it was first applied in combination with fluorescence in situ hybridization
for identification of the microorganisms as described by Lee et al. (1999)
and Ouverney and Fuhrman (1999), MAR has been revived and applied in
numerous approaches and combinations. The combined technique has
been described with several acronyms, such as MARFISH, STARFISH,
and MicroFISH (Cottrell and Kirchman, 2000; Lee et al., 1999; Ouverney
and Fuhrman, 1999), but the basic principle is identical.
This article describes the microautoradiographic technique and how to
apply it for investigating the physiology and ecology of cultured or uncultured single prokaryotic cells directly in complex microbial consortia. We
focus on two important aspects: incubation conditions, which are critical
for the type of information to be obtained about the physiology of the

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microorganisms investigated, and protocols for performing MARFISH,

which has to be done very carefully. The basic theory of MAR has not been
included, but it can be found elsewhere (e.g., Carman, 1993; Rogers, 1979).
Type of Information Obtained by MARFISH

MAR has been used in combination with various simple staining techniques but without combination with FISH to characterize microbial activity in
mixed communities (Andreasen and Nielsen, 1997; MeyerReil, 1978; Tabor
and Neihof, 1982). However, the information obtained cannot be related to
specific microorganisms without them having a very atypical activity or morphology. In some studies the number of active cells assimilating a specific
compound (e.g., leucine, thymidine, acetate, glucose, Nacetylglucosamine,
cAMP) or a mixture of radiolabeled organic substrates (e.g., amino acids) is
obtained. Also, the number of phytoplankton or autotrophic bacteria incorporating labeled bicarbonate can be quantified. By using specific incubation
conditions and inhibitors it is possible to enumerate various functional bacterial groups in more detail. In activated sludge, for example, denitrifiers, sulfate
reducers, polyphosphateaccumulating bacteria, and methanogens have
been enumerated using MAR in combination with 40 ,6diamino2phenylindole, dihydrochloride (DAPI) staining for all procaryotes (Nielsen and
Nielsen, 2002a,b).
When MAR is combined with FISH, the method becomes much more
useful for detailed studies of the ecophysiology of probedefined microorganisms, identification of MARpositive bacteria, and determination of
degradation pathways in microbial communities. Examples of information
that can be obtained are presented next.
Ecophysiology of ProbeDefined Microorganisms
Investigations into specific probedefined microorganisms have provided
information about which organic substrates that can be taken up (e.g.,
Ouverney and Fuhrman, 1999; Kong et al., 2004), autotrophic or mixotrophic
activity (e.g., Gray et al., 2000; Daims et al., 2001; Nielsen et al., 2000), activity
under various electron acceptor conditions and response to inhibitors (e.g., Ito
et al., 2002), and uptake of inorganic phosphate (e.g., Lee et al., 1999; Kong
et al., 2004). Also, quantitative kinetic parameters such as cellspecific uptake
rates and substrate affinity constants (Ks) can be obtained (Nielsen et al.,
2003). Information about the physiology of microorganisms of interest may be
used to design efficient isolation strategies.
Partial Identification of MARPositive Bacteria
If unknown bacteria degrade a certain labeled compound, e.g., an
environmental pollutant (Yang et al., 2003), the identity can be partly or

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fully resolved by applying various FISH probes. An example is the partial

identification of Fe(III) reducers in activated sludge (Nielsen et al., 2002).
If the specific identity of the organism is desired, such information can
greatly reduce the work with analysis of clone libraries and gene probe
design (Kong et al., 2005).
Overview of Degradation Pathways of Organic Matter in a
Microbial Community
By investigating an ecosystem with a number of labeled substrates
assumed to be central to the degradation of organic matter and combining
this with a population structure investigation, it is possible to obtain important information about structure and function of an entire ecosystem.
As an example, it was shown that planktonic Archaea play an important
role in amino acid turnover in marine systems (Overney and Fuhrman,
2000; Teira et al., 2004). Kindaichi et al. (2004) studied interactions between
nitrifying bacteria and heterotrophic bacteria in autotrophic nitrifying
biofilms, where they identified several groups of heterotrophs that could
grow on compounds excreted by the autotrophs.
The Microautoradiographic Procedure

