Silver Impregnation

Please read Ammoniacal silver solutions and Safe working with silver.

Although staining with dyes is the most common way to make tissue structures identifiable microscopically, it is not the only way this may be done. Depositing metals onto or into structures is also very common. Silver is the metal most commonly employed, although gold, iron and copper have also been used. In spite of being a standard practice, it is still not completely understood in some respects. Differing silver impregnations depend on differing processes, and some of these still appear to be somewhat arbitrary, while others can be almost fully understood. To illustrate, the precipitation of silver onto aldehydes by reduction from a methenamine silver solution (Gomori's or Jones' methods) is fairly easy to comprehend, but the impregnation of the various cells and their processes in brain is still largely a mystery.

Silver impregnation is often spoken of as if it were a single, unvarying process, i.e. depending on a single chemical basis which underlies all the different impregnation procedures, but that is probably not the case. The various methods for different structures may manipulate silver deposition by different mechanisms and be based on different chemical underpinnings. The only general underlying principle is that finely divided silver is deposited and appears as dark brown or black deposits.

When discussing silver impregnations, it is usual to refer to structures and substances as being argentaffin or argyrophil. Silver deposition is explained as being argentaffin if the tissue structure causes silver to be deposited without using a chemical reducing agent, and argyrophil if a chemical reducing agent is used. Thus, melanin impregnated from an ammoniacal silver solution is said to be argentaffin and, using the same silver solution in conjunction with dilute formalin to impregnate reticulin, would be said to be argyrophil. There is nothing incorrect about this, although it is of limited usefulness in practice. To these two we should, perhaps, add a third, "induced argentaffin", i.e. the chemical production of an argentaffin material which is then used to reduce silver compounds and blacken structures without using a supplementary reducing agent. The previously mentioned Gomori and Jones procedures are examples of this, both of which use an oxidising agent (chromic and periodic acids respectively) to produce aldehydes which then reduce ammoniacal or methenamine silver solutions without any additional chemical reducing agents.

Discussions on silver impregnation often also include mention of reagents explained as similar to mordants or accentuators in dye staining, referring to them by the names of "sensitisers" and "accelerators", although the distinction between the two is not made clear. The fact is that the role played by many of these compounds is not fully understood and the mechanism of how they assist in selective deposition of silver is really not known. An example of a sensitiser would be the use of aqueous ferric ammonium sulphate (iron alum) to treat sections being stained by the Gordon and Sweet method for reticulin just prior to treating with the ammoniacal silver solution. In other methods an aqueous silver nitrate solution may be used for the same purpose. It is more difficult to give an example of an accelerator. Perhaps the clearest would be the use of pyridine in some methods for neuroglia. It is difficult, of course, to satisfactorily define a substance when its function is obscure.

Argentaffin
Silver diaminohydroxide is easily reduced and can be used to detect reducing substances in sections since silver is deposited where it is reduced. This is the basis for the standard Masson-Fontana method for melanin, enterochromaffin and lipofuscins, although other substances may also be demonstrated. Drury and Wallington point to the presence of tyrosine and phenolic compounds as being responsible for this, but the explanation is vague. Methenamine silver solutions, described in the next paragraph, may also be used for these substances.

Induced argentaffin
Oxidation of sections with periodic or chromic acids will produce aldehydes, which are also reducing substances and will also cause the precipitation of silver at their location. In their case, however, the unstable silver solution is usually made with methenamine, forming a similar compound to that with ammonium hydroxide. Methenamine is also known as hexamine or hexamethylenetetramine. The silver solution it forms is used at mildly alkaline pH with a borate buffer or borax. This is because the silver reduction takes some time, often longer than an hour, and may involve an increase in temperature. Strongly alkaline solutions coupled with increased temperatures are both factors which cause sections to detach from glass slides. Methenamine silver solutions are less alkaline than ammoniacal silver solutions and are less likely to cause sections to detach, even at higher temperatures. Even so, some methods use an ammoniacal silver solution for these extended reductions with satisfactory results.

