Silver Staining - Principles, Procedure and Applications.

1. SILVER STAINING
Principles, Procedure & Applications
Presenter: Moderator
Dr.Varughese George Dr.Garima Anandani

2. Contents
• Introduction
• The Theoretical Basis of Silver Staining Techniques
• Classification of Silver Stains
• Theory and Application of Argentaffin Stains
• Theory and Application of Argyrophil Stains
• Theory and Application of Silver Impregnation Stains
• Theory and Application of Oxidation Reduction Silver Stains
• Theory and Application of Silver Autometallography
• Technical Problems Associated with Silver Stains
• Use of the Microwave for Silver Stains
• Control Tissues for Silver Staining

3. INTRODUCTION
• Introduced by Kerenyi and Gallyas to detect trace amounts of proteins in
gels.
• Selectively alters the appearance of microorganisms , neuroendocrine cells,
basement membrane and pigments in microscopy of histological sections.
• Perfected by Camillo Golgi for the study of the nervous system.

4. Classification of Silver Stains

5. The Theoretical Basis of Silver Staining Techniques
General Aspects
• range in color from brown to gray-black.
• identifes molecules with strong reducing groups (eg, melanin).
• stains carbohydrates and carbohydrate moieties (glycoproteins)
• localizes cytoplasmic and extra-cytoplasmic structures (secretory granules
of neuroendocrine cells)

6. Argentaffin Stains (Silver Reduction Stains)
• Reduces ionic silver when
the silver salt is in a basic
solution results in a
• Deposit of metallic silver at
the site of reduction
Reduction

7. Theory of Argentaffin Stains
• Melanin & certain other compounds have sufficient reducing strength to reduce ionic
silver to metallic silver in a basic solution (ammoniacal silver).
• The excess ionic silver is solubilized by reaction with sodium thiosulfate.
• Other strong reducing groups and molecules that can reduce silver include
– Aldehyde groups
– Lipofuscin
– Hemosiderin
– Melanosis coli pigment
– Ortho & Parapolyphenols,
– Urates & uric acid.
• On longer exposure or under conditions that accelerate chemical reactions, weaker
reducing compounds gradually reduce silver and thus may become more evident :
– substances in the nuclei, such as chromatin
– unidentified factors in red blood cells, in eosinophils, in nucleoli etc.
• Tissues to be stained with argentaffin stains should not be fixed in alcohol containing
fixatives as they solubilize the argentaffin & decrease staining intensity.

8. Staining of the Nucleolar Organizer Region (NOR)
• The genes controlling the production of ribosomes (rDNA) are located in the
nucleolus.
• Non-histone proteins associated with these genes stain with argyrophil like
methods.
• Increased nucleolar activity  increased in silver stained structures in the
nucleus.
• Malignant tumors, poorly differentiated tumors often have greater numbers,
more variation in the numbers, and atypical distributions of NORs.
• Determination of the number of NORs per nucleus may be useful in the diagnosis
of tumors and prediction of the behavior of histologically similar tumors

9. Application of Argentaffin Stains
• Stains melanomas by the Fontana-Masson technique (not specific).
• Stains amelanotic melanomas that cannot be stained by the Fontana-Masson
technique by utilizing acid silver solutions.
• Stains argentaffin secretory granules in midgut carcinoid tumors.
• Stains cells of the adrenal medulla & some paragangliomas.

10. Fontana-Masson method for melanin
• Ammoniacal silver solution is used to treat tissue sections.
• The argentaffin substances within the tissue not only bind the silver
ions in the solution, but also reduce them to metallic silver.
• Gold chloride is used to tone the metallic silver from brown to black.
• Nuclear fast red is the counterstain of choice for this stain

11. Fontana-Masson method for melanin
Melanin pigment in cells of malignant melanoma, Fontana-Masson stain
Melanin : black
Nuclei : red

12. Fontana-Masson method for neuroendocrine tumors
Neoplastic cells demonstrating positive reaction by Fontana–Masson
stain (A) with susceptibility to bleaching (B).