Overview of the Method

The original procedure in MARFISH for incubations of a microbial
sample is outlined in Fig. 1. The sample is incubated under defined conditions for some time, typically a few hours, to ensure that a sufficient
amount of radiotracer is taken up by the active cells. Several factors should
be considered in the design of a proper sample incubation: type and specific
activity of the radiotracer, concentration of unlabeled substrate, concentrations of electron acceptors (oxygen, nitrate, or others), inhibitors, biomass concentration, temperature, and incubation time. It is advisable to
measure substrate consumption and incorporation into the biomass to
ensure optimal incubation conditions. After incubation, the samples are
fixed in paraformaldehyde or ethanol (to allow FISH analysis of gram
negative and grampositive cells, respectively) and washed to remove
surplus radiotracer. Homogenization, cryosectioning, dilution, or concentration of the sample before it is mounted on a cover glass, a glass slide, or a
polycarbonate filter may be necessary for optimal visualization of the
MARFISH signal. For FISH, the standard protocols can be followed using
5(6)carboxyfluoresceinNhydroxysuccinimide ester (FLUOS), and Cy3
labeled oligonucleotide probes before the radiationsensitive emulsion is
applied. The emulsion on glass slides is exposed some days before being

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analysis of microbial hydrogen metabolism

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FIG. 1. Experimental overview of the procedure for conducting a microautoradiography

experiment on a sample from a complex microbial community.

developed using standard photographic methods. MARpositive and negative bacteria can be examined by combining bright field or phase contrast
with epifluorescence microscopy or confocal laserscanning microscopy.
Sample Pretreatment
The sample should preferably be collected just prior to incubation, as
storage may alter the activities of the indigenous organisms in the sample.
Depending on the nature of the sample and the subject of investigation,
storage for 12 days at 4
may be applied. Also, based on the composition
and nature of the system investigated, it might be advisable just prior to
incubation to aerate the sample to ensure the removal of easily degradable
organic matter. When examining anaerobic activity, this preincubation step
can be replaced with an anoxic step to remove nitrate or other electron
acceptors. In a few special cases, the background level of electron donor or
other secondary metabolites might be too high to ensure a proper design of
the incubation, and special care, such as washing the sample, might be
necessary (Gray et al., 2000). When examining certain systems such as
aggregates (e.g., biofilms), physical pretreatment (homogenization and/or
solubilization) might be needed to avoid diffusional limitations of substrate
or electron acceptor (Nielsen and Nielsen, 2005).
Sample Incubation
Sample incubation has to be in accordance with the type of information
expected from the MAR experiments, and there are several important

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TABLE I
IMPORTANT PARAMETERS TO CONSIDER FOR OPTIMAL INCUBATION OF SAMPLES FOR
MAR EXPERIMENTS
Parameter
Type of radiotracer

Addition of unlabeled substrate

Specific activity of the labeled

compound in sample (Ci/mol)
Electron acceptor(s)

Incubation time
Inhibitors
Volume and biomass concentration

Consider for optimal incubation

Energy of tracer (high energy gives lower resolution
of MAR)
Labeling position in tracer (different atoms in
substrate molecule may be transformed differently)
Final substrate concentration (labeled unlabeled
substrate). If possible, it should be well above half
saturation constant (Ks) during the entire
incubation period
High specific activity aims at cells with low activity or
low yield (incorporation) and vice versa
Presence of oxygen, nitrate, nitrite, sulfate, or others.
Avoid toxicities and depletion. Maintain the level
during incubation
As short as possible (a few hours) and no longer than
the time of depletion of substrate/electron acceptor
Inhibition of other influencing metabolisms
Ensure sufficient number of cells

parameters to consider regarding incubation of the sample with radiotracer

(Table I). Different information can be obtained by MAR (see earlier
discussion), and it is important to detail this question and determine how
to examine a specific question in a given system. The radiotracer is chosen
based on the isotope (typically 3H or 14C labeling of substrates) and often
on the location of the labeled atom(s) in the organic molecule (e.g., C1 or
C2 labeling of acetate). Labeling should preferably be on an atom, which is
incorporated directly into the biomass. Because the energy of the isotope is
related inversely to the resolution of the resulting MAR image, the lower
the energy, the higher the resolution. In contradiction to this, isotopes with
higher energy yield higher sensitivity and higher dynamic range valuable to
quantitative examinations. Our experience is that tritium is generally preferred due to the higher resolution, better than 1 m (see example in
Fig. 3). If additional studies of transformation of organic matter are carried
out by quantitative analyzing CO2 production and incorporation of carbon
into the biomass, 14C labeling is preferable. However, instead of using the
same samples for MAR and transformation studies, it is often better to run
separate parallel incubations, as lower tracer amounts are needed for
transformations studies compared to MAR incubations.
Each radiotracer can be purchased with different specific radioactivities
(Ci/mmol) and concentrations (Ci/ml). It is, however, not very important

[14]

