Heavy metal negative staining, a now widely used
routine technique to prepare biological material for
imaging in the transmission electron microscope
(TEM), was introduced almost 50 years ago (Brenner
and Horne, 1959; for reviews, see Oliver, 1973; Horne,
1991). Suitable specimens may either be in solution or
in suspension and thus include the whole spectrum of
structural organization of biomolecules, ranging from
monomeric and oligomeric proteins [e.g., RNA polymerase
(Fig. 1a) or glutamine synthetase (Fig. 1b,
bottom)], to protein polymers [e.g., cytoskeletal filaments
or the multistranded helical cables of glutamine
synthetase (Fig. 1b, top)], and eventually to large multicomponent
supramolecular assemblies [e.g., nuclear
pore complexes or bacteriophages (Fig. 1c)]. Compared
to other electron microscopic techniques, negative
staining is remarkably simple and can easily
provide reliable structural information as quickly as
2min after preparing the specimen down to resolutions
of 2.0nm (reviewed by Bremer et al.
, 1992) and
below (Harris, 1999).
|FIGURE 1 From molecules to replicating molecular machines-examples of negatively stained preparations.
(a) Monomers of Escherichia
coli RNA polymerase holoenzyme. (b) Glutamine synthetase from E. coli. (Top) Helical filaments are formed in the presence of millimolar
amounts of Co2+ ions. (Bottom) The intact enzyme molecule is composed of 12 identical 50-kDa subunits that associate with 622 symmetry: i.e.,
end-on views display a typical blossom-like appearance with six-fold symmetry, whereas side views reveal twofold symmetry. (c) E. coli bacteriophage
T4: organization into a distinct prolate icosahedral head and a contractile helical tail to which a base plate with extended fibers is
attached at the bottom are evident. RNA polymerase (a) and glutamine synthetase
(b) were stained with 0.75% uranyl formate; and bacteriophage
T4 (c) was stained with 1% uranyl acetate.
Scale bar: 100nm.
|FIGURE 2 Schematic representation of a "negatively
stained" model specimen. (a) The schematic view
represents a cross section through
a model specimen
(light grey) that has been embedded in an ideal negative
(dark grey). (b) The schematic view in a was
adding the pixel values along the vertical
axis. The projection profile of the "negatively stained"
model specimen is represented by a thick
whereas the projection profile of the "unstained" model
specimen is depicted by a thinner grey trace. In the case
of the "negatively
stained" specimen, its projection profile
was inverted, i.e., multiplied by-1, to compare it directly
with the corresponding profile of the
As shown schematically in Fig. 2a, a negatively
stained specimen is embedded in a microcrystalline
heavy atom salt replica that portrays its molecular
architecture. Because heavy metal atoms (e.g., U, V, W,
Au, Mo, Pt, Pb, Os) scatter electrons much more
strongly than elements constituting the biomolecules
(i.e., C, H, O, N, P, S), the contrast of negatively stained
specimens (Fig. 2b) is much higher than that of
unstained specimens and reversed (hence "negative"
staining). In addition, the heavy metal salt replica stabilizes
the specimen against collapse and distortion
and substitutes, at least partially, for the aqueous environment
typical for biomolecules. It also serves as a
radiation protectant in the sense that it is more radiation
resistant than biological matter (cf. Aebi et al.
1984; Bremer and Aebi, 1992).
II. MATERIALS AND
Negative staining requires only minor investment
for instrumentation and supplies. For routine applications,
the following items are needed.
- Specimen support grids (Fig. 3a): For example,
from Pelco International (www.pelcoint.com). Typically
200-400 mesh/in, copper grids, 3.05 mm in diameter
and 0.7 mm thick, coated with a specimen support
film (mostly either a carbon/collodion composite film,
or a thin carbon film). The specimen grids should be
stored dust free, e.g., in a petri dish (Fig. 3b) that is kept
at relatively low humidity.
- Precision forceps (Fig. 3c): For example, from
Electron Microscopy Sciences (www.emsdiasum.com).