13. Fontana-Masson method for Cryptococcus
Numerous FMS-positive yeast are seen in this case of
cryptococcosis caused by C neoformans

14. Fontana-Masson method for Blastomycosis
Without the diagnostic broad-based budding (center) seen
in this case of blastomycosis, this FMS-positive yeast could
easily be confused with C neoformans

15. Problems Associated with Fontana-Masson & Other Argentaffin Stains
1. A positive reaction is not specific for melanin.
2. Formalin & glutaraldehyde will react with argentaffin stains as well as other
molecules like enterochromaffin cells, adrenal medullary cells & protein molecules in
which fixation with glutaraldehyde has added aldehyde groups without forming a
methylene bridge group.
3. Endogenous pigments, especially brown-black pigments could give incorrect
interpretation.
4. Some procedures require exposure of the sections to the alkaline silver solution for
up to 24 hrs - sections may separate from the glass slides.
5. Glassware used in staining procedures must be free of contamination, particularly
protein residue and chloride ions, that may precipitate the silver.
6. Dissolution of argentaffin molecules by organic solvents, especially alcohol, before
additive crosslinking fixation may give false-negative results.

16. Advances in Argentaffin Stains
• Studies performed by Staples and Clark indicated that reducing the concentration
of silver nitrate to 1% will produce staining equal to that achieved with the
original Fontana-Masson technique.
• There are less precipitate forms & less chance of section loss caused by excess
ammonium hydroxide in the silver solution.
• Multiple antibodies like HMB45 and S100 can be used in conjunction with
immunohistochemistry to aid in the identification of melanomas and carcinoids.
• Many carcinoids stain with antibodies specific for S100, neuron specific enolase
• (NSE), chromogranin A, synaptophysin & neurofilaments.

17. Argyrophil Stains (Silver Absorption Stains)
• The silver ions (Ag'), once absorbed,
must be converted to elemental
silver by exogenously added reducing
agents.
• Depends upon weak physiochemical
interactions of tissue structures with
either basic or acidic silver solutions.
• Rapidly absorb silver ions from
solutions of silver salts.
• Proteins rich in histidine are involved
in this absorption.

18. Theory of Argyrophil Stains
• Specific cells with peptide-containing granules are stained by argyrophil methods,
including the Grimelius & related stains.
• Most of the granules that absorb silver are contained in cells that are postulated
to arise embryologically from the neural crest area.
• Most of these cells have the ability to take up precursors of amino acids, such as
tyrosine, and to remove the carboxyl groups from these molecules to form
amines.
• The system was initially designated as the Amine Precursor Uptake and
Decarboxylase (APUD) system.
• Used to identify subpopulations of cells of the Dispersed Neuroendocrine System
(DNS) which replaces the APUD system.

19. Theory of Gremelius Stains
• The probable mechanism of argyrophil staining is the rapid absorption of silver
salts by certain types of neurosecretory granules.
• The non-absorbed silver is washed from the specimen.
• The absorbed silver can be "fixed" in place by reducing the absorbed silver ion
(Ag+
) to elemental silver (Ag° ) with an exogenously added reducing agent.
• The reducing solution for Grimelius stain is the hydroquinone-sodium sulfite
solution (Bodian reducer)  reduction fixes the silver in its elemental form to
those structures that have absorbed the colorless silver ions and produces a
brown to black color.
• All argentaffin cells will stain by argyrophil procedures because the silver ions are
reduced and deposited as elemental silver at endogenous reducing (argentaffin)
sites.

20. Related Stains for Neuroendocrine Tumors
• Cells of the adrenal medulla & the neuroendocrine cells of the midgut
(enterochromaffin cells), turn brown when exposed in the fresh state to
potassium dichromate, chromic acid, or iodates.
• Giemsa method can be used to stain "chromaffin" granules a greenish-yellow
color
(The chromaffin reaction is thought to be associated with indoleamines)
• Antibody that aids in the identification of neuroendocrine and neuroectodermal
tissues is NSE, confirmed by chromogranin A or B & synaptophysin.