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which one is chosen, as it often only constitutes a minor part of the total
amount of substrate in the incubation (because of added nonlabeled substrate). Only if the final concentration during the incubation must be very
small (only in systems with very low microbial activity, so the substrate is
not depleted during incubation), a high specific activity of the radiotracer is
needed so that the amount added to the sample can be kept low without
losing the wanted level of radioactivity. In other cases, the purchased tracer
is adjusted with unlabeled substrate to obtain the specific activity and
concentration wanted.
In order to optimize the incubation conditions for each type of microbial sample, it is usually important to test different amounts of the specific
activity of the tracer, substrate concentration, biomass concentration, and
incubation time. Usually, the first step is to fix the incubation time, which is
dependent on the system, but typically is between 1 and 6 h. If the incubation time is too long (too many hours, days), there is a risk of population
growth and of uptake of metabolites by other bacteria than the target
population.
The second step is to decide on a proper substrate concentration (unlabeled plus labeled substrate). The concentration should be sufficient to
avoid substrate depletion during incubation. Levels well above the value
of the halfsaturation constant (Ks) for the target organisms should usually
be selected to get maximum uptake of all target bacteria and are thus highly
dependent on the system investigated. However, Ks is hardly known in most
systems, so a substrate concentration range based on microbial activity is
often used. For highly active systems (biofilms, wastewater treatments systems, etc.), a concentration in the upper range of normal concentrations for
the actual environment is selected, typically 0.12 mM. For systems with low
activity (e.g., many marine environments), 150 nM or sometimes lower
concentrations are used (e.g., Cottrell and Kirchman, 2000; Ouverney and
Fuhrman, 2000). Given this, the third step is to adjust the biomass concentration in such a way that the substrate is not depleted during the incubation
period. In highrate systems we usually apply a biomass concentration of
0.11 g suspended solids/liter or 0.060.6 g organic matter/liter. This is based
on our experience with substrate uptake rates in the range of 0.52 g substrate/g organic matter/hour. Dilution of biomass is made by filtered water
from the same sample. A high dilution would ensure that the substrate is not
depleted during incubation, but means that a larger sample volume is
needed in order to get enough biomass to perform the subsequent MAR
procedure. Normal incubation volumes are 2 to 5ml samples. Larger
incubation volumes increase the need for radiotracer accordingly.
The tracer must be diluted properly with unlabeled substrate to get an
overall specific activity of the tracer, which ensures that only active cells

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physiological ecology

TABLE II
TYPICAL SETUP FOR TWO MARFISH EXPERIMENTS OF A HIGHACTIVITY AND A LOWACTIVITY
SYSTEM FOR THE USE OF ACETATE UPTAKE UNDER AEROBIC CONDITIONS
Parameter

Low activity
3

High activity
3

[ H]acetate (Na)
5064,000 mCi/mmol
To a final concentration of
0.550 nM
104106 Ci/mol

[ H]acetate (Na)
5064,000 mCi/mmol
To a final concentration of
2 mM
2 Ci/mol

Incubation time
Temperature
Inhibitors
Biomass concentration

Oxygen (provided by airspace

in bottle)
624 h
20

None
106107 cells/ml

Volume
Exposure time

5 ml
314 days

Oxygen (provided by
airspace in bottle)
3h
20

None
0.5 g organic matter/liter
109 cells/ml
2 ml
34 days

Type of radiotracer
Specific activity (tracer)
Addition of unlabeled
acetate
Specific activity (overall in
sample)
Electron acceptor

take up sufficient substrate during the incubation in order to be visualized

as MARpositive cells. The amount of tracer required to detect one active
cell is approximately 1015 Ci (3105 Bq), if the sample is exposed 46
days during the MAR procedure.
Table II shows two typical experimental conditions: a highly active
microbial system (activated sludge) and a low activity system (marine
sample). The length of the incubation must be adjusted to ensure a measurable uptake and should be based on knowledge obtained from control
experiments from the same system. For anaerobic experiments it is important to apply strict anaerobic techniques, e.g., by removing oxygen from the
bottles by flushing/evacuation several times with oxygenfree nitrogen.
Trace amounts of oxygen in highrate systems can be removed by introducing a preincubation step with an additional electron donor, but without
addition of a radioactive tracer. The substrates and tracers are added using
strictly anaerobic techniques for anaerobic experiments. A problem often
encountered is the presence of unwanted nitrate or nitrite, which can also
be removed similarly to oxygen by introducing a preincubation step. This
also works best in highrate systems. All sample vials should be sufficiently
mixed during incubations by horizontal placement on a rotary table.
A preincubation step with an unlabeled substrate should always be
applied for all incubations with electron acceptors other than oxygen (e.g.,
nitrate, nitrite, sulfate). An anaerobic preincubation of 12 h is important, as

