Fungus, Grocott Methenamine Silver (GMS) Stain Kit

FUNGUS, GROCOTT METHENAMINE SILVER (GMS) STAIN KIT INCLUDES:  

COMPLIMENTARY POSITIVE CONTROL SLIDES: Enclosed are two complimentary unstained positive control slides for the initial verification of staining techniques and reagents.  Verification must be documented by running one Newcomer Supply complimentary positive control slide along with your current positive control slide for the first run. Retain the second complimentary control slide for further troubleshooting, if needed.

Individual stain solutions and additional control slides may be available for purchase under separate part numbers.

Additionally Needed:

For storage requirements and expiration date refer to individual bottle labels.

APPLICATION:

Newcomer Supply Fungus, Grocott Methenamine Silver (GMS) Stain Kit procedure, with included microwave modification, is the best staining method to demonstrate a variety of fungal organisms including: Pneumocystis, Aspergillus, Blastomyces, Candida and Histoplasma.

METHOD:

Fixation: Formalin 10%, Phosphate Buffered (Part 1090)
Technique:  Paraffin sections cut at 4 microns.
Solutions:  All solutions are manufactured by Newcomer Supply, Inc.

All Newcomer Supply Stain Kits are designed to be used with Coplin jars filled to 40 ml following the provided staining procedure.  Some solutions in the kit may contain extra volumes.

PRESTAINING PREPARATION:

1. 1. If necessary, heat dry tissue sections/slides in oven.
   2. All glassware/plasticware must be acid cleaned prior to use.
      1. 1. See Procedure Notes #1 and #2.
   3. Prepare Silver-Methenamine Working Solution and mix well.
      1. 1. Solution C: Silver Nitrate                      20 ml
         2. Solution D: Methenamine Borate         20 ml
   4. Preheat Silver-Methenamine Working Solution to 45°C-60°C in a water bath approximately 20 to 30 minutes before use.
      1. 1. Maintain solution between 45°-60°C to minimize precipitate.
         2. Do not preheat if using Microwave Modification; Step 11.

STAINING PROCEDURE:

1. 5. Deparaffinize sections thoroughly in three changes of xylene, 3 minutes each. Hydrate through two changes each of 100% and 95% ethyl alcohols, 10 dips each.  Wash well with distilled water.
      1. 1. See Procedure Notes #3 and #4.
   6. Oxidize in Solution A: Chromic Acid 5%, Aqueous for 1 hour.

        Microwave Modification: See Procedure Note #5

1. 1. 1. 1. Microwave Solution A: Chromic Acid 5%, Aqueous without slides in a plastic Coplin jar for 1 minute at 60°C.  Add slides to heated Solution A and oxidize for 10 minutes.

1. 7. Wash well in running tap water, rinse in distilled water.
   8. Place in Solution B: Sodium Bisulfite 1%, Aqueous for 1 minute.
   9. Wash for 5 minutes in running tap water; rinse well in distilled water.
   10. Incubate slides in preheated Silver-Methenamine Working Solution (Step #4) at 45°C-60°C or at room temperature, for 12-18 minutes until sections appear paper-bag brown.
       1. 1. Periodically remove control, rinse in warm distilled water, check microscopically for adequate silver impregnation.  Fungi should be dark brown. 
          2. If organisms are not sufficiently dark, return slides to warm silver solution.  Recheck at 2-3 minute intervals until desired intensity is achieved.
          3. Pneumocystis may take longer to stain than other fungus.
          4. Staining at room temperature will require longer incubation.
   11. Microwave Modification:
       1. 1. Incubate slides in a plastic Coplin jar containing Silver-Methenamine Working Solution. Microwave for 5 minutes at 45°C.
          2. Check microscopically for adequate development.
          3. If additional incubation is required, return slides to warm silver solution.  Recheck at 2-3 minute intervals.
   12. Rinse in three to four changes of distilled water.
       1. 1. Never use tap water at this step.
   13. Tone in Solution E: Gold Chloride 0.1%, Aqueous until sections turn gray; 10-30 seconds.
   14. Rinse well in distilled water.
   15. Remove unreduced silver in Solution F: Sodium Thiosulfate 2%, Aqueous for 2 minutes.
   16. Wash in running tap water for 5 minutes; rinse in distilled water.
   17. Counterstain in Solution G: Light Green SF Yellowish Stain 0.2%, Aqueous for 2 minutes.
   18. Dehydrate quickly in two changes each of 95% and 100% ethyl alcohol. Clear in three changes of xylene, 10 dips each; coverslip with compatible mounting medium.

RESULTS:

PROCEDURE NOTES:

1. 1. Acid clean all glassware/plasticware (Part 12086) and rinse thoroughly in several changes of distilled water.
   2. No metals of any kind should come in contact with silver solutions to prevent precipitation of silver salts. Use plastic forceps (Part 5500) or paraffin coated metal forceps.
   3. Drain slides after each step to prevent solution carry over.
   4. Do not allow sections to dry out at any point during procedure.
   5. The microwave procedure was tested using a laboratory-grade microwave oven. This procedure is a guideline and techniques should be developed for use in your laboratory.
   6. If using a xylene substitute, follow manufacturer’s recommendation for deparaffinization and clearing steps.

REFERENCES:

1. 1. Carson, Freida L. and Christa Hladik. Histotechnology: A Self-Instructional Text. 3rd ed. Chicago, Ill.: American Society of Clinical Pathologists, 2009. 239-243.
   2. Grocott, R G, “A Stain for Fungi in Tissue Sections and Smears using Gomori Methenamine Silver Nitrate Technic”. American Journal of Clinical Pathology 25 (1955): 975-979.
   3. Koski, John. “Silver Methenamine Borate (SMB): Cost Reduction with Technical Improvement in Silver Nitrate-Gold Chloride Impregnations.” The Journal of Histotechnology 3 (1981): 115-119.
   4. Sheehan, Dezna C. and Barbara B. Hrapchak. Theory and Practice of Histotechnology. 2nd ed. St. Louis: Mosby, 1980. 245-246.
   5. Modifications developed by Newcomer Supply Laboratory.