It is advisable to bend the jaws of the forceps slightly
inwards to prevent surface tension from trapping
buffers and solutes between the jaws. A tightly fitting
rubber band or piece of plastic tubing allows the jaws
to be fixed in the closed state.
- Glow discharge unit (Fig. 3d). It is used to
render the support film of the specimen grids
hydrophilic and can be built as detailed by Aebi and
Pollard (1987). The specimen grid is glow discharged
on a small glass block coated with Parafilm (Fig. 3d,
- A metal or plastic tray (Fig. 3e) serves as the
"workbench." Drops of water and stain, typically
100 µl each, can be placed on a piece of Parafilm (Fig. 3e, left). Filter or blotting paper (Fig. 3e, right) is required
for removing excess liquid from the specimen grid.
- Micropipettes: 5µl and adjustable to 20-200µl or
- A stopwatch is required to control adsorption and
- A Pasteur pipette drawn out into a capillary and
connected to a suction device such as a water jet pump
serves to remove excess stain from the specimen grid
|FIGURE 3 Materials and equipment required for negative staining. (a) Specimen support grid (e.g., 200
mesh/in, copper grid, 3.05 mm in diameter and 0.7 mm thick) coated with a collodion-carbon composite film
(the support film is particularly evident at the edges). (b) The specimen support grids are stored on filter
paper in a covered petri dish (the cover is removed for clarity). (c) Precision forceps with the jaws slightly
bent inwards are used for all manipulations. (d) The specimen grids are rendered hydrophilic in a reduced
atmosphere of air using a custom-built glow-discharge unit (for the design, see Aebi and Pollard, 1987). The
specimen grids are glow discharged on a small glass block that is covered with Parafilm (see arrowhead). (e)
A plastic or metal tray serves as the "workbench" for staining. The water and stain drops are placed on a
piece of Parafilm on the left, the glass block with a glow discharged grid is seen on right at the back, and a
piece of filter paper is depicted in the front.
The following solutions are required for negative
A. Preparation of Negative Stain Solution
- Specimen solution/suspension
- Deionized or, even better, double-distilled water. Water
is required for washing the specimen grid after
- Negative stain, i.e., heavy metal solution. For instance,
uranyl acetate or sodium phosphotungstate.
For the properties of various negative stains, along
with references on their preparation, see Bremer et al.
(1992). Here, the preparation of uranyl formate, one of
the more delicate procedures, is described as an
(Mind, uranyl salts are radioactive, cf. p xxx)
- Weigh out uranyl formate to obtain a final concentration
of 0.75% (i.e., 7.5 mg/ml).
- Boil an appropriate amount of water to remove
CO2 and to increase the solubility of the uranyl
formate in the boiling water.
- Pour the boiling water into a small beaker containing
the weighed uranyl formate.
- Stir slowly for 5-10min in the dark (i.e., with the
beaker wrapped into aluminum foil).
- Filter the solution through a 0.2-µm membrane
filter [e.g., a Supor 0.2-µm Acrodisc 32 from Gelman
- Adjust the pH to 4.25 by adding 10 N NaOH (about
2µl per 1ml negative stain solution). The uranyl
formate solution should turn into a moderately
intense yellow (Caution: Increasing the pH toward
neutral rapidly leads to precipitation of uranyl
- Stir for another 5 min in the dark.
- Filter a second time.
- Readjust the volume with double-distilled water
to compensate for water loss due to evaporation,
- Store stain in the dark (e.g., in a tube wrapped into
aluminum foil). It will keep stable for 1-2 days.
: Uranyl formate may be difficult to purchase: Try,
e.g., www.pfaltzandbauer.com (Pfaltz & Bauer Inc.,
Waterbury, CT 06708).
