Summary
Silver hexamine staining is also known as (Periodic-acid silver methenamine) PASM staining. The specimen is oxidized by periodate, which exposes the aldehyde groups of the mucopolysaccharides in the cell walls of fungi and other opportunistic organisms, and the aldehyde groups reduce the silver hexamine to black metallic silver. Sodium thiosulfate acts as a fixative for the silver salt that has developed color and removes unreacted silver ions.
Principle
Oxidation of the tissue with periodate exposes the basement membrane of the tissue or the mucopolysaccharides within the fungus to the aldehyde group, which interacts with silver hexamine and reduces it to black metallic silver, and then sodium thiosulfate is used to remove the unreacted silver ions.
Appliance
Mainly used for staining tissue basement membranes and fungi (mainly applied to Cryptococcus neoformans, Trichoderma, Aspergillus, Histoplasma capsulatum, Penicillium marneffei, Actinomycetes, etc.).
Operation method
Fungal Staining--Hexamine Silver Method
Principle
Oxidation of the tissue with periodate exposes the basement membrane of the tissue or the mucopolysaccharides within the fungus to the aldehyde group, which interacts with silver hexamine and reduces it to black metallic silver, and then sodium thiosulfate is used to remove the unreacted silver ions.
Materials and Instruments
① 5% aqueous solution of borax: borax 5 grams, dissolved in 100 ml of distilled water Move 1. Sections are routinely deparaffinized until washed. 2. The sections were then placed in 5% aqueous chromic acid solution for 1 hour and rinsed in tap water for 5 minutes. 3. The sections are then placed in 1% sodium bisulfite for 30 minutes, rinsed in tap water for 3 minutes, and washed in distilled water. 4. Preheat the silver hexamine application solution to 60 °C, and then put the sections into the prepared preheated silver hexamine working solution for 1 hour, and then take the sections out after washing to see if there is any bacterial body. If light brown bacteria appear, it should be observed every 5~10 min to control the coloring depth of the bacteria, until the color is ideal. Then wash with distilled water for 5~6 times. 5. Put the section into 0.1% aqueous gold chloride solution for 4 minutes to adjust the color, and wash with distilled water for 1~2 minutes. 6. Then put the section into 3% sodium thiosulfate solution for 5 minutes. 7. Wash with tap water for 1 minute and distilled water for 1 minute. 8, light green re-staining for 20 seconds, rinse with running water for 20-60 seconds. 9, 95% alcohol and anhydrous essence dehydration, xylene transparent, gum seal. 10. Staining result: black fungus, light green background. Caveat 1. Pick the specimen to make a smear, the smear must be carefully operated, the smear is thin and uniform. The prepared smear should be dried naturally, if it is fixed by heating, the fixation temperature should not be too high, subject to the back of the slide touching the back of the hand, otherwise the morphology of the specimen may be changed to affect the observation results. 2， gold chloride coloring, must be observed under the microscope. 3、The glassware used in the experiment should be soaked with sulfuric acid wash and rinsed with distilled water beforehand and baked dry. For more product details, please visit Aladdin Scientific website.
② 5% silver nitrate aqueous solution: silver nitrate 5 grams, dissolved in 100 ml of distilled water
③ 3% urotropine aqueous solution: urotropine 3 grams, dissolved in 100 ml of distilled water
④ Silver hexamine storage solution: 3% urotropine aqueous solution is poured into 5% silver nitrate solution, white precipitate is produced, and it becomes clear by shaking, and it can be stored for about 6 months at 4 ℃ under low light conditions.
⑤ Application solution: take 25 ml of storage solution, return it to room temperature, add 5 ml of 5% aqueous borax solution and mix it now.
⑥ 5% aqueous chromic acid solution: chromic acid (chromic acid) 5 g, distilled water 100 ml.
(vii) 1% aqueous sodium bisulfite solution: sodium bisulfite (Sodium bisulfite) 1 g, 100 ml of distilled water.
⑧ 0.05% light green dye: light green 0.1 grams, distilled water 200 ml, acetic acid 0.1 ml
⑨ 0.1% aqueous solution of gold chloride: 0.1 g of gold chloride, dissolved in 100 ml of distilled water
⑩ 3% aqueous sodium thiosulfate: 3 g of sodium thiosulfate, dissolved in 100 ml of distilled water