21. Grimelius stain for neuroendocrine tumors
The neuroendocrine nature of the tumor can be identified using silverstains
such as grimelius stain.

22. Problems with Neuroendocrine Argyrophil Staining
• Suffers from the same problems as those listed under the general discussion of
argentaffin- argyrophil reactions.
• Staples and Grizzle have developed a rapid argyrophil procedure (RAP) that
avoids many of the problems cited in prior argyrophil methods.
• Its strength as a screening test for neuroendocrine cells becomes a weakness in
investigations of specific substances such as the ACTH and peptides where
imuunological methods should be used.

23. Advances in Argyrophil Stains
• Staples and Grizzle have recommended changes to the argyrophil techniques used
to demonstrate secretory granules, eg, the Grimelius procedure.
• Changes are based upon detailed studies of the effects of various variables on the
argyrophil reaction.
• The temperature of the solutions, rather than the concentration or duration of
impregnation, primarily controls the reaction in the section.

24. Warthin-Starry Method
• The Warthin-Starry method is used primarily for the demonstration of
spirochetes,
• Principle : bacteria in general and spirochetes in particular have the ability to bind
silver ions.
• Involves impregnation of the spirochetes in the tissue with silver ions, with
subsequent reduction of these ions to metallic silver using a developer containing
hydroquinone.
• The stain demonstrates black spirochetes against a yellow to pale brown
background.
• The spiral morphology of this form of bacteria can be fully appreciated by the use
of this method

25. Warthin-Starry method for spirochetes
Spirochetes : black
Background : golden-yellow
Clusters of spirochetes (arrow) shown on Warthin–Starry stain.

26. Impregnation Silver Stains
• Stains used to impregnate neurites
• Type I (Bodian Stain)
– rely on a micro-argentaffin reaction to
establish submicroscopic foci, or seeds, of
silver followed by continued absorption of
silver on to these seeds
• Type II (Golgi Stain)
– rely on the localized absorption of anions,
such as chromate, that form relatively
insoluble complexes with added silver ions
– The anion (eg, chromate) impregnates the
tissue, and silver is used to aid in
visualizing the reaction by forming
insoluble complexes.
– The silver ions in these complexes are then
reduced to metallic silver by an
exogenously applied reducing agent.

27. Theory and Application of Silver Impregnation Stains
Demonstration of Features of the Nervous System
• Absorption of silver-protein complexes or silver ions under various
conditions has been used to aid in the visualization of neurites as well as
the cell bodies of neurons.
• Used to emphasize pathologic processes such as Alzheimer's disease in
which neurites form distinctive structures known as senile plaques and
neurofibrillary tangles.

28. Theory and Application of Silver Impregnation Stains
• The Bodian stain and its modifications rely on the deposition of silver grains on neurofibrils and
neurofilaments by a silver-protein complex in the presence of metallic copper.
• During the deposition of the silver seeds, cuprous ions are produced that further interact with the
neurofilaments and silver and cause further deposition of silver.
• This initial impregnation step is followed by a reduction step during which the silver seeds are
enlarged with a reducing agent such as hydroquinone-sodium sulfite in an alkaline environment
that reduces silver ions to metallic silver.
• This reduction occurs preferentially at the sites of the silver seeds previously established by the
impregnation step.
• During subsequent gold toning, the initial sites of silver deposition are further enlarged as metallic
silver reduces ionic gold to metallic gold.
• The silver chloride precipitated can be subsequently reduced to metallic silver by oxalic acid.

29. Specific Structures
• The Bodian and Bielschowsky procedures, as well as subsequent modifications
such as the Sevier- Munger technique, can be used to demonstrate axons.
• Silver nitrate impregnation solution and a developing solution containing gum
mastic and silver nitrate reduce background staining and reliably stain dendrites
as well as axons.
• Stains neuropathologic senile plaques and neurofibrillary tangles found in the
brain, especially in the hippocampal area in dementia of Alzheimer's type.

