Visualizing Bacterial Cell Walls
and Biofilms
Cryo-transmission electron microscopy is enabling investigators to
examine native, hydrated structures in bacteria and biofilms
Terry J. Beveridge
he surfaces of microbial cells are perhaps their most important structure
because they are in immediate contact with the external environment.
Microbial cell walls admit nutrients
and release wastes, and yet they must resist
internal turgor pressure and environmental insults and maintain cellular shape. Walls also
help cells adhere to other surfaces and provide
space for specialized structures such as flagella,
pili, spinae, capsules, S-layers, and exopolymeric substances (EPS).
Delving into the native architecture of gramnegative and gram-positive bacterial surfaces
traditionally depended on electron microscopy,
T
Summary
• Advances in cryo-transmission electron microscopy (cryoTEM) are enabling investigators to
examine native, hydrated structures in bacteria
and their associated biofilms.
• Freeze-substitution reveals that the periplasmic
space of gram-negative bacteria is filled with a
gel and confirms that lipopolysaccharide resides
on the outer face of the outer membrane, while
gram-positive cells appear to build new wall in
layers from the inside outward, with older material removed from the outer edge.
• Bacteria can prove difficult to image with
cryoTEM, and doing so depends on the inherent
density of the proteins, lipids, carbohydrates,
and nucleic acids within specimens being denser
than the surrounding layers of frozen vitrified
water.
• Despite technical difficulties, cryoTEM is being
used to visualize structures within bacterial
biofilms.
which uses high vacuums and high electron accelerating voltages. These requirements usually
ensure that only dry specimens can be imaged,
yet living cells and their structures rely on water
and its astounding ability to interact with and
configure molecular ingredients. However, advances in cryo-transmission electron microscopy
(cryoTEM) are helping to overcome this difficulty because they allow investigators to examine native, hydrated structures in bacteria and
their associated biofilms.
Conventional Views of Bacterial Surfaces
Before the 1950s, microbiologists studying bacterial cell features were limited to the use of
well-crafted objective lenses for light microscopy, vibrant dyes, and intelligent experiments. Better views of the cytoplasmic
interior and enveloping structures of bacteria came in the mid-1950s from transmission electron microscopy (TEM) of thin sections. Here, chemical fixation cross-links
essential structural components to maintain
the general architecture of the cell before it is
seen in thin section.
These early preparations sometimes were
breathtaking, revealing a centrally condensed, nonenveloped chromosome surrounded by randomly distributed ribosomes and providing strong support for the
concept of an anucleate (i.e., prokaryotic)
cell (Fig. 1). These images also revealed a
fundamental difference between grampositive and gram-negative bacteria, which
have more complex walls consisting of an
outer membrane, a thin peptidoglycan
layer, and a periplasmic space filled with
periplasm. Gram-positive walls can be 20-
Terry J. Beveridge
holds the Canada
Research Chair in
the Structure, Physical Nature and
Geobiology of Prokaryotes in the Department of Cellular
and Molecular Biology, College of Biological Science, and
the Advanced Food
and Materials Network—National
Centre of Excellence, University of
Guelph, Guelph,
Ontario, Canada.
This article is based
on a presentation at
the symposium,
“Bacterial Sculpture: Peptidoglycan
Metabolism and
Cell Shape,” convened during the
ASM General Meeting in Atlanta, 5–9
June 2005.
Volume 1, Number 6, 2006 / Microbe Y 279
erichia coli and Pseudomonas
aeruginosa is filled with a gel and
that the majority of the lipopolysaccharide resides on the outer face of
the outer membrane (Fig. 2).
Similar analysis reveals a tripartite profile for the Bacillus
subtilis wall (Fig. 3), correlating
with turnover (Fig. 4). A darkly
stained innermost region consists
mainly of new unstressed polymers that are incorporated into the
wall. Immediately above is a
greatly stressed, translucent region
that is subject to a turgor pressure
of about 25 atm and thus is
stretched almost to the breaking
point. The outermost fibrous region is subject to autolysins that
degrade older wall material. As
cell wall materials turn over, condensed material migrates from the
inner region into the middle where
it is immediately stretched, expanding the wall along the longitudinal axis of the cell. The polymers of the middle migrate to the
outer region, where autolysins clip
them into fibers that slough away.
