Von Kossa

Classification: pigment stain 

Mechanism of staining: metallic substitution 

Purpose: stain calcium 

Positive controls: any calcified tissue, especially bone or teeth

Von Kossa

Placenta with minimal calcium shown as black precipitation


REAGENT

REAGENT

PURPOSE

MECHANISM OF STAINING

SOURCE OF ERROR

Silver nitrate

Reacts with phosphate and carbonate groups

 

Metallic Substitution

Silver is substituted in place where calcium was bound. Calcium in the body is mainly bound to phosphate and carbonate radicals

Omitted: No calcium demonstrated.

Too short: Minimal calcium or hard to see.

Too long: Non-specific staining possible.

UV Light or Hydroquinone

Reducer

Light or hydroquinone reduces the silver salts to black metallic silver, macroscopically visible

Omitted: Silver not detected.

Too short: Minimal calcium may not be demonstrated.

Too long: Precipitation.

5% Sodium Thiosulphate (optional):

 Fixative

Remove excess silver and also fixative

HYPO can prevent non-specific blackening but may also render small amounts of calcium; also reduced silver can also be partially-bleached with this step as well

Omitted: Non-specific staining occurs.

Too short: Few black precipitates might occur.

Too long: No effect.

Neutral Red or van Gieson

Counterstain

Stains non-mineralized tissues, and RBCs

Omitted: Tissue not containing calcium not demonstrated.

Too short: Tissue component may be hard too see.

Too long: Poor contrast with primary stain and small amounts of calcium may be obscured.

 

 

 

Von Koss - Positive

Placenta

Nuetral Red is weak and can be corrected by going back to alcohol and re-stain; gross calcium deposits.


Von Kossa - Negative



Placenta


Possibly no calcium present, acid fixative was used, no silver nitrate was added, there was no reduction or reduction time was too short

 


Special Considerations

Avoid acidic fixatives that dissolve calcium. 

Rinse glassware before use with distilled water avoid contamination with free metal ions. Also use plastic forceps or wax coated forceps to handle slides.


References

Officer B. HIML 251 Lecture notes: Silver stains: Gordon and Sweet’s Reticulin and von Kossa, March 25, 2009