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many aerobic bacteria take up labeled substrate the first 0.51 hour after
changing from aerobic to anoxic conditionspossibly because there were
traces of oxygen presentand that they were unable to take up the same
substrate after 1 or 2 h preincubation with unlabeled substrate. A preincubation can also be used to determine whether assimilation of a certain
substrate is taken up for storage or for further metabolic activity, e.g.,
growth (e.g., Andreasen and Nielsen, 2000; Kong et al., 2004; Kragelund
et al., 2005). For instance, some bacteria have a certain capacity to assimilate
and store substrate under anaerobic conditions, but are unable to grow. A
preincubation of 34 h with an unlabeled substrate saturates the storage
capacity, and by adding labeled substrate after this preincubation step, no
uptake can be found under anaerobic conditions. The addition of substrate
only from the start of the incubation period would give positive uptake (and
MAR signal) but not show whether the organisms used the substrate for
storage or actually metabolized the substrate.
Under certain, but rare, conditions, chemical or physical factors can
cause silver grain formation in the absence of radioactivity, also called
chemography, thus a negative control must be included. A pasteurized
sample (heated to 80
for 10 min) is suitable and must be prepared just
prior to the experiment to avoid germination of bacterial spores. Changes
in pH, temperature, and other physicochemical factors should be checked
before and after each type of incubation.
Termination of Incubation, Fixation, Wash, and Staining
Incubation of samples is terminated by fixing the biomass by adding
freshly prepared 8% paraformaldehyde (PFA) solution in phosphatebuffered saline (PBS) (130 mM sodium chloride, 10 mM sodium phosphate
buffer, pH 7.2) directly to the incubation vials (final concentration 4%).
For fixation of grampositive bacteria, a final concentration of 50% EtOH/
PBS is used for termination. In anaerobic incubations the fixative is added
using a syringe and needle. The samples are fixed for 13 h at 4
.
In experiments with sufficient biomass, the samples are pelleted by
centrifugation and are washed three times by resuspension and centrifugation using filtered (pore size 0.22 m) tap water. In cases of limited
biomass, the sample can be concentrated by filtering the sample through
a polycarbonate filter (pore size 0.22 m) and subsequently washing by
filtering prefiltered water through the filter. Biomass attached to the filter
can either be removed from the filter by backwashing or by detachment
using 3aminopropyltriethoxysilanetreated coverslips (see later). Alternatively, the sample can be centrifuged (10,000g for 10 min), and 9095% of
the supernatant is removed carefully by pipetting. This can be repeated a

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number of times until most unbound tracer is washed away. The advantage
of nonfiltered samples is that they can be immobilized easily on cover glass.
Cover glass allows a good visualization through the cover glass so any stain
or hybridization underneath the MAR film emulsion is still fully visible. If
visualizing through the film, the silver grains of MARpositive cells typically block the staining signal. Filtered samples can be visualized by peeling
off the exposed film and then placing it on a glass slide and subsequently
staining it. Another approach uses the adhesiveness of an aminoethoxysilanetreated slide and, by assuring close contact between cells and the glass
surface with clamps, the cells can be transferred from the filter to a cover
glass surface (Cottrell and Kirchman, 2000). Efficient transfer of cells from
a filter surface must be examined in every case by performing controls,
typically carried out by DAPI or acridine orange staining of the cells on the
filter after transfer.
The washed sample biomass can be immobilized on hydrophilic (gelatincoated) glass slides or cover glasses (see later) and air dried on the
bench or in a 60
oven, which in our experience slightly increases the
attachment of single cells to the gelatincoated surface. Gelatin coating is
performed using cover glasses washed thoroughly in boiling water containing dish detergent for 5 min and rinsed in distilled water. After drying, the
slides are dipped in a jar with a 0.1% gelatin, 0.01% CrK(SO4)2 suspension
heated to 70
in which it is allowed to stand for 5 min before air drying
overnight at room temperature in a vertical position.
Most often around 20 l of the fixed, washed biomass is placed on the
slide/cover glass and then dispersed on the glass surface using the side of a
pipette tip. The remaining biomass from the sample can be stored at 20

in 50% EtOH/PBS until later use. Stored samples containing PBS should
be rinsed carefully in distilled water after immobilization to avoid autofluorescence from precipitated salt crystals and filmdamaging effects.
Before immobilization of the biomass to the coverslips, the samples may
be treated to improve visualization of single cells. If aggregateforming
bacteria are to be investigated, the sample must often be homogenized or
sectioned into thin slices by cryosectioning to be able to distinguish the
source of the MAR signal. The sample is embedded in cryosectioning
material (TissueTek OCT; Miles, Elkhart, IN) and frozen before slicing
it into a suitable thickness (a few micrometers and up to 20 m) and
transferring it to a glass surface. Embedding material can be washed
away easily prior to further treatment without any significant loss of cells.
Alternatively, gentle use of a glass tissue grinder can often disrupt the
microbial aggregate without destroying the microcolonies and without lysing the cells. Planktonic cells and filamentous bacteria outside the aggregates can be investigated without any further treatment. Special staining

