GROCOTT Staining Kit – Microwave procedure

Download Catalouge : 043823W_en

Description

Product for the preparation of cyto-histological samples for optical microscopy.
To demonstrate fungi in tissue sections.

PRINCIPLE
In most fungi, cell walls are made of chitin, a polymer of N-acetylglucosamine together with polymers of D-glucose and Dmannose, proteins and lipids. Chromic acid reacts with glycolic and glycoaminic groups in polysaccharide chain oxidising them to aldehydic groups and thus breaking the chain itself. These newly formed aldehydic groups reduce silver chloride, which is part of the silver-methenamine complex, to metallic silver and make it visible.

WARNING
For good results, follow these rules:
– Avoid to infect section and slide with no pathogenic fungi (handle only with gloves, do not expose preparation to air)
– Use always fresh distilled water.
– Use only clean glasswork.
– Avoid deposit of dust on section.
– Avoid contact between solutions and metal objects (tweezers…)

METHOD
1) Bring section to distilled water.
2) Put on the section 10 drops reagent A: leave to act 20 minutes.
3) Wash in running tap water for a few seconds.
4) Put on the section 10 drops of reagent B for 1 minute.
5) Wash in tap water for 5 minutes.
6) Rinse in 4 changes of distilled water.
7) In a plastic Coplin Jar 50 ml, put 40 ml of distilled water and add 30 drops of reagent C, 15 drops of reagent D and 20
drops of reagent E. Stir briefly with a glass stick previously washed in distilled water.
8) Put the section in the Coplin Jar and incubate in microwave oven at 500 watt for 1 minute.
9) Take out the Coplin Jar from oven and let it cool for 5 minutes.
10) Wash in 6 changes of distilled water.
11) Put on the section 10 drops of reagent F for 3 minutes.
12) Wash in distilled water.
13) Put on the section 10 drops of reagent G for 5 minutes.
14) Wash in tap water.
15) Put on the section 10 drops of reagent H for 30 seconds.
16) Dehydrate through ascending alcohols, clear in xylene and mount

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GROCOTT Staining Kit – Microwave procedure