30. Bielschowsky technique
• Tissue sections impregnated with
a 20% silver nitrate solution and
then treated with an ammoniacal
silver solution to which
formaldehyde has been added.
• Nerve endings, neurofibrils,
neurofibrillary tangles, and
neuritic plaques are all stained
black. Bielschowsky silver stain showing the
processes of basket cells in the cerebellum.

31. Problems associated with Impregnation Procedures
1. Techniques for detection of spirochetes often employ pyridine, a highly toxic carcinogen.
2. Substances that are argentaffin and argyrophil positive will stain with silver impregnation
procedures.
3. Control tissues are difficult to obtain for some procedures.
4. Citrus replacements for xylene may cause leaching of silver after adequate staining has
been obtained
5. Impregnation methods require that the sections to be stained be of different thicknesses,
depending on what is to be demonstrated.
e.g, methods to demonstrate neurofibrillary tangles and senile plaques in brain specimens
require consistent thicker sections (>10 pm), whereas methods to demonstrate bacteria
require thin sections (3 pm or less) to reduce background staining.
1. Problems inherent to silver procedures.

32. Advances in Impregnation Procedures
• Reduced time required for silver staining - usually accomplished by rapidly
increasing the temperatures of solutions using microwave techniques.
• Decreased concentration of the silver solutions used, reduced costs and
background staining.
• increased sensitivity of silver staining methods.
• Murdock and Fratkin have utilized a 10% silver nitrate- 4% gelatin solution for
selective impregnation and demonstration of senile plaques and neurofibrillary
tangles.
• Garvey reported good results with a 0.5% solution of ammoniacal silver nitrate
with gum mastic-hydroquinone as the reducing solution.

33. Silver Oxidation-Reduction Stains or
Combination Stains
• rely on the production of reducing
sites (typically aldehyde groups) by
treatment with strong oxidizing
agents.
• The extent of oxidation is determined
by the exposure, time and strength of
the oxidizing agent, causing
compounds to form varying numbers
of reducing sites.
• Examples -
– Grocott modification of the Gomori
methenamine-silver (GMS) for fungus,.
– the Jones stain for demonstration of
basement membranes.
– Gomori reticulum stain

34. Theory and Application of Specific Stains: Oxidation
Reduction Silver Stains (Combination Silver Stains)
• Macromolecular carbohydrates and glycoproteins are oxidized to convert the chitin or cellulose
polysaccharides in the fungal walls to aldehydes.
• Chromic acid and other strong oxidizers used to demonstrate fungal walls.
• Periodic acid and potassium permanganate demonstrate reticulin fibers and basement membranes.
• In stains used to demonstrate fungi, strong oxidation reduces background staining of substances
containing fewer carbohydrate components because some aldehyde molecules are oxidized to carboxyl
groups that are weaker reducing agents than aldehyde groups.
• Insufficient oxidation causes reticulin fibers and basement membranes to stain strongly and, thus,
increases the background staining of fungal stains.
• Aldehyde groups that can reduce ionic silver to elemental silver are formed from adjacent hydroxyl
(glycol) groups at, prior to oxidation, have little reducing activity.
• This reaction produces reducing activity in the sugar moieties of complex molecules, such as glycoprotein
in basement membranes, just as in chitin or cellulose molecules in fungal walls.

35. Theory and Application of Specific Stains: Oxidation
Reduction Silver Stains (Combination Silver Stains)
• Many of the combination stains may not completely destroy all the endogenous
reducing groups so that these methods may partially stain cells with argentaffin
activity.
• Combination stains should not be used to stain cells with argentaffin or argyrophil
activities because these activities will be at least partially destroyed by the
oxidation step.
• Using PAS and combination silver stains is advantageous to block free aldehyde
groups before the oxidation step that creates new aldehyde groups.
• Aldehyde blocking reagents include sodium thiosemicarbazides & borohydrides.
• Another approach is to use nonoxidized adjacent sections as controls to identify
argentaffin sites.