Fig. 1. A portion of a conventionally embedded B. subtilis showing a polar cap and some of the
While electron microscopists
longitudinal wall (or sidewall) where cell wall turnover occurs (arrow denoting the cell wall points
use freeze-substitution techniques,
to the junction of the polar and side wall). Notice that a periplasmic space is not seen and that
the cell wall seems to consist of a continuous amorphous matrix. The condensation of the DNA
other researchers used neutron
is thought to be artifactual. Scale bar, 500 nm.
scattering, modeling, and chroFig. 2. A portion of a freeze-substituted Pseudomonas aeruginosa PAO1 that shows the O-side
matographic analysis to develop a
chains of the lipopolysaccharide on the outer face of the outer membrane (large arrow) as well
better picture of the thickness, oras the periplasmic gel within the periplasmic space (smaller arrow). (Image was kindly
dering, and complexity of the baccontributed by Ryan Hunter of my laboratory.) Scale bar, 50 nm.
teria cell wall. However it is orgaFig. 3. Cell wall of a freeze-substituted B. subtilis showing the three regions of the wall that can
be attributed to cell wall turnover. A periplasmic space is not seen. (This image was published
nized, its polymeric network needs
in V. R. F. Matias and T. J. Beveridge, Mol. Microbiol. 56:240 –251, 2005, and is reproduced here
to be strong and elastic. Bacterial
with consent of the authors and journal.) Scale bar, 50 nm.
cell wall modelers fall into two
camps, one claiming that such
fold thicker than the peptidoglycan in grampolymers are organized horizontally, while
negative bacteria and frequently possess secondmembers of the other camp favor a vertical
ary polymers, such as teichoic and teichuronic
scaffold. Tantalizing atomic force microscopy
acids, attached to a peptidoglycan network.
(AFM) images of Staphylococcus aureus D2H
Conventional embeddings can be misleading
reveal newly made walls consisting of concentric
when fixation fails to preserve all structural
rings (Fig. 5), thus favoring the horizontal armacromolecules. Moreover, when water is rerangement model.
moved, most structures shrink and macromoleFrozen Hydrated Thin Sections
cules reconfigure. However, cryo-techniques can
of Vitrified Bacteria
preserve higher-order structures, especially in
the enveloping layers. Freeze-substitution reveals
Recent images of frozen, hydrated sections of
that the periplasmic space of bacteria such as Eschbacteria are revolutionizing our views of their
FIGURE 1–3
280 Y Microbe / Volume 1, Number 6, 2006
cellular structures. Although such
FIGURE 4
images were published as early as
the 1980s, demands for specialized
instruments and technical expertise
delayed progress. Recently, the technique has been used to examine the
nucleoid and ordering of DNA in
Deinococcus radiodurans to explain
its resistance to ionizing radiation.
Meanwhile, investigators interested
in bacterial envelopes are using this
approach to reexamine gram-negative bacteria such as E. coli K-12 and
P. aeruginosa PAO1 as well as grampositive species such as B. subtilis
This diagram shows three regions of the B. subtilis cell wall,
168 and S. aureus D2H (Fig. 6 – 8).
determined from images like those in Fig. 3. Region #1 is where
newly made wall polymers are found and is densely stained.