So far the explanations for depositing silver on reducing agents, including generated aldehydes, has been straightforward and is fairly well understood. It is a simple oxidation-reduction reaction involving an unstable silver solution. The compound in the silver solution is reduced to metallic silver, or some other insoluble silver compound, and deposited on the reducing agent in very finely divided form. Finely divided silver appears black or dark brown and that is what we see. However, there is a possibility that the silver deposited is something other than metallic silver itself. Some kind of oxide has been suggested, although evidence as to its exact nature is lacking.

Argyrophil
The remaining silver impregnation techniques are less well understood. These are the various methods for demonstrating reticulin, and those techniques used for neurological elements such as nerve cells and their processes and the various glia cells and processes.

For some time the selectivity of silver impregnations for reticulin was explained as being due to production of aldehydes in the reticulin fibres by permanganic acid treatment, also known as a Mallory bleach, which is a common step prior to applying the sensitiser and silver solution. However, many impregnations are successful, more or less, even when this step is omitted or if an aldehyde block is applied after the Mallory bleach and before impregnation, casting doubt on it as the explanation. The Mallory bleach can either enhance or reduce staining and impregnation intensity depending on whether the mild oxidation frees up staining and impregnation sites, or blocks them. Some are of the opinion that the Mallory bleach reduces deposition of silver onto background structures, making reticulin stand out due to higher contrast. It is not clear what material in tissues is being oxidised by permanganic acid and what its actual role in metallic impregnations is.

To some extent the explanations for the observed selectivity of silver impregnations for reticulin are speculative, and a full explanation of the chemical interactions taking place is lacking. The following should be understood within that context.

The most commonly used methods for reticulin impregnation follow the pattern below, or something similar:–

  1. Mild oxidation of the tissue, often with permanganic acid made by mixing potassium permanganate and sulphuric acid, or with potassium permanganate alone. It is possible that this oxidation step produces binding sites on the fibre and makes the reticulin fibres more reactive to the sensitiser. That is not a very satisfactory statement and makes the explanation somewhat ill defined, but it is not possible to be more definitive that that. It is also possible that this oxidation decreases binding sites on tissues other than reticulin and, as a consequence, also decreases the amount they reduce the silver solution, causing the impregnation of reticulin to have greater contrast in comparison. It could also be that both processes are taking place at the same time.
  2. Bleaching the discolouration produced by the permanganate, usually with an aqueous solution of oxalic acid, which is also a mild oxidising agent. It should be noted, however, that there is no suggestion that the oxalic acid further oxidises tissue components and it is applied solely to remove the brown discolouration produced by the permanganate.
  3. Treating with a "sensitiser". Often this is iron alum (ferric ammonium sulphate), although other sensitisers, including silver nitrate, have also been used. It is not clear what happens, but the commonest explanation is that the (ferric) iron combines with binding sites freed up by the permanganate oxidation and attaches to the reticulin fibre predominantly, with much lower amounts attaching to other components. This iron alum is then washed off, but only for long enough to remove the excess. Too much washing removes it from the reticulin fibres and results in a poorer quality impregnation.
  4. Applying ammoniacal silver solution for a short time, usually about thirty seconds. It is thought that the silver compound attaches to the reticulin fibres in both a focal and non-specific fashion. The focal attachment is to the iron bound to the fibres, and the non-specific attachment is to the reticulin fibres in general, although the specifics are never spelled out. There is also some non-specific attachment to the tissue in general. The excess ammoniacal silver solution is then rinsed off sufficiently to ensure there is no free silver compound remaining and the only silver salts present are bound to the tissue, but not for long enough to remove any of the bound silver salts. At this stage most of the silver compound is not reduced, but isolated foci of reduced silver may be present, perhaps at the locations where the iron attached during sensitising. At this stage the tissues often have a very pale, light brown, slightly translucent appearance.
  5. Reducing with a chemical reducing agent, usually 10% formalin in tap water. It is important that formal saline or neutral buffered formalin not be used as the buffer salts and sodium chloride interfere with the reduction. Plain formalin diluted with water is needed. This chemically reduces the silver attached to the tissue. Since most of it is attached to reticulin fibres, these are darkly impregnated on a much paler background. It is thought that the silver attached focally in association with the iron may act as nodes for the reduction in much the same way as light sensitised silver halide atoms in photographic film act as nodes for reduction of the whole silver halide grain during development with photographic developers, which are chemical reducing agents.
  6. Following chemical reduction with formalin, excess chemicals are removed and the impregnation stabilised with water washes or treatment with sodium thiosulphate.