36. Fungal Stains Based on the Oxidation-Reduction Silver Stains
• Stains used to screen for fungi, Pneumocystis, and other organisms should
permit rapid, easy decisions as to whether or not organisms are present in
tissue.
• This requires a stain with good contrast, ie, strong staining of the
organism with a clean background.
• Grocott's modified Gomori Methenamine-Silver stain (GMS) uses a
strong oxidizer (chromic acid) to accomplish this.

37. Problems with the GMS and Related Stains
1. Non-specific staining
– Proper oxidation must be used to reduce the staining of reticulum and
other carbohydrate moieties that have a weaker reaction with silver than
do oxidized fungal walls.
– Underoxidation will not generate adequate aldehyde formation to
produce adequate staining.
1. Overstaining produces loss of detail and non-specific silver
deposition.
2. Understaining may lead to a misdiagnosis when only rare
organisms are present
– e.g Histoplasmosis
– When the tissue is understained, few organisms may not be visualized,
resulting in a false-negative diagnosis.

38. Grocott Methenamine (hexamine)- Silver for fungi and
Pneumocystis spp. organisms
• The cellular walls of fungi are very thick and contain much more
carbohydrate.
• Chromic acid creates dialdehyde groups from the carbohydrates of the
fungi cell walls with overoxidation and destroys the carbohydrate
structures in the tissue section.
• It utilizes a light green counterstain, resulting in fungus cell walls that are
various shades of black to taupe in a light green background

39. Grocott Methenamine (Hexamine)- Silver method for fungi and
Pneumocystis spp. organisms
GMS stain for Pneumocystis jiroveciGMS stain for Cryptococcus neoformans

40. Advances in GMS and Related Stains
• Churukian and Schenk have developed an oxidation- reduction silver
staining method that utilizes microwave heating and can be applied to
unprocessed and unfixed specimens.
• This method is based on a 5% periodic acid oxidizing solution followed by
an ammoniacal lithium carbonate silver solution.
• It also can be used on deparaffinized sections, but the tissue adheres
better to slides if 5% chromic acid is substituted for 5% periodic acid.
• Helps in quick diagnosis of fungal and Przeumocystis carinii infections.

41. Reticulin Stains
• Fine. delicate fibers that make up the bulk of the supporting framework of the
liver, spleen, and lymph nodes.
• Stains to demonstrate reticulin fibers can be used to emphasize the pattern of
reticulum of certain tumors.
• Certain glands (eg, pituitary) and tissues (eg, liver) normally have a definite pattern
after reticulum staining.
• Disruption of this pattern suggests an abnormal proliferation of cells (ie, a tumor
or adenoma) or characteristic inflammatory destruction.

42. Theory of Reticulin Stains
• Young collagen fibers or small collagen bundles together compose a structure known
as reticulum are oxidized by permanganate.
• The fibers contain a carbohydrate component that can be oxidized to aldehyde groups.
• The excess permanganate is removed by oxalic acid or potassium metabisulfite - this
control the extent of oxidation.
• Ferric ammonium sulfate is then used to sensitize tissue for reaction with the
ammoniacal silver solution.
• The silver solution at a basic pH is reduced to elemental silver by the interaction with
aldehyde groups, and elemental gold replaces this elemental silver in the toning
reaction.
• The non-reacted silver and gold are removed with sodium thiosulfate.

43. Gomori’s method for reticulin fibers
• Glycols are first reduced to dialdehydes by the use of an acid.
• The tissue is next sensitized to accept metallic silver ions with an
ammoniacal silver solution.
• The silver ions that are attached on and around the dialdehyde groups are
then reduced to metallic silver.
• Finally, the tissue sections are toned from brown to black by replacing the
metallic silver with metallic gold.
• sodium thiosulfate is used to remove any remaining unreacted silver in
the tissue to prevent darkening of the slide over time.
• The end result is that reticulin fibers are stained black against a clear
background.