[14]

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247

procedures (such as FISH, DAPI, gram, and Neisser staining) can be made
at this point.
Fluorescence in Situ Hybridization
The MARFISH combination can be performed by hybridizing the
sample directly on the cover glass. Hybridizations of the fixed cells are
carried out using fluorescently labeled oligonucleotides targeting specific
ribosomal RNA sequences. We recommend the use of probes labeled with
the sulfoindocyanine dye Cy3, which usually gives a high fluorescence
intensity, but FLUOS can also be used. In our experience, Cy5 (far red)
has a too small Stoke shift and can easily be mistaken with the reflection of
the emitting wavelength from the silver grains.
A sequential dehydration in series of ethanol solutions (50, 80, and 96%
EtOH; 2 min in each) allows a better visualization of thick samples, a better
mobilization of the probe through the cell walls, and helps in removing
traces of embedding material when cryosectioning is applied. After the
cover glasses have dried, 8 l of hybridization buffer with the required
formamide concentration is mixed with 1 l (50 ng/l) of each probe in the
center of the cover glass. The sample is then hybridized at 46
for 1.5 h in a
moisture chamber (e.g., a 50ml plastic Falcon tube containing a tissue paper
moistened with the applied hybridization buffer), taking care to place the
cover glass horizontally, maintaining the probe at the center of the cover
glass. Unspecifically bound probe is washed away by dipping the cover glass
into washing buffer (48
for 15 min). Salt crystals are avoided by gently
submerging the cover glass in distilled water prior to air drying. After any
desired staining (e.g., DAPI), the glasses are allowed to air dry.
Catalyzed Reporter DepositionFISH (CARDFISH)
In many natural ecosystems, the activity level of the microorganisms is so
low that the normal FISH procedure provides fluorescence levels too weak
to be detected. The identification of such cells can instead be examined by
the significantly more sensitive CARDFISH technique (catalyzed reporter
depositionFISH). The CARDFISH procedure can, after some modifications, be combined easily by MAR, and such a combination has revealed
that MAR can be used to determine the activity of cells with low ribosomal
content (J.L. Nielsen et al., unpublished results; Teira et al., 2004).
Modifications of the CARDFISH procedure can be carried out by
immobilizing the cells on a cover glass surface instead of filters. The fixed
and washed sample is transferred to a gelatincoated cover glass and
allowed to dry completely at 50
. To avoid loss of cells during the process
of hybridization, a thin layer of lowmelting agarose gel (0.2%) is placed

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onto the sample by a pipette and is processed further through this agarose
layer. Permeabilization is carried out depending on the nature of the cells
and should be optimized in every case. Typically, a treatment in lysozyme
(10 g/liter in 50 M EDTA, 50 M TrisHCl, pH 7.5) for 30 min at 37

