[Histonet] Grocott's Methenamine Silver Stain

[Paul Bradbury]

   Hi Kathleen,
   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
   C.
   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,
                               2mL borax.
   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.
   The method:
   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
   acid)
   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
   again.
   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.
   Good luck,
   Paul
   Kamloops, BC, Canada