44. Gomorri's Silver Impregnation Staining Technique for Reticulin Fibers
Reticular fibres : Black or dark brown
Nucleus : Grey
Other tissues : According to counterstain

45. Problems Associated with Reticulin Stains
• Background staining in tissues with high carbohydrate content may mask the
pattern of the reticulin fibers.
• Background staining may be increased by staining of glycogen, especially in liver
sections controlled by diastase digestion of liver sections prior to reticulum
staining.
• Other problems typical of silver procedures may occur.

46. Advances in Reticulin Stains
• Staples and Clark have reported that 1 % silver nitrate can be used to replace the
10% silver nitrate in the preparation of the ammoniacal silver solution for
reticulum procedures.
• This method yields sections with little of the background staining usually
associated with the reticulum procedure.
• The reticular fibers appear to be more sharply delineated from the rest of the
tissue section.
• A rapid microwave method to demonstrate reticulin fibres in plastic sections has
also been developed by Leong and Pulbrook.

47. Basement Membranes and the Jones Periodic
Acid-Methenamine (PAM) Silver Stain
• This stain is used to demonstrate basement membranes are most commonly used to
investigate glomerular lesions in the kidney.
• Deposits of immunoglobulin that may disrupt the basement membranes of endothelial
cells also cause puncture "holes" or other "non-staining regions" in basement
membranes when examined with silver stains.
• immunoglobulin deposits such as IgA may apparent splitting of basement membranes.
• Carbohydrate moieties of the basement membrane are oxidized to aldehydes
• by the periodic acid solution.
• Aldehyde groups reduce the methenamine silver to elemental silver at the basement
membrane.
• Excess silver is removed by a reaction with sodium thiosulfate.

48. Periodic acid-methenamine silver microwave method
for basement membranes
The stain uses methenamine to form a complex with silver,
which is then reacted with dialdehyde groups formed by the
reduction of glycol units in the basement membrane.

49. Periodic acid-methenamine silver microwave method
for basement membranes

50. Advances in Silver Staining of
Basement Membranes
• Most advances in staining methods specific for basement membranes involve
speeding up the staining process with a microwave oven while attempting to
control the consistency of these rapid methods.
• Most modifications are based on the Jones technique.
• The methenamine silver solution is stable under short term microwave heating
• although some microwave methods have replaced methenamine silver with
ammoniacal silver.
• Any problems due to inadequate washing of slides after oxidation or inadequate
cleaning of glassware are magnified in microwave staining.

51. Metallic-Metallic Interactions with Silver
(Silver Autometallography)
Immunogold method
• used to localize antigens in tissue.
• relies on the deposition of gold particles
to detect sites of antibody-antigen
interaction.
• tiny (5-20 nm in diameter) gold particles
are conjugated to antibodies in the place
of labeling enzyme (peroxidase /alkaline
phosphatase) or are incorporated into
avidin-biotin complexes.
• Intensification with silver results in the
deposition of a shell of metallic silver at
the sites of gold particles.
• used to amplify deposits of metallic silver
and compounds of mercury or zinc
present in living tissues.

52. Gold Toning Enhancement of Silver Stains
• Used to modify the color of silver deposits.
• In a solution containing Cl ions, each Ag ion formed immediately precipitates as AgCl at
the site at which the Ag+
ion is produced.
• AgCl is a very insoluble complex that "coats" the sites where the silver has been
deposited  whitish appearance.
• If gold chloride is used to tone sections, a complex of Au° and AgCl marks the sites
where Ag+
-Ag° seeds formed initially.
• Additional intensification of these sites is possible by using a reducing agent such as
oxalic acid.
• In some cases, the silver chloride is removed by thiosulfate.

