0038-9 153/SS/6302-0097$02.00/0 STAIN TECHNOI~~GY Copyright @ 1988 by The Williams & Wilkim CO.

Vol. 65. No. 2 P d in U.S.A.

PALLADIUM TONING OF SILVER-IMPREGNATED RETICULAR FIBERS

HELEN KRIKEUS AND ALLEN A. SMITH, Widener University, Chester, Pennsylvania 19013

Fajersztajn (1 901) introduced gold toning of silver impregnations of nerve fibers. When Maresch (1905) adapted silver impregnation to the study of reticular fibers, he retained the gold toning procedure.

Amateur photographers printed their own pictures in those days, and they usually toned the prints with gold to prevent fading (Bielschowsky 1902). Gold toning was thus the obvious way to make histological silver impregnations darker and more permanent. Photographic demand made gold chloride as readily available as film and no more costly.

Gold chloride is no longer cheap or widely available. Hoarding has increased the price of gold by an order of magnitude, and the stability of modern photographic papers has narrowed the interest in gold chloride to metallurgists and histologists.

Fajersztajn (1901) did report that platinum toning was as permanent as gold, but he preferred the “Cassius purple” background produced by gold toning. Since modern histologists photograph rather than draw their prepa- rations, the purple background is no longer an advantage.

The price of platinum has risen with the price of gold, but the price of palladium has not. Moreover, the use of palladium salts in industrial catalysts and in the plating of calculator contacts has made them almost as widely available as gold salts. These considerations led to our devising a palladium toning method for silver impregnations (Smith and Krikelis 1986).

Finding that the toning of Holmes’ nerve fiber stain had more stringent requirements than the toning of Fontana’s argentaffin cell stain led us to question the universal applicability of ou r procedure. We therefore tested our palladium toning procedure on a variety of reticulum stains.

MATERIALS AND METHODS A Swiss strain white mouse was killed by cervical dislocation. The spleen

was removed and fixed for 48 hr in neutral buffered formalin (Lillie 1954), washed 24 hr in water, dehydrated in alcohols, cleared in xylene, and em- bedded in Paraplast (Shenvood Medical Industries, St. Louis, MO). Four- micrometer sections were cut on a rotary microtome and attached to albumin- ized slides.

97

Bio

tech

His

toch

em D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

New

cast

le U

pon

Tyn

e on

12/

22/1

4Fo

r pe

rson

al u

se o

nly.

98 STAIN TECHNOLOGY

Paired slides were then stained with Laidlaw’s silver carbonate stain for reticulum (Sheehan and Hrapchak 1980), Snook’s u m y l nitrate-dhmmine silver reticulum stain (Sheehan and Hrapchak 1980), and Gomori and Burt- ner’s ferric chloride-methenamine silver stain (Lillie 1954). We brought the methenamine silver solution to 60 C in a water bath before immersing the slides in it, and we shortened the incubation time to 45 min.

One slide from each pair was toned for 5 min in HAuCls, 0.2% in deionized water (Samuel 1953), the other for 10 min in KnPdCls, 0.05% in 4 N HCI. Both preparations were dehydrated, cleared, and mounted in HSR (Hartman- Leddon Co., Philadelphia, PA). The preparations were photographed on Kodak Plus-X film with a Zeiss Photomicroscope.

RESULTS

The results of palladium toning and gold toning were similar. Laidlaw’s silver carbonate gave the best picture, both visually and photographically (Figs. 1 and 2). The black reticular fibers stood out against their pale back- ground after both toning procedures. The pale purple background produced by gold toning was more pleasing to the eye, but we found that the pale g r a y background produced by palladium toning made photomicrographic negatives that were easier to print.

Snook’s uranyl nitrate-diammine silver stain gave heavy black reticular fibers on a g r a y background with both toning procedures (Figs. 3 and 4).

Gomori and Burtner’s methenamine silver stain gave a high contrast picture with black reticular fibers, darkly stained nuclei, and no hint of cytoplasmic staining (Figs. 5 and 6). We found the black palladium-toned nuclei less distracting than the mixture of black and purple nuclei produced by gold toning.

The timing of the methenamine silver procedure was critical. Reticular fibers were hardly stained after 40 min in the solution; they were well stained after 45 min. After 50 min, the component fibers of the trabeculae could no longer be resolved.

DISCUSSION

A 0.05% solution of potassium hexachloropalladate in 4 N HCI tones Gridley’s reticulum stain as well as gold chloride does (Smith and Krikelis 1986). It is not surprising that palladium is also as effective as gold in toning Snook’s reticulum stain.

