323
A General-Purpose Method of Silver Staining
By A. PETERS
(Frovijhe Department of Zoology, University of Bristol)
SUMMARY
A method of silver staining for paraffin sections has been described. Sections should
be fixed in either Nonidez fixative, 4 % formaldehyde, or 4 % formaldehyde saturated
with mercuric chloride. The sections are impregnated for 16 hours in 1/20,000 silver
nitrate at pH 8 or 9 and developed in a glycine physical developer after the reducible
silver has been removed with a 2% solution of sodium sulphite.
The effect of pH on impregnation has been described. A spectrum of staining was
obtained in which nerve fibres began to stain appreciably at pH 7, cell nuclei at pH 8,
cell cytoplasm at pH 9, and connective tissue at higher pH values. Therefore, impregnation should be carried out at pH 8 to obtain a good staining of nerve fibres and at
pH 9 if some staining of cell bodies is also required.
I
N recent years, a number of methods for the silver staining of paraffin
sections of nervous tissue have been described. Probably the most important have been those of Holmes (1947), Romanes (1950), and Samuel (19536).
Although these methods vary in detail, they have the common factor that
impregnation is carried out at a controlled pH in a dilute solution of a silver
salt. Holmes and Samuel used silver nitrate and Romanes used silver chloride.
While Holmes and Romanes employed a hydroquinone-sulphite developer,
which reduced the silver taken up by the sections during impregnation,
Samuel removed the reducible silver with a sodium sulphite solution and
developed in a physical developer. Thus, in Samuel's method the silver which
is reduced to produce the final staining picture is derived from the developing,
solution and not from the silver combined with the section during impregnation (see Peters, 19556). By this means the development process can be
controlled to a greater extent than is possible with the chemical developers,
such as hydroquinone-sulphite.
The staining method to be described in this paper has been evolved as a
result of a series of experiments on the mechanism of silver staining (Peters,
1955 a, b, c, and d). Some observations on fixation and the effect of pH of the
impregnating solution will also be considered, because these two factors play
an important part in the production of the final staining picture.
Fixation
Rowe and Hill (1948) considered the use of various fixatives before staining by the method of Holmes (1947). They concluded that Susa, chloral
hydrate, and mercury-formol fixatives gave the best results, but they pointed
out that chloral hydrate fixatives produce severe shrinkage of the tissue.
A series of simple fixing agents, including picric acid, potassium dichromate, chromic acid, 70% alcohol, and formalin were tested. The results showed
[Quarterly Journal of Microscopical Science, Vol. 96, part 3, pp. 323-328, 1955.]
324
Peters—A General-purpose Method of Silver Staining
that of this group, only formalin- and alcohol-fixed tissue gave consistently
good staining results. However, while alcohol-fixed material gave rise to a
good staining picture, the fixation was poor. As a result of these and other
experiments, it was concluded that the best staining was produced after fixation in Nonidez fixative (25 g. of chloral hydrate in 100 ml. of 50% alcohol
(Nonidez, 1939)) in 4% formaldehyde, and in 4% formaldehyde saturated
with mercuric chloride.
While the Nonidez fixative produced some shrinkage of the tissue, it had
the advantage that, after staining, the nerve elements stood out clearly against
the other tissue elements. Formalin produced better fixation than the chloral
hydrate, but the staining of the nerve fibres was not so well differentiated.
The addition of mercuric chloride to the formalin has the effect of increasing
the depth of staining of the nerve fibres and suppressing the staining of the
background, so that the contrast was improved. When mercury-formol is
used, the precipitate which is formed during fixation must be removed from
the tissue with 2% iodine in 70% alcohol before impregnation in the silver
solution. The excess iodine should not be removed from the sections with
sodium thiosulphate, because, unless the thiosulphate is completely removed,
it interferes with the silver staining.
The pH of the impregnating solution
The effect of pH on the staining of the cerebellar region of the rat's brain
was determined over the pH range 4-5 to 11-2. Sections were impregnated in
a 1/20,000 solution of a silver salt for 16 hours at 37° C. A solution of silver
nitrate was buffered at pH 4-5 and 5-6 with sodium acetate / acetic acid buffer
and at pH 7-0, 8-o, and 9 0 with borax / boric acid buffer. At pH 9-4, 9-9, 10-3,
10-7, and 11-2 a 1/20,000 solution of the silver diammine complex was employed and the pH of the solution was controlled by the addition of either
sodium carbonate or ammonia. (Silver nitrate could not be used at these high
pH values because of the formation of silver hydroxide.) The sections were
either developed in 1% hydroquinone / 10% sodium sulphite or in the glycine
physical developer.
After development in the hydroquinone-sulphite developer, there was an
increase in the density of staining from pH 4-5 to 9-0. When the pH was
controlled by the addition of sodium carbonate to the diammine complex,
staining increased from pH 9-4 to n-2, but when the pH was adjusted with
ammonia, there was a fall-off in the intensity of staining with an increase in
the pH. The addition of ammonia probably reduced the ionization of the
diammine complex and thereby suppressed the release of free silver ions
available for staining. Thus, the solution was stabilized, and any tendency for
it to reduce or combine with proteins of the nervous tissue was retarded
(Peters, 1955a). At higher pH, when the greatest concentration of ammonia
was added to the solution, there was the lowest concentration of silver ions
available for staining.