53. Theory and Application of Specific Stains: Silver
Autometallography (Metallic-Metallic Interactions)
• The development of immunogold staining methods for electron microscopy was
followed by the development of procedures to intensify this staining so that
antibody-antigen complexes could be localized with the light microscope.
• The initial silver lactate solution method was difficult to control beacause the
sections had to be developed in darkness as light (electromagnetic radiation)
catalyzed formation of metallic silver in solution and increased background
staining.
• This method can also be used to detect and localize heavy metals in tissues or to
detect DNAIRNA hybridization sites.

54. Theory of Autometallography
• Metallic seeds like gold and silver provide surfaces that catalyze the reduction of
ionic silver to metallic silver in the presence of a strong reducer such as
hydroquinone.
• The larger the initial metallic seed,
the greater is the surface area
the faster is the reaction.
• It is better to slow the reaction so that more controlled staining occurs.

55. Problems Associated with Silver Autometallography
• When silver autometallography continues too long, micrometallic centers
begin to form in solution.
• As the surface areas of the microparticles increase, the process of
catalyzed reduction is accentuated and silver deposition + precipitation
can become prominent.
• Slides should be stained vertically to reduce background from this
precipitation.
• If solutions are not pure, glassware is not clean, or metal utensils or trays
contaminate the solutions, microprecipitation can become severe and
even the dilute silver solutions can become depleted of silver ions,
reducing their staining capabilities.
• Upon prolonged staining, unidentified substances may leach from tissue
secretions to form catalytic sites in the solution or catalytic sites may form
within tissues.

56. Technical Problems Associated with
Silver Stains
Silver solutions are very susceptible to degradation.
– proteins and ions attached to the walls of glassware may react with
silver solutions.
– ions (eg, chloride in non-distilled water) may precipitate silver; and
silver solutions may react with any metal used in the staining
procedure, such as forceps or slide holders.
– all glassware should be chemically cleaned with commercial cleaning
agents, concentrated nitric acid, or bleach and rinsed in distilled
water;
– solutions should be made with distilled water;
– No metal should be allowed to contact the silver solutions.

57. Technical Problems Associated with
Silver Stains
• separating the true reacton from non-specific precipitation.
• difficulty in obtaining standard preparations of certain reagents.
• instability of reagents.
• control of staining time, pH, and temperature.
• variability of staining that may change with the method of fixation and
type of tissue.
e.g, argentaffin staining is reduced in tissues fixed with fixatives
containing alcohol.
• high cost of reagents.
• difficulty in keeping sections on the slides.
• using improper or inappropriate control tissue and collecting.
• adequate control tissue.
• older solutions may present a safety problem, especially an explosion
hazard.

58. Technical Problems Associated with
Silver Stains
• As stock solutions of methenamine silver age, there is an increase in nonspecific silver
staining and an increase in rapid granular silver deposition artifact during
impregnation.
• High temperatures could cause nonspecific silver deposition.
• Oxidizing solutions may lose their oxidizing power on aging.
• Fixation in glutaraldehyde may also cause non-specific staining with the silver solution
- although glutaraldehyde increases crosslinking, it also may produce localized reactive
aldehyde groups in the tissue that may persist even through processing.
• Short exposures to formalin may have similar effects.
• Use of control slides to judge the quality of staining and to aid in the interpretation df
staining is important.

59. Use of the Microwave for Silver Stains
• Many special stains, including silver impregnation techniques, have been modified
to include the use of heat to decrease the staining time.
• Heat causes the molecules of the solutions to move faster, increasing the
molecular movement of dyes or metals across tissue sections and decreasing the
staining time.
• Stains that may take hours in a convection oven at 60°C can be performed in
minutes in solutions preheated in a hot waterbath at 85°C and are complete in less
than 1 minute in the microwave oven.
• Much of the time required for techniques done in convection ovens is necessary
only for solutions to reach optimum temperatures.
• Once the optimal temperature is reached, staining proceeds quite rapidly.