Our standard staining procedure for soluble proteins
and their supramolecular assemblies is illustrated
in Fig. 4. As might be appreciated, this
procedure is quick, simple, and straightforward.
|FIGURE 4 Negative staining. (a) A ~5-µl drop of sample solution/suspension is placed onto a glowdischarged
specimen support grid that is held horizontally by a pair of forceps and allowed to adsorb for
30-60 s. (b) The specimen grid is then turned vertically and (c) allowed to gently touch the filter paper, which
(d) removes excess liquid. (e) The adsorbed specimen is then washed by carefully placing it sideways onto
a drop of water for i s, blotted as shown in b-d, and washed a second time. The actual staining is performed
similarly by first washing the specimen grid on a drop of negative stain as described in e, blotting and repeating
this step once more, this time leaving the specimen grid for 10s on the drop of negative stain solution.
(f) After blotting off excess stain, the residual stain is removed by gently moving a capillary (drawn-out and
shaped from a Pasteur pipette) around the edge of the grid. The capillary is connected to a low-vacuum
suction device (e.g., a water jet pump).
- Glow discharge specimen grids in a unit such as
shown in Fig. 3d, e.g., for 15 s (for details, see Aebi and
- To the freshly glow-discharged specimen grid,
apply a 5-µl drop of the sample solution/suspension
(Fig. 4a). Let it adsorb for 30-60 s, and then blot off the
drop as illustrated in Figs. 4b-4d. For blotting, hold the
specimen grid vertical (Fig. 4b) and allow it to touch
the filter paper gently (Fig. 4c). The drop will be
removed by capillary forces (Fig. 4d).
- Next, wash the specimen grid on a drop of
double-distilled water by carefully lowering it sideways
onto the surface of the drop (Fig. 4e). After ls,
carefully remove the specimen grid from the water
drop, blot as shown in Figs. 4b-4d, and repeat this
washing regimen once more.
- Finally, stain the specimen for 1s by lowering the
specimen grid onto a drop of negative stain solution
as described for the washing regimen (Fig. 4e) and
then blot the specimen grid as described in Figs. 4b-4d.
Repeat this step once; this time, however, leave the
specimen grid for 10-15s on the negative stain drop
(as illustrated in Fig. 4e) before blotting.
- After blotting off excess stain (Figs. 4b-4d),
further reduce the residual stain layer to just a thin (i.e.,
50-100nm thick) liquid film prior to air drying. This
should be achieved in a controlled fashion; a drawnout
and shaped into a slightly bent capillary Pasteur
pipette, connected to a suction device such as a water
jet pump, is moved gently around the edge of the specimen
grid (Fig. 4f). The preparation is now ready for
inspection in the EM.
The quality of a negatively stained preparation (i.e.,
the interpretable structural detail captured on the EM screen, depicted on an electron micrograph, or monitored
by a video or slow-scan camera attached to the
EM) depends primarily on the penetration properties
(e.g., size, charge, and hydrophobicity) of the heavy
metal salt used for negative staining and on the physicochemical
properties (e.g., solubility, charge, and
hydrophobicity) of the specimen. Because some negative
stains have a relatively acidic pH, whereas others
are neutral or moderately basic and because they may
be neutral, anionic, or cationic (for review, see Bremer et al.
, 1992), negative staining is quite versatile and can
be tailored to specific needs.
When visualizing structures comprising components
of different electron densities, e.g., colloidal
gold-labeled proteins, the electron density of the heavy
metal may become crucial. While 2- to 3-nm Au particles
associated to bacteriophage T4 polyheads, i.e.,
aberrant tubular assemblies constituted exclusively of
the major viral head protein gp23, are barely discernible
from the uranyl acetate acetate-stained
protein units (Fig. 5a), 3-nm Au-labled immunoglobulin
G binding to the appropriate haemaglutinin antigen
(Fig. 5b) and even 1- to 2-nm Au-labeled Fab
fragments binding to haemaglutine assemblies (Fig.