The superiority of palladium toning of Gomori and Burtner’s methenamine silver stain for reticular fibers is surprising. The difference between the two toners seems to be that gold readily forms colloids (Lewis et al. 1943) whereas palladium does not (Shvo et al. 1981). Methenamine silver is selective enough to distinguish between two classes of nuclei, leaving much heavier deposits in one than in the other. Gold toning not only preserves this distinction but converts it to a distinction between black deposits of massive gold and purple deposits of colloidal gold. Palladium toning converts all silver deposits to massive palladium black.

Bio

tech

His

toch

em D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

New

cast

le U

pon

Tyn

e on

12/

22/1

4Fo

r pe

rson

al u

se o

nly.

FIG. 1. Mouse spleen stained with Laidlaw's diammine silver carbonate. Cold toning. X 200. FIG. 2. Mouse spleen stained with Laidlaw's diammine silver carbonate. Palladium toning.

FIG. 3. Mouse spleen stained with Snook's uranyl nitratediammine silver hydroxide. Cold

FIG. 4. Mouse spleen stained with Snook's uranyl nitratediammine silver hydroxide. Palla-

FIG. 5. Mouse spleen stained with Gomori and Burtner's femc chloride-methenamine silver.

FIG. 6. Mouse spleen stained with Comori and Burtner's femc chloride-methenamine silver.

x 200.

toning. X 150.

dium toning. x 150.

Gold toning. x 100.

Palladium toning. X 100.

99

Bio

tech

His

toch

em D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

New

cast

le U

pon

Tyn

e on

12/

22/1

4Fo

r pe

rson

al u

se o

nly.

100 STAIN TECHNOLOGY

The purple color that gold imparted to the background of Laidlaw’s reticulum stain was Fajersztajn’s and Bielschowsky’s reason for choosing gold over platinum toning. It does seem to be less tiring to scan sections with a colored background than sections with a gray background. However, the photomicroscope tended to underexpose the gold-toned sections. Photomi- crographic negatives of palladium-toned sections were much more likely to give good prints.

The hydrochloric acid extends the useful life of the palladium toning solution to about six months. Gold chloride already has hydrochloric acid present in the crystals; properly, it is chlorauric acid.

Unlike gold chloride crystals, potassium hexachloropalladate crystals are not hygroscopic. This makes them easier to handle. Potassium hexachloropal- ladate costs per gram about one-half as much as gold chloride, or one-eighth as much per milliliter of toning solution (Aesar 1987, Alfa 1986, Fluka 1986). No toxicity data are available for tetravalent palladium. Since it is readily

reduced to divalent palladium, its toxicity is probably similar-highly toxic by injection but only slightly toxic by mouth (Sax 1979). Palladium ions, like nickel ions, are suspected of being carcinogenic. They do not appear to penetrate the skin. The authors have had skin contact with both potassium hexachloropalladate crystals and the toning solution without noticeable effect.

REFERENCES

Aeror Catalog. 1987. Johnson Matthey Inc., Seabrook, NH. Al/a C-. 1986. Morton Thmkol Inc., Danvers, MA. Bieluhowsky, M. 1902. Die Silberimp-tion der Axencylinder. Neurol. Zent. 21: 579-584. Fajmtajn, J. 1901. Ein neues Silberimpregnationsverfahren als Mittel zur Farbung der Axen-

F l h Catalog. 1986. Fluka Chemical Corp., Ronkonkoma, NY. L i l k , R. D. 1954. Hirtqbothol~gic Technique and Practical Histochemist7, 2nd ed. McCraw-Hill,

New York, NY. Lewis. W. K., Squires. L. and Broughton, G. 1943. Industrial Chonict?y of Colloidal and Amorphous

MclmioLr. MacMilhn, New York. NY. p. 97. Mucsch, R. 1905. Ubcr GitterfaJcrn der Leber und die Verwendbarkeit der Methode Bielschow-

sky^ zur Damellung feinster Bmgewebsfibrillen. Zentralbl. Allg. Pathol. Pathol. Anat. 16: 641449.

cylinder. Neurol. Zcnt. 20: 98-106.

Sunud. E. P. 1953. Gold toning. Stain Technol. 28: 225-229. Sax, N. I. 1979. Dangerouc PrOprttiCs of Industrial Materials, 5th ed. Van Nostrand Reinhold, New

Shechul. D. C. and Hraphak, B. B. 1980. The07 ond Practice of Histotechnology Mosby, St. Louis,

Smith. A. A. and Krikelis, H. 1986. Palladium toning in histology. Proc. Pa. Acad. Sci. 60: 147-

Shvo. J., Thomas, D. W. and Laine, R. M. 1981. Modelling heterogeneous catalysts with

York, NY. p. 883.

MO.

150.

homogeneous catalysts. J. Am. Chem. SOC. 103: 2461-2463.

Bio

tech

His

toch

em D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

New

cast

le U

pon

Tyn

e on

12/

22/1

4Fo

r pe

rson

al u

se o

nly.