Clearly, this explanation is not satisfactory but, compared to the explanation for the impregnation of nervous tissue elements, it is quite informative! Impregnations for nerve fibres and glia cells are largely empirical. They work, often quite well, but the chemical basis for them is not understood. There is no point having a detailed description of the processes as they are largely still a mystery. Generally, a close adherence to the published method for each technique is strongly recommended and will give the most reliable results. Even so, sometimes the impregnations fail for no apparent reason, and experience often plays an important role.

As well as for the argentaffin materials previously mentioned, silver impregnation may also be used for tissue elements other than reticulin and nervous tissue, amyloid and spirochaetes being two examples. Very little is known about the chemical basis for these, either. It is not even known whether there are similarities in the chemical underpinnings for each of the impregnatable structures, let alone what those underpinnings are. All we can really say with certainty is that after some chemical treatment an unspecified material in each impregnatable structure will do one of the following:-

  1. Reduce an unstable silver compound to metallic silver or some other silver compound which is deposited in finely divided form within or on the structure,
  2. Preferentially combine with the ammoniacal silver solution and then be reduced by an external reducing agent to metallic silver or some other silver compound which is deposited in finely divided form within or on the structure,
  3. A combination of both.

Toning
Many silver impregnations produce dark brown structures on a lighter brown background rather than black on a grey background. This is often all that is required and in many cases identification of the structure involved is quite clear. However, some microscopists prefer a pure black deposit on a grey background. The process known as "toning" brings this about. Toning involves treating an impregnated section with what we usually call "gold chloride". This is also known as "yellow gold chloride" or sodium gold chloride (NaAuCl4), and is the sodium salt of chloroauric acid (HAuCl4). Chloroauric acid itself is called "brown gold chloride" and is not used for toning, but may be required for some other impregnation techniques.The gold chloride used for histological toning is not the same as auric chloride (Au2Cl6), which is not used in histotechnology.

The process of toning involves the replacement of some of the silver atoms with gold atoms. Finely divided gold generally appears purple. A purple overlay on a brown object makes it look black, and that is simply what toning does. Usually this takes from a few seconds (reticulin) to a few minutes (some brain cells). If applied for a long time, the colour change goes beyond black on grey and the deposited silver becomes purple. There is no right or wrong about toning, it is simply the application of a gold chloride solution until the colours of the impregnated structures are the colour the microscopist prefers. There is no change to what structures are demonstrated, only a change in colour and, perhaps, contrast.

Fixing
In order to reduce the likelihood of non-specific deposition of residual silver and gold onto impregnated sections, it is customary to treat them with reagents which remove unreduced salts from the tissue. The reagent used is invariably a 2-5% solution of sodium thiosulphate. This is often called "hypo", a term derived from an old name for it (sodium hyposulphite), and it is the same chemical used in silver based photography for stabilising or "fixing" the image. Its effectiveness is based on the simple removal of unreduced silver compounds by dissolving them into the thiosulphate solution. The sections are then thoroughly washed to remove all traces of sodium thiosulphate so as to avoid any possibility of dissolving away silver or gold deposited onto tissue structures, thereby bleaching or fading the impregnation.

 

Reference

Drury, R A, and Wallington, E A, (1967).
Carleton's histological technique., Ed. 5., p. 110.
Oxford University Press, London, England.

Wallington, E A,, (1965),
The explosive properties of ammoniacal-silver solutions.,
J Med Lab Technol, v 22, page 220-3.

Wikipedia articles
Gold chloride Chloroauric acid
 


 

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