5c), are still well discernible when stained with
, a less electron dense and intrinsically
neutral negative stain (Baschong and Wrigley, 1990). A
priori, the optimal stain for a particular specimen has
to be chosen and evaluated by trial and error.
|FIGURE 5 Negative staining of Fab-gold and antibody-gold complexes: (a) Bacteriophage T4 polyheads
labeled with Fab-Au2.5nm unstained (left) and stained with 1% uranyl acetate. (b) Au3-labled immunoglobulin
G bound to bromelain-treated haemagglutinin. (c) Au1-2nm-labeled Fab bound to haemagglutinin rosettes.
Arrow: Au1-2nm colloid. (Adapted from Baschong & Wrigley, 1990
In our laboratory, starting in a first trial with an
uranyl salt [we still favour uranyl salts and formate
over acetate for it is slightly more stable in the electron
beam (cf. Bremer et al.
, 1992)] or with a tungstate (e.g.,
NaPT, i.e., sodium phosphotungstate) proved successful
with a wide variety of specimens, including
cytoskeletal, membrane and soluble cytosolic proteins,
and their supramolecular assemblies (for examples,
see Aebi et al.
, 1983, 1984, 1988; Bremer et al.
1992, 1994; Hoenger and Aebi, 1996). Within this respect, it may be noteworthy that uranyl acetate can
also act as a primary fixative: It was observed to stabilize
bacteriophage T4 polyheads against dilutioninduced
dissassembly in an aldehyde-like manner
(Baschong et al.
, 1983; W. Baschong unpublished
results) and was more recently documented to act as a
primary fixative for skeleton muscle (Fassel and
With tightening safety rules for the use of radioactive
substancesmuranyl salts are radioactivem
ordering, handling, and disposal of uranyl salts
usually have specific guidelines (check with your
safety responsible). In turn, the availability of uranyl
salts may become more restricted, while novel readymade
commercial methylamine-vanadate (Tracz et al.
, 1997) and methylamine-tungstate-based stains
(Shayakhmetov et al.
, 2000) have been introduced
(www.nanoprobes.com, Nanoprobes Inc., Yaphank, NY
11980-9710). As a consequence, nonradioactive metal
salts such as tungstates, molybdates and vanadates
may be given preference. The performance of such
alternatives is best evaluated by trial and error, as
illustrated in Figs. 6a-6e by staining bacteriophage T4
polyheads with uranyl formate (a) and prospective
alternatives, i.e., with NH4
(d), and RhCl3
(e), and liposomes with
methylamine vanadate (f).
|FIGURE 6 Comparative series of prospective negative stains used on bacteriophage T4 polyheads:
(a) with 1% uranyl formate, (b) with 2% NH4MoO4, (r with half-saturated NH4VO4, (d) with 1% Na2WO4,
and (e) with 2% RhCl3. (f) Liposomes stained with metylamine-vanadate, courtesy of C. Prescianotto-Baschong.
The negative staining protocol described earlier is
suitable for most specimens and stains. With delicate
proteins, an additional washing step of the grid with
water or sample buffer prior
to applying the specimen
may be included to wet the specimen support film and
to minimize surface tension and/or denaturation of
the specimen. The optimal sample concentration,
which obviously has to be determined experimentally,
should yield well-dispersed and evenly distributed
particles adsorbed to the specimen support film. It is
determined by the size, shape, and adsorption properties
of the biomolecules or supramolecular assemblies under investigation, but also by the washing and
staining regimen employed. For oligomers or polymers
(e.g., such as filaments), crystalline, or paracrystalline
specimens, a higher concentration than for
individual biomolecules is required. As a rule of
thumb, a few micrograms per milliliter for single particles
(see Fig. 1a and bottom of Fig. 1b) and a few
hundred micrograms per milliliter for supramolecular
assemblies such as filaments (see top of Fig. 1b) or
virus particles (see Fig. 1c) usually yield a reasonable
particle density on the EM grid. The ionic conditions
of the sample buffer and the presence of detergents,
while strongly interfering with specimen adsorption,
may have little effect on the quality of the negative
stain replica surrounding the biomolecules, as long as
a sufficient number of washing steps (usually two to
six for most compounds) are employed prior to applying
the negative stain solution to the specimen.