followed by treatment with achromopeptidase (60 U/ml in 0.01 M NaCl,
0.01 M TrisHCl) (at 37
for 30 min) is required for the horseradish
peroxidase (HRP)labeled probe to enter the cells. The cover glass is then
washed in distilled water and treated with 0.1 M HCl for 10 min and
washed again in distilled water. The hybridization is then performed by
mixing 2 l HRP probe (final concentration 0.05 M) in 200 l hybridization buffer [0.9 M NaCl, 20 mM TrisHCl, pH 7.5, 10% (w/v) dextran
sulfate, 0.02% (w/v) sodium dodecyl sulfate, 1% blocking reagent (Boehringer Mannheim, Mannheim, Germany)] and formamide according to the
probe used. Hybridization takes place at least 2 h at 35
followed by a 10
min washing step at 37
in hybridization buffer. At this step, it is important
to keep the slides moist throughout the hybridization and amplification
procedure. Amplification of the tyramide signal is carried out by incubating
the slide in a 50ml tube with PBS and 0.05% Triton X100 for 15 min at
room temperature. After removal of excess buffer, the slide is incubated
for 30 min at 37
in the dark after the addition of 5 l Cy3tyramide
(1 mg/ml) and amplification buffer [10% (w/v) dextran sulfate, 2 M NaCl,
0.1% (w/v) blocking reagent, and 0.0015% H2O2 in PBS]. The slide is
then washed in 50 ml PBS containing 0.05% Triton X100 and is subsequently rinsed in distilled water, both at room temperature. Following this
procedure, the slide is processed with film emulsion as described later.
Autoradiographic Procedure
The following steps are performed in a darkroom with safe light for
black and white film development procedures. The film emulsion (LM1,
Amersham Biotech) (usually diluted by the addition of 1 part distilled
water and 9 parts film emulsion) is placed in a water bath at 43
and is
shaken gently every 2 min for 10 min to avoid air bubbles in the emulsion.
The emulsion is poured carefully into a dipping vessel to a level of approximately 4 cm. The glass slides/cover glasses are dipped carefully in the
emulsion for 5 s. The slides are then placed vertically on a folded tissue
paper for 5 s, and the side without the sample is cleaned with a piece of
tissue paper. The slides are placed horizontally on a plastic tray, and the
emulsion is allowed to solidify for 2 h in the dark before they are placed in
a slide box with waterfree silica gel. The box is covered with aluminum foil
and exposed at 4
. The exposure time is normally between 2 and 10 days,
depending on the sample, labeling, incubation, etc. To ensure sufficient

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exposure time for both active and less active cells to appear MARpositive,
variable exposure length must be compared regarding the number of
MARpositive cells. For development of the film, the slides are placed in
the developer (Kodak D19, 40 g/liter) for 3.0 min. They are drip dried
before being placed in distilled water for 1 min. The slides are placed in
fixer (Nathiosulfate, 300 g/liter) for 4 min. To remove the fixer, the slides
are washed in distilled water for 3x3 min. The slides must air dry at least 4 h
before microscopic examination. Storage in a refrigerator preserves the
slides for at least 1 year.
Microscopic Evaluation
The microscopic evaluation of MAR can be performed using either
brightfield microscopy (either DIC or phasecontrast microscopy work
ideally) or a laserscanning microscope (LSM) using the transmittent light
mode. We use two different systems: an upright microscope stand, Zeiss
Axioskop 2 plus and a Zeiss LSM 510 Meta (Carl Zeiss, Oberkochen,
Germany) mounted on an inverted Axioplane microscope stand with UV
laser (Coherent Enterprise; Coherent, Inc., Santa Clara, CA), an ArKr
laser (458 and 488 nm) and two HeNe lasers (543 and 633 nm). MAR
signals are best visualized by the laserscanning microscope without oil, but
in order to avoid fading of the fluorescence signal, we normally add a small
drop of antifading agent (e.g., Citifluor or Vectashield ) under a cover glass.
An inverted microscope stand facilitates visualization, whereas an upright
microscope requires mounting and immobilization of the coverslide onto a
slide using tape. LSM images can be obtained using the standard software
package (Zeiss LSM 510 v. 3.02), and epifluorescence images are captured
using a chargecoupled device camera (CoolSnap HQ; Photometrics,
Tucson, AZ). Confocal LSM images yield higher intensities of the fluorescence signal and increase the resolution by removing outoffocus fluorescence. The MAR signal cannot be recorded confocally, but is best
visualized by applying the transmittent mode. By using a normal epifluorescence microscope without an inverted stand, the cover glasses are placed
upside down on a glass slide coated with the antifading agent. In this way,
the silver grains are underneath the cells rather than covering them,
making it easier to visualize the FISH signal.
In order to find the optimal exposure times, several extra glass slides
should always be included for daily inspection of the silver grain densities.
In this case, it is easiest to combine DAPI staining with the inspection of
the MAR signal by switching between bright field and fluorescence microscopy. Enumeration of the number of MARpositive cells and the amount
of silver grains should be evaluated in order to ensure a proper exposure

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time. Too brief an exposure will only yield the most active organisms in the
sample, whereas too lengthy exposures will increase background signals,
thus decreasing resolution of the MAR technique.
Usually, the amount of silver grains on top of the cells is assessed
in qualitative terms as MARnegative or MARpositive. A clear MAR
positive result should be easy to distinguish from the background level, and
we often use at least four to five silver grains located on top or close to a
single cell as a guideline (with background levels below 0.01 silver grains
per m2). Examples of MAR combined with the normal FISH procedure
and the CARDFISH procedure for identification of the microorganisms
are shown in Fig. 2. Furthermore, a real quantification of the MAR signal
can give valuable information about the substrate uptake by the individual
cells (Nielsen et al., 2000; Pearl and Stull, 1979). A proper quantification,
however, requires procedures that ensure prevention of leakage from the
cells and use of internal standards. The procedure for quantitative MAR
(QMAR) is described in detail elsewhere (Nielsen et al., 2003).
Example of Application: Dual Uptake of Substrates

