Grocott Methenamine silver Flashcards
1 Oxidation
expose reactive sites that will bind to silver
-suppress staining of other sites
-type of oxidizer determines specificity
preheated chromic acid - polysacc to aldehydes in fungal wall
over oxidizes everything BUT the wall so it can stain with silver
2 Bleaching
Removes any discoloration caused by the oxidizer
Sodium bisulphite
3 Sensitization*
not required in argentaffin reactions like this one
4 Impregnation
to deposit silver at the exposed reactive sites
Stock Methenamine-Silver nitrate solution
Working Methenamine-Silver nitrate solution
25ml, 25ml, 2 ml
has Sodium borate as buffer
5-Reduction*-
Target tissue components are capable of auto-reduction (from colourless state to black deposits).
-Auto-reduction occurring during silver reactions are termed argentaffin reactions
6 Toning -
gold in place of silver to remove any brown colour in other tissue components (collagen).
The mechanism of staining is Ion exchange or metallic substitution
Gold chloride
7- Fixation -
application of sodium thiosulphate. Before this application, the technologist must check the slides microscopically to ensure impregnation is neither too light, nor too dark.
TOO LIGHT: Stay longer in the heated reagent
TOO DARK: can’t correct; must repeat
Once tissue is treated with HYPO, argentaffin silver staining errors cannot be corrected
8 Counterstain-
colour to tissue components that are non-target.
Nuclear fast red and light green
Eosin and van Gieson
What is the
classification
target tissue
mechanism of staining
staining principle
positive controls
for GMS
classification- Microorg stain
target tissue- fungi
mechanism of staining -Silver impregnation
staining principle -Induced Argentaffin metallic impregnation
positive controls- anything with fungi
What do you have to have for this stain
-tissues fixed with formalin
-40-50c water water
-chemically cleaned glassware and rinse in DW before use
What color is chromic acid
Bright burnt orange – good
Brown – discard
Rehydrating alcohols during dewaxation steps must be fully removed.
If not, the residual alcohol will reduce the chromic acid (will turn brown).
Ensure complete removal of alcohol – prior to oxidation in chromic acid. Why?
alcohol will reduce the chromic acid and it will turn brown
If not microwaving, use the water bath to heat up silver solution instead of oven. why?
faster for the staining solution to achieve the required temperature
Do not overheat the silver solution. Why?
Most of the silver will adhere onto the glass (creating a “mirror” film)
Use chromic acid as oxidizer. Why?
ensure that the fungal wall will be properly oxidized to expose binding site and the rest of the tissue components will get over-oxidized and become unstainable with silver.
What problem will under–oxidation present
It will not open up the binding sites on the fungal, protozoa wall. But the non-target binding sites will be opened increasing non-specific staining.
Previously stained tissue sections can be stained with GMS. Why?
Chromic acid is such a strong oxidizer that it will take out all other stains previously applied and will leave only the fungal and protozoa walls available for silver staining.
Avoid glutaraldehyde fixative. Why?
since the target tissue is made up of aldehyde units, tissues fixed with glutaraldehyde will have increased binding sites for silver staining, thereby increasing non-specific staining.
high temps cause
non-specific staining. Wipe silver film around the tissue and on the back of the glass slide before coverslipping.