Heavy metal salts may not be inert but may interact
physically or chemically with the specimen. Such
sample-stain interactions may not explicitly produce
"preparation artifacts" but sometimes unveil biologically
significant information about the specimen going
beyond purely structural aspects. For instance, human
epidermal keratin filaments (cf. Aebi et al.
, 1983), after
negative staining with uranyl formate (UF; pH 4.25),
appear rather compact and featureless with a fairly
uniform width (Fig. 7a). By contrast, when the same
filaments are stained negatively with sodium phosphotungstate
(NaPT; pH 7.0), they partially unravel
and thereby unveil their protofibrillar substructure (Fig. 7b). This local unraveling becomes even more
pronounced after washing the filaments briefly with 10
phosphate buffer (NaPi
; pH 7.0) prior to staining
them negatively with UF (Fig. 7c). Most likely, the inorganic
) acts as a modulator of the lateral
interaction of the protofilaments and/or protofibrils
constituting the filament (cf. Aebi et al.
, 1983, 1988). In
fact, a similar response to different negative stains has
also been observed with F-actin filaments where inorganic
phosphate appears to modulate the relative
strength of the intersubunit bonds along
the two long-pitch helical strands of actin subunits
defining the filament (cf. Bremer et al.
, 1991, 1994;
Bremer and Aebi, 1992).
A. Poor Specimen Adsorption
|FIGURE 7 Negatively stained human epidermal keratin filaments. (a) Keratin filaments in 10mM Tris,
pH 7.5, stained negatively with 0.75% uranyl formate (UF; pH 4.25) appear relatively featureless and compact
with a fairly uniform width. (b) In contrast, when the same keratin filaments are stained negatively with 2%
sodium phosphotungstate (NaPT; pH 7.0) instead, they locally unravel and exhibit their protofibrillar substructure.
(c) Even more dramatic unraveling into their protofibrils occurs when the adsorbed filaments are
washed briefly with 10mM phosphate buffer (NaPi; pH 7.0) prior to staining them with 0.75% UF, pH 4.25.
Scale bar: 100 nm.
B. Patchy, Poor, or Bad Staining
- Specimen support film is wrongly charged: Highly
negatively charged proteins or DNA do adsorb poorly
to support films exhibiting a net negative charge as
produced by glow discharge in a reduced atmosphere
of air. By contrast, glow-discharging specimen grids in
a reduced atosphere of pentylamine yield a net positive
charge of the support film (cf. Aebi and Pollard, 1987).
- Specimen support film is not properly glow discharged:
Try longer glow discharge, and glow discharge
only one grid at a time and use it immediately.
C. "Bubbling" of Stain Upon Irradiation
- Specimen concentration is too high: A high density
of particles adsorbed to the specimen support film
renders quantitative and/or controlled removal of
excess stain more difficult, thereby resulting in inhomogeneities
in the staining even in particle-free areas.
Try lower specimen concentration.
- Stain is not sufficiently removed: Try removing
stain more quantitatively by suction with a capillary as
illustrated in Fig. 4f.
- Suboptimal stain for the specimen: Try a different
- Suboptimal wetting properties of the specimen
support film: Try longer glow discharge times or switch
to a different sample buffer, assuming that this does
not compromise the specimen.
- Incompatibilities between stain and buffers: For
instance, uranyl salts precipitate in the presence of
inorganic phosphate. Hence avoid using phosphate
- Buffer contains high molar salt or detergent: Try
including more washing steps.
