- Catalog order # K7264 - 4oz kit - $70.00
- K7268 - 8oz kit- $107.00
-
Gomories Methenamine IMEB Microwave Silver Stain Kit Procedure (GMS)
APPLICATION
Mammalian Tissue
DESIGNED TO SHOW
Black oxidized Silver Deposits on opportunistic organisms and specific tissue elements
PURPOSE
For the detection of Fungus, Pneumocystis, Mucin, and Basement Membrane
SPECIMEN REQUIREMENTS
Fixation: Tissue: Any well fixed tissue, however, it is suggested to use formalin fixed tissue.
Sections: Cut Paraffin sections at 3-4 microns.
COMPONENTS AND REAGENTS
1. Methenamine Solution - 4 oz / 120 ml
Solution should be stored in Refrigerator
2. Stabilized Silver Nitrate Sol. - 4 oz / 120 ml
Solution should be stored in Refrigerator
3. Sodium Borate Solution - 4 oz / 120 ml
4. Chromic Acid Solution - 4 oz / 120 ml
5. Sodium Bisulfite Solution - 4 oz / 120 ml
6. Gold Chloride Solution - 4 oz / 120 ml
7. Sodium Thiosulfite Solution - 4 oz / 120 ml
8. Light Green SF Counterstain - 4 oz / 120 ml
9. Control Slides1 stained and 3 unstained
* Working Methenamine Silver Solution
Add 10 ml of Concentrated Methenamine Solution to 40 ml of Distilled Water. Mix. Add 10ml of Concentrated Stabilized Silver Nitrate Solution and shake or stir for 1 minute. Add 2 ml of Sodium Borate Solution and stir for 1 minute. Store in the dark until used.
PROCEDURE
A conventional microwave is used for heating solutions
1. Deparaffinize sections and hydrate to deionized water.
2. Treat sections in Chromic Acid Solution for 10 minutes
3. Rinse sections under tap water.
4. Treat sections in Sodium Bisulfite Solution for 1 minute.
5. Wash sections quickly through two changes of distilled water, 30 seconds each.
6. Treat sections in *Working Methenamine Silver Solution for 5 minutes in microwave using Low power.
(Additional treatment in heated solution is necessary if sections have not turned dark brown within the 5 minute incubation).
7. Treat sections in Gold Chloride Solution for 45 seconds.
8. Rinse sections briefly in tap water for 2 minutes.
9. Treat sections in Sodium Thiosulfate Solution for 1 minute.
10. Rinse sections in tap water for 2 minutes.
11. Stain sections in Light Green Counterstain for 30 seconds with agitation. Increasing time will increase intensity.
12. Dehydrate sections through three changes of Reagent Alcohol.
13. Clear sections through two changes of Xylene or Xylene substitute.
14. Coverslip with Clearslip.
RESULTS
Pneumocystis C.-Black to Gray
Fungus-Black to Gray
Mucin-Black to Gray
Basement MembraneBlack to Taupe
BackgroundGreen
NOTES
1. Distilled water is recommended to be used on all the procedure steps. Pending on water source, it is suggested to use deionized or steam distilled water, because natural or processed water qualities in different regions may have an effect on staining results. Always use Distilled Water when manufacturing your Working Methenamine Silver Solution.
2. Working Methenamine Silver Solution should be made fresh before use. Working Methenamine Silver Solution has a shelf life of 4 months without the addition of the Sodium Borate Solution. This solution must be stored in the refrigerator. With the addition of Sodium Borate, the working solution should be stored in the dark and has a shelf life of 2 hours. Black precipitate will develop on the bottom of the incubation jar. This is a normal event.
3. It is strongly suggested to use a waterbath instead of an oven. A waterbath will heat the Working Methanamine Solution more rapidly than your conventional lab oven. If you use an oven, increase your time to 60 minutes.
4. **Different microwaves provide different power sources. 500W conventional microwaves take longer to heat than industrial microwaves. A basic rule to remember is to stop heating the solution when it begins to boil. Heating the solution further will cause loss of liquid, tissue removal from the slide and possibly damage to the microwave and harm to the technician. Delayed heating will not provide consistent results. Increase your power output or time if necessary.
It is strongly suggested to use a laboratory approved microwave that provides heating and ventilation for fumes.
REFERENCES
1. AFIP, Manual of Histologic Methods, McGraw Hill Book Co., 1968, pp. 230
2. Molecular Cell Biology, James Darnell, Harvey Lodish., Scientific American Books., 1981
Dev. 8/97
