Re: [Histonet] Grocott's Methenamine Silver Stain
Just a few thoughts about your lack of reaction with the GMS.
Basically, what you are trying to show here is the neutral
mucopolysaccharides in the capsule of the fungal elements. They wil
stain intensely with a PAS, however, so will a whole bunch of other
stuff (glyogen, contents of some goblet cells, basal lamina, etc), but
fungal elements are morphologically quite easy to distinguish. The
oxidation step in the PAS involves a relatively short treatment in 1%
periodic acid, this will convert any available 1:2-glycols to aldehyde
groups, which in turn react with the Schiff reagent ... consequently,
a whole bunch of stuff reacts positively.
The GMS will demonstrate only structures which are strongly
PAS-positive. Instead of oxidizing in periodic acid, thr GMS uses
chromic acid (a much more powerful oxidizer). Many of the weaker
reactive groups over-oxidize and become non-reactive with the
subsequent silver solution. Glycogen, neutral mucopolysaccharides in
goblet cells, and fungi remain reactive ... and should react with the
silver solution. The GMS is a pretty reliable procedure.
Try using 10% chromic acid for 10 minutes (I developed this
modification when I was teaching Histology and had to be able to
perform the whole method within a two hour lab period). It works great
and several labs have adopted it for use ... the whole method can be
done in less than 30 minutes! The 10% chromic acid solution is stable
for several months.
How old is your stock methenamine-silver solution? Do you keep it in
the fridge? If in doubt, make up some fresh and store it at 4 degrees
The borax (sodium tetraborate) acts as a buffer the make the working
silver solution slightly alkaline (and unstable).
Mix the working solution as follows:
25mL of methenamine-silver stock solution,
25mL distilled water,
Preheat the silver solution to 56 degrees. Heat the solution just
before you need it, not too soon or all the silver will precipitate
out before it has chance to react with the fungal elements in the
section. The silver impregnation step should require 6-10 minutes. You
will see the section begin to turn :golden-light brown) as the silver
reacts with the tissues.
Before and after the silver step, rinse the sections with several
changes of distilled water. Use plastic forceps to avoid getting any
metallic ions into the silver solution.
1. Take sections down to water
2 Oxidize in 10% chromic acid for 10 minutes
3 Wash in water for 1 minutes
4. Treat with 0.5-1% sodium bisulphite (gets rid of excess chromic
5. Wash section in several changes of distilled water (cleanliness
is a virtue)
6. Place sections in pre-warmed methenamine-silver working
solution, keep temperature at 56 degrees.
7. Watch them like hawk ... within 4-5 minutes the sections will
start to turn golden.
8. When they are light brown, take the control out, rinse in
distilled water and look at it microscopically. Fungal hyphae should
appear black on a light gold background.
9. If the fungi are still brown, return the section to the warm
silver solution and give it another 30 seconds or so. Then check it
10. When you are happy with silver impregnation, remove all sections
from the silver solution and wash thoroughly in distilled water.
11. Treat sections with 1-2% sodium thiosulphate for 1 minute.
12. Wash again
13. Treat sections with gold chloride (whatever strength you have on
hand) for 1-2 minutes,
14. Wash yet again,
15. Counterstain with light green.
16. Wash (last time)
17. Dehydrate, clear and mount.
Kamloops, BC, Canada
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