In some studies the simultaneous uptake of two substrates of probe

defined populations has been investigated under in situ conditions (Kong
et al., 2004). Such investigations can be carried out by adding both substrates simultaneously, but with only one (and different) compound labeled
in parallel incubations. If the probedefined cells are MARpositive under
both conditions, the cells are able to take up both substrates simultaneously. However, in cases where specific probes are not available or if only a
minor part of the population is active, this approach does not work.
Instead, it is possible to use dual labeling by applying a weak and a strong
isotope for labeling of the substrates. Because the half distance for production of silver grains is different for the two isotopes (less than 1 m and 23
m for 3H and 14C, respectively), it is possible to see whether a certain cell
has taken up 3H or 14Clabeled substrates or if it has taken up both.
Figure 3 shows such an example. Filamentous bacteria and single cells
from an activated sludge system were investigated for simultaneous uptake
of [3H]acetate and [14C]propionate. Both filamentous bacteria, hybridizing
with the probe for Candidatus Meganema perideroedes (belonging to
the Alphaproteobacteria, Nielsen et al., 2003), and many unidentified single
cells were able to take up both substrates simultaneously. However, several
unidentified single cells and some filamentous bacteria hybridizing with a
probe for Cytophaga (CF319a) could assimilate only acetate and not propionate. In order to obtain such images as shown in Fig. 3, the specific
activity of the two labeled compounds and the exposure time must be

[14]

251

analysis of microbial hydrogen metabolism

FIG. 2. Microautoradiography (MAR) combined with fluorescence in situ hybridization

(FISH) or catalyzed reporter deposition FISH (CARDFISH). Images AC and DF show
the same microscopic view of an activated sludge sample (Grindsted wastewater treatment
plant, Grindsted, Denmark) and a river water sample (North Pine Dam, in southeast
Queensland, Australia). (A) MAR image after incubation of the sample with [14C]propionate
under aerobic conditions. Note the presence of a MARpositive filament and a few single cells
(seen as areas covered by a thick layer of silver grains). (B) The same microscopic field as A,
but recorded for fluorescence after hybridization with a Cy3labeled probe specific for
Meganema perioderoedes (probe Gr1Cy3) and a FLUOSlabeled bacterial probe (probe
EUB338FLUOS). (D) Water sample incubated with [3H]thymidine under aerobic conditions, and (E) hybridization of CARDFISH after hybridization with a Cy3labeled probe
specific for Actinomycetes (probe HG1654Cy3). (C and F) Overlay of AB and DE,
respectively. All images were recorded by laserscanning microscopy (LSM510 Meta, Carl
Zeiss, Jena, Germany). Bar: 10 m.

optimized. In the actual case, the concentrations were 0.7 mM and 20 Ci/
ml for acetate and 1.4 mM and 20 Ci/ml for propionate, incubation time
was 2 h, and exposure time was 17 h.
Example of Application: Heterotrophic Assimilation of

14

CO2

A new variation of the MAR method has been developed, the HetCO2
MAR (Hesselse et al., 2005). This approach is based on the fact that most
heterotrophic bacteria assimilate CO2 in various carboxylation reactions
during biosynthesis. Typically 110% of the biomass carbon has been derived

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[14]

FIG. 3. Detection of simultaneous uptake of two substrates by bacteria from an activated

sludge sample (Grindsted wastewater treatment plant, Grindsted, Denmark) using dual
labeling of [3H]acetate and [14C]propionate. Images A and B show MARpositive filaments
and single cells, respectively, after incubation with [3H]acetate. Images C and D show MAR
positive cells after uptake of [14C]propionate, whereas images E and F show the uptake of
both [3H]acetate and [14C]propionate. Note that silver grains are located up to 23 m from
MARpositive cells with the more energetic isotope 14C, whereas the less energetic 3H isotope
only forms silver grains up to 1 m from the cell and thus yields a higher resolution. By
incubating with a relatively higher final specific activity of the 3Hlabeled substrate it is
possible to see cells taking up both labeled substrates simultaneously. Bar: 20 and 10 m in A,
C, and E and in B, D, and F, respectively.