To produce such images, cells are
Region #2 consists of older, less dense wall material that is
immersed in a cryoprotectant such
stretched by the cell’s turgor pressure. Region #3, containing the
as dextran or sucrose before being
oldest wall material, is actively being solublized by autolysins. Wall
turnover progresses from inside to outside.
frozen so rapidly that the surrounding water vitrifies. Molecular motion stops so quickly that native
low to be differentiated from the surrounding
structures cannot deteriorate and cells are emice (Fig. 7). Yet, the plasma and outer membedded in noncrystalline, or amorphous, ice that
branes, the periplasm, and the peptidoglycan
resembles a glass. The cells are thus physically,
layer can be seen, and their overall arrangement
not chemically, fixed, and the vitrified specimens
is the same as that seen in conventional and
are sliced into 50-nm frozen hydrated sections,
freeze-substitutions even though the dimensions
which are viewed in a cryo-transmission eleco
are different.
tron microscope at approximately ⫺140 C.
The 25-nm thickness of the cell walls of many
Unlike other types of TEM that depend on
gram-positive
species accommodates about 25
heavy metal staining agents, frozen hydrated
stacked
layers
of peptidoglycan. Some species
sections are not stained because doing so would
differ
slightly
in
the peptide stem of the Ndestroy their vitrified state. Hence, bacteria are
acetylmuramic
acid
of the glycan strand. For
extremely difficult to visualize and imaging deinstance,
S.
aureus
contains
lysine instead of
pends on the inherent density of the proteins,
diaminopimelic
acid,
pentaglycine
bridges belipids, carbohydrates, and nucleic acids within
tween
peptides,
and
variable
interpeptide
bondspecimens being denser than the surrounding
ing
percentages.
These
differences
may
account
layer of frozen water (Fig. 6). Because images of
for differences in elasticity and environmental
these bacteria reflect how macromolecules disreactivity between species.
tribute within the cell, higher magnifications
The walls of B. subtilis can be differentiated
reveal the polymeric organization of the cell
from
the surrounding ice. When images are
wall. In gram-negative bacteria, the asymmetry
scanned
by a densitometer, the mass goes from
of lipids in the outer membrane becomes apparhigh
on
the
inner face to low on the outer face,
ent (Fig. 7). The outer face of the bilayer consubstantiating
the idea that cell wall turnover
taining lipopolysaccharides (LPS) has more phosgoes
from
inside
to outside (Fig. 3 and 4). This
phorus and mass per unit volume, and thus
transition
is
not
seen
in frozen hydrated sections
shows up as a darker line because of its greater
of S. aureus cell walls, which tend not to turn
contrast than does the inner face consisting of
over (Fig. 8).
phospholipids.
Remarkably, there is a periplasmic space beUnlike freeze substitutions (Fig. 2), the O-side
tween the plasma membrane and cell wall in
chains of the LPS on “smooth” gram-negative
both B. subtilis and S. aureus (Fig. 8). The denspecies such as P. aeruginosa PAO1 cannot be
seen in frozen sections because their mass is too
sity within the periplasm is low, suggesting to us
Volume 1, Number 6, 2006 / Microbe Y 281
FIGURE 5–7
Fig. 5. Atomic force image of newly made cell wall surface derived from a septum in S. aureus. Note how the new polymers are arranged
in concentric ridges that disappear as the wall matures. (This image was originally published in J. Bacteriol. 186:3286 –3295, 2004, and is
reprinted with the permission of the journal and the authors.) Scale bar, 50 nm.
Fig. 6. Frozen hydrated (or cryo-) section of an intact P. aeruginosa PAO1 cell, revealing the membranes in the cell envelope as well as the
ribosomes and DNA fibers. (This image was published in J. Bacteriol. 185:6112– 6118, 2003, and is reproduced with consent of the journal
and the authors.) Scale bar, 200 nm.
Fig. 7. A high magnification of cell envelope of P. aeruginosa PAO1 revealing how well the membranes and periplasm can be seen by their
differential densities in a cryo-section. Note that the outer face of the outer membrane is darker than other membrane faces because of the
greater density of the LPS. However, the LPS O-side chains are more widely dispersed and of lower density, and thus cannot be seen. PM,
plasma membrane; PS, periplasmic space containing periplasm; PG, peptidoglycam layer; OM, outer membrane. (This image was published
in J. Bacteriol. 185:6112– 6118, 2003, and is reproduced with the consent of the journal and authors.) Scale bar, 50 nm.
