In the histology world, the mere mention of a “silver stain” may be the cause of panic and uncertainty with regard to the performance of the stain, and the quality of the final resulting microscope slide. All other special stains, with few exceptions, are relatively easy and straightforward to perform; not so with silver stains.
Silver stains can be categorized into (a) stains to visualize substances, such as calcium, melanin and reticulin and (b) stains for microorganisms, such as fungi and spirochetes. The goal of all silver stains is the same: to get metallic silver to precipitate out at the staining site, and then replace it with gold to provide the final, stabilized, black reaction product.
The specific procedures for these stains are outside of the scope of this blog. However, the following stain procedure outline explains the steps used.
Oxidation enhances subsequent staining by the silver solution. Oxidizers include phosphomolybdic acid, potassium permanganate and periodic acid.
Sensitization usually employs a metal salt to help bind silver from the silver solution. The original sensitizer for Wilder’s silver technique and the Steiner and Steiner stain is 1% uranyl nitrate. However, Margeson and Chapman pioneered the substitution of zinc formalin for the original radioactive uranyl nitrate solution, which is also a strong oxidizer.
Silver impregnation solution contains metallic silver in a solution. The idea is to have the silver carrying solution composed such that the silver ions will move from the solution, bind to the tissue section and then precipitate out in metallic form.
The reduction step in the reaction involves providing electrons, in the form of substances such as hydroquinone and formaldehyde, to chemically make the silver ions precipitate out into visible metallic silver. This allows the structures in question to be visualized in dark black staining.
Toning of the sections in gold chloride is a chemical reaction whereby the metallic silver is replaced by metallic gold, which is very stable and maintains the black color product.
The use of sodium thiosulfate, or “hypo”, helps to remove any unbound silver remaining from the toning reaction. This is followed by counterstaining, usually either with nuclear fast red or fast green, for a proper final color rendition.
Thus, with all of these simple, straight forward steps, what could possibly go wrong? Let’s begin at the beginning and work it through.
Use acid clean glassware for all containers and Coplin jars. Why? This will prevent the presence of any unwanted ions on the glass surfaces (including the slides themselves) causing non- specific precipitation of the silver. You will see this as a black or mirror precipitate on the inside of the Coplin jar, or on the surface of the microscope slide. If this does happen, the unwanted silver can be removed (see procedure at the end of this article).
Use plastic forceps to handle all slides — not metal. Metal present in metal forceps may cause the silver to precipitate out. Back in the old days, before plastic forceps, we simply dipped the tines of metal forceps into paraffin, allowed it to cool, thereby preventing the metal from contacting any of the solutions.
Pay attention to the slides when they are in the silver solution – especially if the solution is a hot, heated solution. The point at which the silver solution itself may precipitate out is a fine one. The slides and staining solution should be monitored closely during these incubations. Also, if you are monitoring the slide under a microscope, make sure to rinse the slide in distilled water before viewing, thereby preventing the foundation of black silver precipitate forming on the microscope stage or slide. Your coworkers especially will appreciate this.
- Sheehan and Hrapchak. Theory and Practice of Histotechnology. Second ed. CV Mosby Co. St. Louis. pp 181-183. 1980.
- Chapman CM. Dermatopathology: A Guide for the Histologist. Copyright 2003.
- Chapman CM and Dimenstein IB. Dermatopathology Laboratory Techniques. Copyright 2015. In press.
- Margeson LM, Chapman CM: The use of zinc formalin as a sensitizer in silver stains for spirochetes. J Histotech, 19:135-138, 1996.
Questions and comments may be directed to:
Clifford M. Chapman, MS, HTL(ASCP), QIHC
Bioscience Solutions Group, LLC
SILVER PRECIPITATE REMOVAL
Sometimes during the course of performing a silver stain (i.e. Fontana-Masson, Warthin-Starry, Modified Steiner, Methenamine silver, reticulum, etc.) the silver solution may precipitate on the surface of the microscope slide in a non-specific manner. This procedure describes a method that can be used to remove non-specific silver precipitate.
Fixation. Formalin fixation; avoid alcohol containing fixatives since alcohol dissolves argentaffin granules.
Sectioning. Cut paraffin sections at 5 microns.
Include a known positive control with each run. Use tissue with argentaffin containing cells, such as stomach, intestine and/or skin.
1% Potassium Ferricyanide
Potassium ferricyanide 1.0 gm
Distilled water 100 ml
5% Sodium Thiosulfate
Sodium thiosulfate 5.0 gm
Distilled water 100.0 ml
Silver Removal Working Solution
1% Potassium ferricyanide 10 ml
5% Sodium thiosulfate 40 ml
CAUTION: Potassium ferricyanide is toxic. Wear safety glasses, chemical resistant gloves, and an apron. Work under a hood. Always pour the sodium thiosulfate into the container first, and then slowly add the potassium ferricyanide.
PROCEDURE: Use chemically clean glassware.
- If a microscope slide is covered with precipitated silver after the silver impregnation step it will appear either as black precipitate, or have a silver mirrored appearance.
- Remove the slide from the silver solution and rinse in distilled water.
- Immerse in the Silver Removal Working Solution for 10 min.
- Rinse in distilled water.
- Return the slide to a fresh preparation of the silver solution from which it came.
- Continue the procedure.
The precipitated silver will be removed by the solution, rendering the slides clear. They can then be stained by the selected procedure.
- Sheehan: Theory and Practice of Histotechnology. Second edition.
15: 276-277. Columbus, Batelle Press, 1980.