MICROWAVE MODIFICATION OF
BIELSCHOWSKYS METHOD FOR NERVE FIBERS
FIXATION:
10% buffered neutral formalin.
TECHNIQUE:
Paraffin sections cut at 8 um.
SOLUTIONS:
1.0% Silver Nitrate Solution
Silver nitrate ---------------------------------------------------------------------Distilled water --------------------------------------------------------------------
0.4 gm
40.0 ml
Prepare fresh.
5% Silver Nitrate Solution
Silver nitrate ---------------------------------------------------------------------Distilled water --------------------------------------------------------------------
0.1 gm
2.0 ml
Store in a refrigerator at 3-6O C.
10% Nitric Acid Solution
Nitric acid, approximately 70% -------------------------------------------------Distilled water --------------------------------------------------------------------
1.0 ml
9.0 ml
Prepare fresh.
Developer Solution
Formaldehyde, 37-40%, reagent grade ------------------------------------------0.4 ml
Distilled water -------------------------------------------------------------------4.0 ml
Citric acid -----------------------------------------------------------------------0.2 gm
10% Nitric acid ------------------------------------------------------------------0.1 ml
Prepare fresh.
1% Ammonium Hydroxide Solution
Ammonium hydroxide, 28% --------------------------------------------------Distilled water -------------------------------------------------------------------2% Sodium Thiosulfate Solution
-1.0 ml
99.0 ml
Sodium thiosulfate --------------------------------------------------------------Distilled water --------------------------------------------------------------------
2.0 gm
100.0 ml
Staining Procedure:
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
Deparaffinize and hydrate to distilled water.
Place slides in 40 ml of 1.0% silver nitrate solution in a plastic Coplin jar and microwave
at power level 3 (180W) for 1 minute. Dip the slides up and down several times and
allow them to remain in the warm solution (50˚ C) for 15 minutes.
Place slides in distilled water.
Pour the warm 1% silver nitrate used in step 2 in a 125 ml flask. Add 28% ammonium
hydroxide, drop by drop with constant shaking, until the initial precipitate disappears
and the solution turns clear. Then add 5% silver nitrate, drop by drop with constant
shaking, until the solution becomes slightly cloudy.
Pour the ammoniacal silver solution prepared in step 4 into a plastic Coplin jar. Place
slides in this solution and microwave at power level 3 (180W) for 1 minute. Dip the
slides up and down several times and allow them to remain in the warm solution (60˚ C)
for 15 minutes.
Place slides in 1% ammonium hydroxide solution for not more than 20 seconds.
Add three drops of developer to the ammoniacal silver solution used in step 5. Quickly
mix with a glass rod and immediately place the slides in the solution for about 3 minutes
or until the tissue sections turn brown. The solution will turn a greyish color and a
mirror of silver will form on the sides of the Coplin jar and sometimes on the slides, but
not on the tissue sections.
Place slides in 1% ammonium hydroxide solution for not more than 15 seconds.
Rinse in three changes of distilled water.
Wipe off the mirror of silver from both sides of the slides taking care not to damage the
tissue sections.
Place slides in 2% sodium thiosulfate solution for 30 seconds.
Rinse in four changes of distilled water.
Dehydrate in graded alcohols.
Clear in three or four changes of xylene.
Mount with synthetic resin.
Staining Results:
Axones ---------------------------------------------------------------
brown to black (usually sharply
linear)
cytoplasmic neurofibrils ----------------------------------------------brown to black
Neurofibrillary tangles and plaques of Alzheimers disease ------------dark brown or
black
Granulovacuolar bodies ---------------------------------------------black
Neurons and background -------------------------------------------varying shades of brown
Neuromelanin -------------------------------------------------------black
Lipofuscin -----------------------------------------------------------brown or black
Comment:
This staining method differs from Bielschowskys method in that it requires much less silver
nitrate. In Bielschowskys method at least 8.0 gm of silver nitrate is used as compared to only 0.5
gm of silver nitrate in the described method.
We have found that neurofibrillary tangles and plaques of Alzheimers disease stain better with
the described procedure than they do with Bielschowskys method. Our method is also less time
consuming requiring only about 40 minutes to perform.
When solutions are heated with microwave irradiation there can be up to 15˚ C difference in
temperature between the top and bottom portion of the solutions. Therefore, in order to equalize
the temperature of the solutions the slides are dipped up and down in steps 2 and 5 of the staining
procedure. This assures uniformity of staining results.
It is essential to use acid cleaned glassware, rinsed in double distilled water.
The shelf-life of silver nitrate can be greatly increased by storing it in a refrigerator at 3-6˚ C.
References:
Mallory, F.B.: Pathological Technique. Hafner Publishing Co., New York, 1968, pp. 158-160.
Ketring, J.L. et al.: Modification of the silver impregnation technique of
Bielschowsky for use in glycol methacrylate-embedded brain tissue. Arch. Pathol.
Lab. Med. 113:196-198, 1989.