Grocott Methenamine Silver Set, GMS

SET INCLUDES:                                                                                                     

Additionally Needed For Fungus Stain, Grocott Methenamine Silver, GMS:

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

APPLICATION:

Newcomer Supply Grocott Methenamine Silver Set, GMS with included microwave modifications, provides the silver staining solutions for the Fungus Stain, Grocott Methenamine Silver (GMS) procedure.  This is one of the best staining methods for demonstrating a variety of fungal organisms including: Pneumocystis carinii, Aspergillus, Blastomyces, Candida and Histoplasma.

When staining for Pneumocystis with other fungal organisms, running a separate control specific for Pneumocystis (Part 4556) is recommended.

METHOD:

Fixation: Formalin 10%, Phosphate Buffered (Part 1090)

Technique:  Paraffin sections cut at 5 microns

Solutions:  All solutions are manufactured by Newcomer Supply, Inc.

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

PRESTAINING PREPARATION:

1. All glassware/plasticware must be acid cleaned prior to use.
   1. See Procedure Notes #1 and #2.
2. Prepare Silver-Methenamine Working Solution and mix well:
   1. Solution A: Silver Nitrate                        20 ml
   2. Solution B: Methenamine Borate            20 ml
3. Preheat Silver-Methenamine Working Solution to 45°C – 60°C approximately 20-30 minutes before use.
   1. See Procedure Notes #3 and #4
   2. Save for use in Step #9.
   3. Do not preheat if using Microwave Modification; Step 10.

STAINING PROCEDURE:

4. 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. See Procedure Notes #5 and #6.
5. Oxidize in Chromic Acid 5%, Aqueous (Part 10341) for 1 hour.

        Microwave Modification: See Procedure Note #7

1. Oxidize slides in a plastic Coplin jar containing Chromic Acid 5%, Aqueous (Part 10341) and microwave for 1 minute and 20 seconds at 60°C.

6. Wash well in running tap water; rinse in distilled water.
7. Place in Sodium Bisulfite 1%, Aqueous (Part 13821) for 1 minute.
8. Wash for 5 minutes in running tap water; rinse well in distilled water.
9. Incubate slides in preheated Silver-Methenamine Working Solution (Step #3) at 45°C-60°C or at room temperature, for 12-18 minutes until sections appear paper-bag brown.
   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.
10. Microwave Modification: See Procedure Note #7
    1. Incubate slides in a plastic Coplin jar containing Silver-Methenamine Working Solution (Step #2) and microwave for 1 minute at 70°C. 
    2. Check microscopically for adequate development.
    3. If additional incubation is required, return slides to warm Silver-Methenamine Working Solution.  Recheck at 2-3 minute intervals.
11. Rinse in three to four changes of distilled water.
    1.   Do not use tap water at this step.
12. Tone in Gold Chloride 0.1%, Aqueous (Part 11285) until sections turn gray; 20 seconds to 1 minute.
13. Rinse well in distilled water.
14. Remove unreduced silver in Sodium Thiosulfate 2%, Aqueous (Part 13888) for 2 minutes.
15. Wash in running tap water for 5 minutes; rinse in distilled water.
16. Counterstain in Light Green SF Yellowish Stain 0.02%, Aqueous (Part 12204) for 2 minutes.
17. 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. Acid clean all glassware/plasticware (Part 12086) and rinse thoroughly in several changes of distilled water.  Cleaning glassware with bleach is not equivalent to acid washing.
2. Plastic (Part 5500), plastic-tipped, or paraffin coated metal forceps must be used with any silver solution to prevent precipitation of silver salts.  No metals of any kind should be in contact with any silver solution. Only glass thermometers should be used.
3. Preheating Silver-Methenamine Working Solution to 45°C-60°C prior to incubation is suggested for timely silver development. A water bath can be used for preheating.  Begin preheating the silver solution approximately 20-30 minutes before use.
4. Staining slides at higher temperatures will cause the development reaction to happen faster, but may also cause precipitate to form in the working silver solution and deposit on the slides.  Maintaining the silver solution between 45°C-60°C will help to minimize precipitate.
5. Drain staining rack/slides after each step to prevent solution carry over.
6. Do not allow sections to dry out at any point during staining procedure.
7. The suggested microwave procedure has been tested at Newcomer Supply using an “EB Sciences”, 850 watt microwave oven with temperature probe and agitation tubes.  This procedure is reproducible in our laboratory.  It is nonetheless a guideline and techniques should be developed for your laboratory which meet the requirements of your situation. Microwave devices should be placed in a fume hood or vented into a fume hood, according to manufacturer’s instructions, to prevent exposure to chemical vapors.
8. If using a xylene substitute, closely follow the manufacturer’s recommendations for deparaffinization and clearing steps.

REFERENCES:

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 4.3 (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.