As long as the free silver ion concentration in the impregnating solution was
Peters—A General-purpose Method of Silver Staining
325
constant, the intensity of staining, on development with hydroquinone-sulphite, increased with the pH. As the pH was raised, there was a tendency for
the deposited silver to become coarse, although this factor was not important
until about pH 10-7.
Development with the glycine physical developer showed that with a constant silver ion concentration in the impregnating solution, the formation of
TABLE I
The effect of pH on silver staining
Intensity of staining
pH
Nerve
fibres
Nuclei
Cytoplasm
Connective
tissue
4-S-S-6
+
+
+
+
7'o
++
+
80
+++
++
-j-
-!-
90-99
++
++
+
+++
++
-f
+
++
++
+++
+
+
+++
+ + T-
+ faint; + + distinct;
deep.
103
107
112
-f-
J
L-
Comments
Staining too light to
show details.
Fibres begin to stain.
Staining still very
light.
Fibres
contrasted
against light background.
Quite good staining of
all nerve elements.
Fibre staining rather
coarse.
Cell nuclei appear as
outlines.
Staining
generally coarse.
Few details visible;
staining very coarse
and homogeneous.
silver nuclei increased with an increase in the pH value of impregnation (see
Peters, 1955a).
The effect of pH on the staining of the various nerve elements and connective tissue is shown in table 1. An interesting point brought out by this
table is the spectrum of nerve-element staining which is obtained as the
pH is raised (Silver, 1942). Thus, fibres and nuclei begin to stain at the lower
pH values, while the cytoplasm only begins to stain appreciably at pH 9-0.
Moreover, the specific staining of the cytoplasm persists to a higher pH than
that of the nerve fibres and cell nuclei. Some of the specificity is lost at higher
pH as a result of a staining of the connective tissue.
It can be seen from table 1 that the best pH for impregnation is 8-o to 9-0,
because over this range the deepest staining of the nerve fibres and nuclei
is obtained. In general, the staining of the nerve fibres is deepest at pH 8-o
and there is little staining of the other tissue elements at this pH. At pH 9-0
the staining of the cell nuclei is deeper, but there is some reduction in the
intensity of staining of nerve fibres. However, a more complete general
326
Peters—A General-purpose Method of Silver Staining
staining of the nerve elements results at pH 9-0 than at pH 8-o. At all other
pH values the staining is either too light or too unspecific.
In their methods, Holmes (1947) impregnated at pH 8-5, Romanes (1950)
at pH 9-0, and Samuel (19536) at pH 678.
A possible explanation for the spectrum of staining lies in the physical state
of the proteins at the different ! pH values. Silver (1942) suggested that the
spectrum effect was caused by a variation in charge on the cellular components,
so that parts of the cell stain at different pH values and have an 'optimum
magnitude of charge to absorb the nascent colloidal silver'. Although it is
doubtful if Silver's theory of staining is generally correct (see Holmes, 1947,
and Samuel, 1953a), the pH effect may well be due to a difference in charge
on the proteins at different pH values. A further possibility is that the sites
of formation of the silver nuclei change with pH; this would lead to the
developed silver being deposited at different sites over the pH range. Such a
change in the sites of formation of nuclei could be attributed to a change in the
redox potential of the cell proteins with the pH value (see Peters, 1955a).
Thus, the number of silver nuclei formed at a particular site would depend on
the value of the redox potential at that site.
Method of staining
(1) Fix blocks of tissue in either Nonidez fixative (1939) or 4% formaldehyde or 4% formaldehyde saturated with mercuric chloride.
(2) Mount paraffin sections on slides with albumen, dewax, and take to
water. (If mercury-formalin has been used for fixation, remove the precipitate
from the sections with 2% iodine in 70% alcohol.)
(3) Impregnate sections in the following solution in an incubator for 16 hours:
1 ml. of 1% silver nitrate, 180 ml. of distilled water, and 20 ml. of o-i M
boric acid / borax buffer at pH 8 or 9. The standard buffer solution is
made by mixing solutions of o-i M boric acid and 0 1 M borax until the
required pH, as indicated by a glass electrode, is attained.
Impregnate at 37° C. for material fixed in chloral hydrate and at 56° C. or
370 C. for formalin and mercury-formalin material.
(4) Transfer sections to 2% sodium sulphite for 5 minutes to remove the
reducible silver (Samuel, 1953a). .
(5) Wash in several changes of distilled water.
(6) Develop the sections in the following glycine-containing physical developer until the required depth of staining is attained. Sections should be
examined at intervals during development. The usual time is 2-5 minutes.
glycine 1-25 g.\
I
Na,SO,(anhyd.)
*5 * 2 0 m l .
5% gelatine (powdered B.P.) 25 ml.
distilled water
225 ml./
o-i M citric acid / sodium citrate buffer at pH 6-3 . 20 ml.