D. Disintegration of Specimen During
- Strong recrystallization or water content of stain is
too high: Try "prebaking" specimen at a low magnification
(e.g., at 1000×) for 3-5s before using higher
E. Significant Background of Monomers
and/or Small Oligomers with a Specimen
Containing Supramolecular Assemblies
- Specimen is instable in water (washing steps!):
Wash and preequilibrate the specimen with sample
buffer instead of water. Replace the conventional
buffer with a buffer that will dry off by sublimation
when in the electron microscope, such as ammonia
acetate. Alternatively, use such a volatile buffer at least
for the washing steps. Mild cross-linking with, e.g.,
0.05-0.25% glutaraldehyde for 2min on ice (quench
with 1% glycine, pH 7.0, or with 1% freshly made
NaBH4) may also stabilize the specimen.
- Specimen is in steady-state equilibrium with
monomers and/or oligomers: Pellet the specimen (e.g., in
a tabletop or air fuge at 100,000g), discard the supernatant,
resuspend the pellet in sample buffer, and
immediately prepare the grid. Alternatively, fix, e.g.,
the protein assembly, with 4% formaldehyde while in equilibrum and purify on a sucrose gradient (Baschong et al., 1991).
We thank Rosmarie Suetterlin and Daniel Mathys
for their help. This work was supported by the Canton
Basel-Stadt and the M.E. Müller Foundation of
Aebi, U., Fowler, W. E., Buhle, E. L., and Smith, P.R. (1984). Electron
microscopy and image processing applied to the study of protein
structure and protein-protein interactions. J. Ultrastruct. Res
Aebi, U., Fowler, W. E., Rew, P., and Sun, T.-T. (1983). The fibrillar
structure of keratin filaments unraveled. J. Cell Biol
Aebi, U., Haener, M., Troncoso, J. C., and Engel, A. (1988). Unifying
principles in intermediate filament structure and assembly. Protoplasma 145
Aebi, U., and Pollard, T. D. (1987). A glow discharge unit to render
electron microscope grids and other surfaces hydrophilic. J.
Electron. Microsc. Tech
Baschong, W., Baschong-Prescianotto, C., Engel, A., Kellenberger, E.,
Lustig, A., Reichelt R., Zulauf, M., and Aebi U. (1991). Mass
analysis of bacteriophage T4 proheads and mature heads by
scanning transmission electron microscopy and hydrodynamic
measurements. J. Struct. Biol
Baschong, W., Baschong-Prescianotto, C., and Kellenberger, E.
(1983). Reversible fixation for the study of morphology and
macromolecular composition of fragile biological structures. Eur.
J. Cell Biol
Baschong, W., and Wrigley, N. G. (1990). Small colloidal gold
conjugated to Fab fragments or to immunoglobulin G as highresolution
labels for electron microscopy: A technical overview. J. Electron. Microsc. Tech
Bremer, A., Henn, C., Engel, A., Baumeister, W., and Aebi, U. (1992).
Has negative staining still a place in biomacromolecular electron
microscopy? Ultramicroscopy 46
Bremer, A., Henn, C., Goldie, K. N., Engel, A., Smith, P. R., and Aebi,
U. (1994). Towards atomic interpretation of 3-D reconstructions
of F-actin filaments. J. Mol. Biol
Brenner, S., and Home, R. W. (1959). A negative staining method for
the high resolution electron microscopy of viruses. Biochim.
Biophys. Acta 34
Fassel, T. A., and Graeser, M. L. (1997). Uranyl acetate as a primary
fixative for skeletal muscle. Microsc. Res. Tech
Harris, J. R. (1999). Negative staining of thinly spread biological particulates. Methods Mol. Biol
Horne, R. W. (1991). Early developments in the negative staining
technique for electron microscopy. Micron Microsc. Acta 22
Oliver, R. M. (1973). Negative stain electron microscopy of protein
macromolecules. Methods Enzymol
Shayakhmetov, D. M., Papayannopoulou, T., Stamatoyannopoulos,
G., and Lieber, A. (2000). Efficient gene transfer into human
CD34+ cells by a retargeted adenovirus vector. J. Virol
Tracz, E., Dickson, D. W., Hainfeld, J. F., and Ksiezak-Reding, H.
(1997). Paired helical filaments in corticobasal degeneration: The
fine fibrillary structure with NanoVan. Brain Res