that it consists of a dilute brine of molecular
constituents. The periplasmic space is only about
22 nm thick, and is easily deformable. Conventional embeddings and freeze-substitutions of B.
subtilis rarely show this space (Fig. 1 and 3). My
colleagues and I earlier suggested that the
periplasm of gram-positive bacteria resides in
the interstices of the cell wall network. Subsequently, using frozen hydrated sections, we visualized the periplasmic space as a separate entity that lies between the plasma membrane and
the inner face of the wall. A similar periplasmic
space is also found in Enterococcus hirae, suggesting that it is a common feature of grampositive bacteria.
Visualizing Structural Details in Bacterial
Biofilms Proves Challenging
Biofilms are difficult to study because they consist of extremely soft matter (EPS) interspersed
with bacteria, materials that they have shed such
as flagella, pili, and membrane vesicles (MVs),
and other harder materials such as biominerals.
Although biofilms grow large enough to be seen
without magnification, confocal microscopy
282 Y Microbe / Volume 1, Number 6, 2006
with fluorescent probes is especially useful for
distinguishing molecular networks within biofilms. Ratiometric dyes are helping to probe
chemical conditions such as pH within biofilms.
However, because no form of light microscopy is capable of discerning molecular arrangements within biofilms, higher-resolution microscopy is necessary. Although AFM comes to
mind, it visualizes only the topography of samples, and its cantilevers deform highly hydrated
materials such as EPS. And, although some researchers rely on scanning electron microscopy
(SEM), especially variable pressure SEMs, this
technique falls short of depicting minute ordering within biofilms.
Hence, we prefer TEM, even though chemical
fixatives do not fully penetrate biofilms and
organic solvents collapse the EPS. There are also
problems using cryoTEM. Because EPS is 90 –
95% water, its density is so low that it cannot be
distinguished from external water. Thus a frozen
hydrated section of a biofilm resembles a frozen
hydrated section of planktonic cells. Both show
bacteria interspersed with low-density material.
Thin sections of freeze-substituted biofilms
FIGURE 8 –9
Fig. 8. High magnification of the cell envelope of S. aureus showing the plasma membrane (PM), the periplasmic space (PS), and the cell
wall matrix (CW) in a cryo-section. This image was supplied by Valerio Matias of my laboratory. Scale bar, 50 nm.
Fig. 9. Image of freeze-substituted cells in a P. aeruginosa PAO1 biofilm showing the remarkable preservation of the cells, their LPS (with
O-side chains; large arrow), and the exopolymeric substance (EPS; smaller arrows). This image was published in J Bacteriol. 187:7619 –7630,
2005, and is reproduced with consent of the journal and the authors. Scale bar, 500 nm.
are, so far, the best cryoTEM method for deciphering finely ordered structures. Specialized
equipment can vitrify samples to a depth of
10 –50 ␮m, making it possible to see a biofilm
from top to bottom. For gram-negative cells,
high magnifications can differentiate the O-side
chains of LPS from the surrounding EPS matrix
(Fig. 9). Even within this matrix, differences in
polymeric arrangements can be detected, from
densely packed to loosely packed fibers.
However, freeze-substitution has a major
drawback because only thin (50-nm) slices of a
biofilm can be directly imaged. Tomography of
thicker sections using 200-kV or higher-voltage
microscopes may soon provide better three-dimensional pictures of biofilms.
Comparison of Freeze-Substituted and
Frozen Hydrated Sections
Although these two cryoTEM techniques both
provide accurate representations of bacteria,
their enveloping layers, and biofilms, they yield
different results. The frozen-hydrated sections
technique depends on intrinsic density differences among cellular constituents, whereas the
freeze-substitution technique requires structures
to be stained by heavy-metal contrasting agents.
Moreover, frozen-hydrated sections reveal
where cellular mass resides, while freeze-substitution reveals chemical reactivity.