1 % silver nitrate solution
.
.
.
.
.
1 ml.
Peters—A General-purpose Method of Silver Staining
327
(7) Wash in running tap water for 10 minutes.
(8) Dehydrate, take through absolute alcohol to xylene, and mount in
Canada balsam.
The stock solution of the developer is quite stable. To prepare this the
glycine and sodium sulphite are dissolved, by warming, in about 100 ml. of
distilled water and the warm gelatine solution is added immediately. The
volume is then made up to 250 ml. The optimum pH for development may
vary slightly with the sample of gelatine; initial tests should be carried out with
citrate buffers over the range pH 6-o to 6-5. However, once the pH value has
been determined for any particular sample of gelatine, no further tests are
necessary. The citrate buffer controls the pH value at the site of development;
on either side of the optimum pH the silver deposition is rather coarse.
In general, no toning is necessary, because the fibres show up black against
a green background and therefore give a good contrast.
The reducible silver may be removed with a citrate buffer at pH 3-2, but
the sodium sulphite is more convenient to prepare. Unless the reducible silver
is removed, the initial development is rapid and tends to be somewhat unspecific.
The temperature of impregnation varies with the fixative and the type of
tissue. For example, formol-fixed rat cerebellum produced the best staining
picture at 56° C. and formol-fixed human cerebellum at 370 C. On the other
hand, material fixed with chloral hydrate never produced specific staining
after impregnation at 560 C.
Whether the sections are impregnated at pH 8 or 9 depends on the nerve
elements that are required in the final staining picture. As has already been
pointed out, at pH 8 the staining of the nerve fibres is deep and that of the
non-nervous elements is light, while at pH 9, although more background is
stained, the nerve-cell bodies stain more intensely.
Deeper staining may be produced by treating the sections with 20% silver
nitrate for 1 hour before impregnation (Holmes, 1947). In the present method
this step is generally unnecessary.
It is recommended that embryonic tissues should be fixed in Nonidez
fixative and dehydrated in the Lang series of alcohols (Lang, 1937).
A less perfect staining of nerve fibres may be achieved by developing the
impregnated sections in one of the following solutions. Here, development
follows step 3 in the above scheme.
(1) hydroquinone.
.
Na2SO3 (anhydrous)
distilled water
.
Warm the solution to 200
(2) chloroquinol .
.
Na2SO3 (anhydrous)
distilled water
.
Use at room temperature.
.
. ig.
.
. 10 g.
.
100 ml.
C. before use.
.
. 1 g.
.
. 4 g.
.
100 ml.
328
Peters—A General-purpose Method of Silver Staining
While the basic method of staining in the above scheme produces good
results, the modifications have been listed because a rigid method of staining
cannot be expected to produce the best possible results with all tissues and
fixatives. Thus, initial trials should be carried out to determine the pH and
temperature of impregnation which gives the best staining with the sections
available.
This method has been used successfully with fish, amphibian, and mammalian tissues, including brain, spinal cord, sciatic nerve, sympathetic ganglia, muscle end-plates, and embryonic material.
Note.—The glycine mentioned in these papers is the compound called
by that name in photography; that is to say, ^>-hydroxyphenylglycine.
I wish to express my sincere thanks to Professor J. E. Harris for his continued interest and advice during the course of this work. The work was
carried out during the tenure of a postgraduate research grant in the University and later a maintenance grant from the Department of Scientific and
Industrial Research.
REFERENCES
HOLMES, W., 1947. In Recent advances in clinical pathology. London (Churchill).
LANG, A. G., 1937. 'The use of M-butyl alcohol in the paraffin method.' Stain Tech., 12,
113-
NONIDEZ, J. F., 1939. 'Studies on the innervation of the heart. I. Distribution of the cardiac
nerves with special reference to the identification of the sympathetic and parasympathetic
postganglionics.' Amer. J. Anat., 65, 361.
PETERS, A., 1955a. 'Experiments on the mechanism of silver staining. I. Impregnation.'
Quart. J. micr. Sci., 96, 84.
•
'9556. 'Experiments on the mechanism of silver staining. II. Development.' Ibid., 96,
103.
J
955C- 'Experiments on the mechanism of silver staining. III. Quantitative studies.'
Ibid., 196, 301.
1955a . 'Experiments on the mechanism of silver staining. IV. Electron microscope
studies.' Ibid., 96, 317.
ROMANES, G. J., 1950. 'The staining of nerve fibres in paraffin sections with silver.' J. Anat.
Lond., 84, 268.
ROWE, M.( and HILL, R. G., 1948. 'The effects of various fixatives on the staining, by Holmes'
method, on axons in the central and peripheral nervous systems.' Bull. Inst. med. Lab.
Tech., 14, No. 4.
SAMUEL, E. P., 1953a. 'The mechanism of silver staining.' J. Anat. Lond., 87, 278.
I953&- 'Towards controllable silver staining.' Anat. Rec, 116, 511.
SILVER,,M. L., 1942. 'Colloidal factors controlling silver staining.' Ibid., 82, 507.