Fungal Stain (Hexamine Silver Method)

Assay-kits

Fungal Stain (Hexamine Silver Method)

Product code: ACMBS-55
Specifications: 20Tests (Box)
Intended use: Intended for qualitative staining for polysaccharide staining in cell walls of fungi and other organisms
Store temperature: 2℃～8℃
Expiry date: 12 Months

More Details

PrincipleMain componentsSample requirementTest procedureResult interpretationMethod limitationPrecautions

Principle

After the specimen is oxidized with periodic acid, the mucopolysaccharide in the cell wall of fungi and other opportunistic organisms exposes aldehyde groups, which reduce silver hexamine to black metallic silver. Sodium thiosulfate fixes the colored silver salt and removes unreacted silver ions.

Main components

Reagent composition	Main ingredient
1. Periodic acid solution	Periodic acid
2. Silver nitrate solution	Silver nitrate
3. Hexamethylenetetramine solution	Hexamethylenetetramine
4. Borax solution	Borax
5. Sodium thiosulfate solution	Sodium thiosulfate
6. Brilliant green solution	Brilliant green

Sample requirement

Pick samples to make smears. Care must be taken when smearing. The smears are thin and uniform. The prepared smear should be naturally dry. If it is fixed by heating, the fixing temperature should not be too high. The back of the slide should not be hot if it touches the back of the hand. Otherwise, the shape of the specimen may be changed to affect the observation result.

Test procedure

A. Working solution preparation: (one part of working solution is about 8.2ml, 8 slides can be stained)

Equipment (self-prepared): disposable test tube, pipette, disposable tip and dropper. 200μl of silver nitrate solution + 3ml of hexamethylenetetramine solution + 1 bottle of borax solution, mixed into one part of working solution.
Specific operation: Add 200μl of silver nitrate solution and 3ml of hexamethylenetetramine solution in a clean test tube, mix thoroughly, then pour into a bottle of borax solution, shake it thoroughly. If you see a little transparent particle, it is a normal phenomenon, please don't filter it. Spare.
(Please prepare in the above order to avoid affecting the effect of the working solution. If the working solution is used up on the day, store it at room temperature, if it is not used up on the day, it must be sealed and protected from light and placed in a refrigerator at 2℃～8℃ store and use within a month).

B. Staining procedure

1. Pick a sample to make a smear, dry it naturally, and then heat and fix it.
2. Add periodic acid solution and oxidize for 15 minutes, wash with distilled water slightly.
3. Place the smear in a stained slide box, and place the slide box in a 62℃ water bath to float the slide box on the water. Add 1ml of working solution to the smear, then cover the lid of the box and the water bath, common fungi stain for 10～15 minutes, Pneumocystis carinii stain for 20～25 minutes. When the specimen shows a yellow-brown reaction, remove it. (※ Note: the working solution cannot contact metal ions, please make sure the temperature of the water bath reaches 62℃, and do not dry the staining solution during staining).
4. After washing with distilled water, then microscopy. If the coloration is not deep enough, please repeat step 3 to continue the reaction.
5. Rinse with running water.
6. Treat with sodium thiosulfate solution for 3 minutes, rinse with running water.
7. Counter-stain with Brilliant Green staining solution for 1minute, rinse with water, dry, and observe under oil microscope.

Result interpretation

The mycelia and spores of the fungus are brown-black or black, the structure is clear and obvious, and the background is green.

Method limitation

Only used for staining for morphological preliminary inspection and observation.

Precautions

1. The heated working solution is only for one-time use, if the prepared working solution cannot be used in time, it should be placed in a refrigerator at 2℃～8℃, protected from light, and used within one month.
2. It is recommended to use a thermometer to measure the temperature of the water in the water bath (up to 62℃). If the temperature is lower than 62℃, the warm-up time needs to be extended, and the water bath should ensure sufficient water volume.
3. When staining with mold and Pneumocystis carinii, it must be controlled under a microscope, otherwise it will be easily confused with other nuclei after staining too deeply. It should be observed every 5 minutes until the morphology of mold or pneumocystis carinii is clear and discernable.
4. The Brilliant Green staining solution should not be overstained, otherwise it may affect the observation. The staining time should be determined according to the actual situation, this step can also be omitted.
5. Freebies (stained slide boxes) can be reused. After the staining is finished, wash it with cleaning solution and water and dry it for next use.
6. This kit must be used by professionals and the interpretation of results.
7. Should carefully read the instruction manual before using. Can only be used before expiration date, and good personal hygiene protection.
8. After use, disposal of waste should comply with the hospital or the environmental protection department requirements.
9. Production date and lot number and expiration date showed on the package.

Reference

1. Lu Hongzhou, Qian Xueqin, Xu Heping, et al. Examination and illustration of medical fungi [M]. Shanghai Science and Technology Press.

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