[14]

analysis of microbial hydrogen metabolism

253

from CO2 fixation. Assimilation of 14CO2 by heterotrophic bacteria can be

used for isotope labeling of active microorganisms in environmental samples
and visualized by MARFISH. The MAR signals are comparable with the
traditional MAR approach based on the uptake of 14Clabeled organic substrates [see the detailed protocol described by Hesselse et al. (2005)]. The
new HetCO2MAR approach appears to differentiate better between substrate uptake (and possibly storage) and substrate metabolism that result in
growth. Another obvious advantage is that isotope labeling of heterotrophic
microorganisms is no longer restricted to the use of commercially available
and often expensive labeled organic substrates, so any organic compound
or mixture of organic substrates can, in principle, be investigated for
potential uptake and metabolism just by focusing at the uptake and incorporation of 14CO2. This novel HetCO2MAR approach expands significantly
the possibility for studying the ecophysiology of uncultivated microorganisms.
Common Methodological Problems

MAR, particularly MARFISH, has some drawbacks and limitations.

Extensive experience with both MAR and FISH is required, preferably
access and experience with a confocal laser microscope, the techniques are
timeconsuming, and it is relatively expensive due to the cost of radiotracers. In some countries, the safety regulations require separate laboratories and equipment for isotope work. It is important to conduct all
experiments in rooms dedicated to isotope work and to follow all local
safety instructions. Get all formal permissions from the local safety officer.
Also, all involved staff should be properly trained and advised. If all safety
instructions are followed, MAR is not a dangerous method.
The availability of suitable radiolabeled substrates can be a problem.
Often, the suppliers can only offer a limited selection of either 3H or 14C
labeled organic compounds, whereas more rarely sold compounds can only
be produced upon request at a special price. However, by screening the
suppliers, most common organic compounds can be purchased at a reasonable price. Also, use of the novel HetCO2MAR approach as described
earlier may circumvent these problems.
Interpretation of a positive or negative MAR signal must always be
carried out carefully. Controls and duplicates (on independent samples)
should always be included. It is also very important to evaluate uptake/no
uptake of different substrates only if comparable treatment has been applied, such as same amount of tracer and cold substrate and same incubation
time and exposure time. If unexpected results are obtained, these must
always be checked for errors in the procedure, particularly related to the

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incubation conditions, and the experiments should be repeated. This is

especially critical for anaerobic conditions.
Conclusion

The method of assessing microbial activity of probedefined microorganisms under in situ conditions as described in this chapter has been used
successfully during the last 56 years in several ecosystems. A wide range of
hitherto uncultured organisms from very diverse environments has been
examined and their activities have been established. Microautoradiography
is a very versatile technique that can be applied in most microbial environments and yields information on the ecophysiology of individual microorganisms. Recent improvements, such as the combination of MAR with
CARDFISH for microorganisms with low ribosome content, quantitative
MAR for obtaining kinetic data, and the detection of duallabeled cells,
offer new options for more detailed studies of microbial consortia. Also,
combinations with other in situ methods, e.g., formation of internal storage
products or measurements of local microenvironments by microsensors,
can provide valuable information. Although new improvements are most
likely under way, the technique today is fully capable of conducting many
detailed and valuable studies of microbial communities.
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Further Reading
Ginige, M. P., Hugenholtz, P., Daims, H., Wagner, W., Keller, J., and Blackall, L. L. (2004).
Use of stableisotope probing, fullcycle rRNA analysis, and fluorescence in situ
hybridizationmicroautoradiography to study a methanolfed denitrifying microbial
community. Appl. Environ. Microbiol. 70, 588596.

[15] Atomic Force Microscopy of Bacterial Communities

By MEGAN E. NU N EZ, MARK O. MARTIN, PHYLLIS H. CHAN, LIN K. DUONG,
ANIL R. SINDHURAKAR , and EILEEN M. SPAIN
Abstract

This chapter discusses atomic force microscopy (AFM) for the benefit
of microbiologists who are interested in using this technique to examine the
structures and dynamics of bacteria. AFM is a powerful technique for
imaging biological samples at the nanometer to micrometer scale under
nondestructive conditions. In order to be imaged with AFM, bacteria must
be supported by a surface, which presents challenges because many laboratory strains of bacteria are planktonic. Still, in nature many bacteria live
at surfaces and interfaces. This chapter discusses the benefits and difficulties of different methods that have been used to support bacteria on
surfaces for AFM imaging and presents two methods in detail used to
successfully grow and image bacteria at solidliquid and solidair interfaces. Using these methods it is possible to study bacterial morphology and
METHODS IN ENZYMOLOGY, VOL. 397
Copyright 2005, Elsevier Inc. All rights reserved.

0076-6879/05 $35.00
DOI: 10.1016/S0076-6879(05)97015-8