60. Use of the Microwave for Silver Stains
• The speed of rapid silver techniques relies upon bringing solutions to a high temperature as rapidly
as possible.
• The rapid heating of solutions by any technique significantly shortens staining times when
compared to convection ovens.
• Microwave energy significantly reduces the time necessary to produce stained sections and has the
added advantage of decreasing background staining.
• Normal impregnation time in the microwave oven for silver nitrate solutions varies from 60 to 75
sec.
• The intensity of the impregnation is controlled microscopically.
• Reactions such a Perls iron reaction, alcian blue stain, the rhodamine and rubeanic acid techniques
for copper may be performed in the microwave in 45 to 50 sec.
• These modifications allow a diagnosis to be made on critically ill patients within 2 hours after a
biopsy is received in the laboratory.

61. Microwave Procedures
Before attempting microwave procedures, the technologist should be aware of several precautions and
checks to ensure accurate results and laboratory safety.
1. Place glass coplin jars in a small container of water and place both in the microwave oven as they
tend to crack when exposed to the heat of the microwave oven.
2. Do not use tight fitting lids to cover solutions in the microwave which could cause the solution to
overheat and boil over in the microwave or a steam burn when the lid is removed.
3. The technologist should use hot pads when transferring the solution from the microwave to the
counter as solutions heated in the microwave are VERY HOT and can cause severe burns.
4. The solution should be heated first and the slide is added after the solution has been removed
from the oven.
5. Care should be taken not to overheat the solutions.
6. Once the solution has been heated, use wooden applicator sticks to thoroughly mix the solution to
prevent hot spots from staining the tissue unevenly.

62. Microwave Procedures
7. A microscope is a must when using the microwave oven for controlled screening and to
check on overstaining which can occur in a matter of SECONDS due to the rapid
staining and impregnation times.
8. Solutions should be discarded after use as they will break down very fast with
microwave procedures.
9. It is necessary to let the microwave cool and dry by leaving the door open between
cycles when doing several procedures consecutively.
10. Before attempting to perform the procedures on biopsy tissue, stain several control
slides to establish the proper times and settings for each individual microwave.
11. For accuracy and even staining, a digital microwave with a revolving platform is the
most reliable.
12. Rectangular or square staining dishes may produce more uniform staining by
preventing overheating in the center of round containers by internal reflection of
microwaves.

63. Control Tissues for Silver Staining
• The purpose of a positive control is to demonstrate that the stain is working with a proper signal to
noise ratio, ie, stain to background ratio.
• Control tissues can best be stored as paraffin blocks or slides.
• Many tumors stain poorly when compared with normal tissues and this must be considered in
choosing and evaluating a control.
• It may be advantageous to have multiple tissues in some control slides for silver stains or
immunologic stains.
• Control tissues for the demonstration of organisms should be matched to the organism expected.
• Ideal blocks - those that contain a tissue infected with Pneuinocystis jiroveci
• and a second piece of tissue containing a fungal "yeast" form, eg, histoplasmosis.
• If a control tissue contains cryptococcal yeast forms, it could be used as a control for the PAS,
mucicarmine and GMS stains.

64. Recommended Control Tissues for
Silver Stains

65. References
• Bancroft, J.D. and Stevens, A.: theory and practice of histological techniques ed.7,
Churchill livingstone inc. 1990. Edinburgh. London,Melbourne and New York.
• C.F.A. Culling, Handbook of Histopathological and Histochemical Techniques, 1974,
671.
• Hull ME, Hunphrey PA, Pfeifer JD. Washington manual of surgical pathology.
Elsevier 2006; Second Edition: Chapter 54.
• Grizzle WE: Theory and practice of silver staining in histo-pathology. J
Histotechnol 19(3):183–195, 1996.
• E Hempelmann & K Krafts (2017): The mechanism of silver staining of proteins
separated by SDS polyacrylamide gel electrophoresis, Biotechnic & Histochemistry,