These two types of samples also tend to have
dimensional differences because frozen-hydrated structures are typically larger and more
robust than freeze-substituted structures. For
researchers in geomicrobiology, this density-site
reactivity correlation could be helpful when
studying how bacteria interact with metal ions
in the environment, with those metals acting as
natural stains. Surface sites on bacteria often act
as nucleation sites for the development of nanomineral phases. The early state of such phases
should be apparent in frozen-hydrated sections
because of their high densities compared to organic components surrounding them.
The Future of CryoTEM in Microbiology
CryoTEM is making strong inroads in molecular biology by providing three-dimensional
structures of specific proteins that are cloned
into bacteria. This technique is particularly important for analyzing noncrystallizable proteins.
However, as particle size and complexity increase, three-dimensional analysis becomes
more difficult. Although few laboratories are
Volume 1, Number 6, 2006 / Microbe Y 283
capable of such complicated analyses, cryoTEM
of viruses can yield breathtaking results depicting protein capsid arrangements, nucleic acidpacking, and receptor sites.
Larger objects such as bacteria are still too
difficult to analyze by such techniques. They are
far too complex to subject to accurate Fourier or
correlation-averaging analyses as single particles. Moreover, unlike viruses, bacterial particles are not quasicrystalline. Paradoxically, bacteria are so small that freezing and cryosectioning them is difficult compared to
mammalian tissues. Thus, bacteria are too large
for single-particle analysis and too small for
uncomplicated cryo-handling.
Nonetheless, I predict that this field will grow
substantially. There are so many questions to
address. How do gram-positive and -negative
bacteria divide? Do gram-negative cells divide
by constriction or by septation? Can we visualize tubulin-like proteins necessary for arranging
division sites by cryoTEM? How do capsule
polymers and S-layer glycoproteins interdigitate
with wall macromolecules? How do flagellar
basal bodies, spinae, and pili interact with enveloping layers? What do intracellular bodies such as
thylakoids, carboxysomes, ␤-hydroxy-alkanoate
bodies, and magnetosomes look like, and where
do they reside in cytoplasmic space? What is the
ordering of DNA during the growth cycles of a
cell? Can we see the highly condensed packingorder of DNA in endospores? Can we see structural differences between archaea and bacteria?
The list of such questions goes on and on. Although only a few laboratories are capable of
conducting cryoTEM analysis of bacteria, this
shortage can be remedied. More difficult is the
lack of appropriate expertise among young researchers. The popularity of genes and molecules
has taken its toll, making electron microscopy a
forgotten art among most young microbiologists.
The scarcity of cryo-laboratories with experience in
microbiology means that few young investigators
are being trained in this field. Yet, I am hopeful.
ACKNOWLEDGMENTS
Valerio Matias and Ryan Hunter in my laboratory are responsible for several of the cryoTEM images in this article. Peter Lau,
Anton Korenevsky, and Oleg Stukalov are doing the same for AFM, while Sarah Schooling is studying biofilms, and Farhana
Islam is our resident geomicrobiologist. Bob Harris, Dianne Moyles, and Anu Saxena address our TEM needs. I thank
Manfred Jericho of Dalhousie University in Halifax, Nova Scotia, for allowing me to reprint Fig. 5. The research reported here
is supported by an NSERC–Discovery grant and funding through the Canada Research Chair Program. Additional funding
comes through the Advanced Food and Materials Network-National Centre of Excellence (AFMnet-NCE), the US-DOENABIR program, and the US-DOE Grand Challenge in Biogeochemistry Program. Microscopy was performed in the NSERC
Guelph Regional Integrated Imaging Facility, which is partially funded through an NSERC–Major Facilities Access grant.
SUGGESTED READING
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Matias, V. R. F., A. Al-Amoudi, J. Dubochet, and T. J. Beveridge. 2003. Cryo-transmission electron microscopy of
frozen-hydrated sections of Escherichia coli and Pseudomonas aeruginosa. J Bacteriol. 185:6112– 6118.
Matias, V. R. F., and T. J. Beveridge. 2006. Native cell wall organization shown by cryo-electron microscopy confirms the
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