Jone's Methenamine Silver

Classification: connective tissue stain 

Purpose: stain glomerular basement membrane 

Staining principle: argentaffin metallic impregnation 

Positive controls: aorta, skin, lung, internal control

Well Stained Slide


Results:




Glomerular basement stains brown-black


REAGENT

REAGENT

PURPOSE

MECHANISM OF STAINING

SOURCE OF ERROR

Periodic acid

 

Mild oxidizer which does not oxidize anything but the basement membrane

 

Exposes reactive aldehyde groups

Omitted: Basement membrane will not be demonstrated.

Too short: Basement membrane may have patchy staining.

Too long: Does not affect the staining.

Methenamine silver

 

Impregnation

(Silver nitrate,hexamine and borax)

Silver binds with the aldehyde groups (reactive groups)

Aldehyde groups will reduce silver

Omitted: Basement membrane will not be demonstrated.

Too short: Patchy staining of basement membrane may be seen.

Too long: Difficulty in seeing the details in glomerulus.

Gold chloride

 

Toning

(optional)

Substitute silver with gold by ion exchange

Removes any brown color in other tissue components

Omitted: Collagen will remain golden brown.

Too long: Production  of red tones due to overtoning. Will start to remove silver staining.

Too short: No effects on the staining.

 

Sodium thiosulphate (hypo)

 

Fixation

Removes unreacted silver

Omitted: Causes non-specific blackening of tissue over time.

Too long: No effects on the staining.

Too short: Causes blackening of tissue over time.

Light green

Counterstain

 

Omitted: No other tissue components demonstrated.

Too long: Basement membrane obscured.

Too short: Other tissues stain lightly and no detail seen.

 

 

 

Poor Stain

Counterstain is too dark. 


Basement membrane is obscured. 


Well Stained Slide

Details of basement membrane of kidney glomerulus are visible.


Well Stained Slide

Details of basement membrane of kidney glomerulus are visible.




Special Considerations

When methemine silver complex is used the aldehydes reduce the silver of the methemine silver and form a metallic silver. Therefore, no extranous reducer is required.

Timing is important when working with silver at 70 degrees celsius, the solution is good for 60 to 75  minutes and this should be done without preheating.

Toning with  gold chloride is an optional step.

Renal tubular basement membrane will be stained black before basement membranes. We are looking for glomerular basement membrane which should be appear brown-black.

No metal forceps should be used when working with this stain.

This stain can not be removed from tissue after fixation step. Before the fixation we can remove the stain with iron alum.


Other Sources of Error

- black precipitates on the slides because of silver solution.

- formation of precipitates due to checking the slides too often.

uneven staining due to cooling the slides too much

-  sharper staining and less background staining may be achieved with  microwave usage. 

Reference

Officer B. HIML251 Lecture notes: Silver Stains. Jone's Methenamine Silver, March 26, 2009