Re: [Histonet] Grocott Stain - quick method

From:"Anthony Reilly"

Hi Patricia
This is a method that takes approximately 30 minutes that has been used
successfully for 10-15 years for urgent biopsies for lung transplant
patients. You will need to determine you own times for the microwaving
of the silver solution.  The important thing is to heat  and mix in
stages to prevent uneven temperatures within your solution which will
result in uneven staining across your section.
1                    REAGENTS ANDEQUIPMENT4.1       Reagents
1.         10% Chromic Acid (AQ)
            Chromium Trioxide                                         -
            Distilled water                                            
     -           50ml.
2.         5% AQ Silver Nitrate
            Silver Nitrate                                             
      -           5g.
            Distilled water                                            
     -           100ml.
3.         3% AQ Methenamine (Hexamine)
    -           3g.
            Distilled water                                            
     -           100ml.
4.         5% AQ Borax (Photographic Grade)
            Sodium Tetraborate                                        -
            Distilled water                                            
     -           100ml.
5.         Stock Methanamine ‑ Silver nitrate solution
            5% Silver Nitrate AQ                                      
-           5ml.
            3% Methenamine AQ                                      -   
6.         Working Methenamine Silver Nitrate Solution:‑
            Borax 5% AQ                                                
 -           2ml.
            Distilled water                                            
     -           25ml.
            Mix and add:‑
            Methenamine‑Silver Stock Solution                -         
7.         1% Sodium Metabisulphite AQ
            Sodium Metabisulphite                                    - 
            Distilled water                                            
     -           100ml.
8.         0.1% AQ Gold Chloride
            Gold chloride                                              
    -           1g.
            Distilled water                                            
     -           1000ml.
9.         2% Light Green (Stock)  
                        Light green                                    
                 -           2g.
                        Distilled water                                
                 -           100ml.
                        Acetic Acid                                    
                 -           1ml.
10.       Light Green Working Solution
                        Light green stock                              
              -           1 part.
                        Distilled water                                
                 -           19 parts
11.              2% Sodium Thiosulphate
Sodium Thiosulphate                                      -          
Distilled Water                                                        
    -           500ml.
 4.2       Equipment
            Panasonic 1000W Microwave Model NN-5782WF
 2                    PROCEDURE
1.                  Bring sections to water
2.         Place sections on staining rack and cover with, or place in
a coplin jar containing a freshly prepared 10% chromic acid solution for
10minutes. This solution may be stored for one week in the
3.         Washthoroughly in running tap water in container in sink 1
4.         Rinse in 1% Sodium Metabisulphite 30 seconds
5.         Wash thoroughly in tap water for a minimum of 2 minutes.
6.         Place slides on rack and rinse well in distilled water
before placing in silver solution.
7.         Place coplin jar in centre of microwave. Microwave on full
power for 20 seconds.  Mix solution well with disposable pipette to
ensure even distribution of heat.  Microwave again on full power for 15
seconds.  Remove slides from microwave and mix thoroughly with
disposable pipette.  Check slides microscopically for end point which
should take 3-5 minutes.  At the end point fungi should be dark brown to
black in colour while background remains golden brown.  
8.         Carefully tip off solution in coplin jar and replace with
distilled water
9.         Place slides on rack and give two more changes of distilled
10.       Tone in 0.1% Gold Chloride 2 minutes
11.       Rinse in distilled water and flood with 2% aq. sodium
thiosulphate ("hypo") 2 minutes (fixing)
12.       Rinse in distilled water
13.              Counterstain with freshly prepared light green working
solution for 20seconds.
            14.       Washslides briefly in tap water.
14.              Dehydrate, clear and mount 
 3                    RESULTS
Fungi                                       -           Black
Background                             -           Green
 4                    NOTES
1.         This method is not specific for fungi but rarely fails to
demonstrate any fungi present. Reticulin fibres and threads of fibrin
will be blackened by this method if overstaining occurs, and must not be
confused with fungi.
Tony Reilly
Chief Scientist
Anatomical Pathology
Pathology Queensland
Princess Alexandra Hospital
Ipswich Rd, 
Woolloongabba Q 4102
Ph: 07 32402412
Fax:0732402930 ( )

>>> "Patricia Karlisch"  20/02/2008 5:07 am >>>
  Does anyone have a quick method to do the Grocott stain.  
  Thank you,
   Pat Karlisch 

Pat Karlisch
Supervisor, Histology, Pathology and Laboratory Medicine
Penn State Milton S. Hershey Medical Center
Mail Code H179
Hershey, PA 17033
Phone (717) 531-6072
Fax: (717) 531- 7741

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