Gram Staining, 20 mL×4 (for Microorganism staining)

Product Information

Introduction

Gram staining is a differential staining method widely used in microbiology, which can be used to distinguish Gram-positive bacteria (G+) from Gram-negative bacteria (G-). The basic principle is based on the different chemical components of bacterial cell walls. After primary staining with crystal violet and mordant with iodine, bacteria form water-insoluble crystal violet-iodine complexes in the cell wall. Gram-positive bacteria have thick cell walls, do not contain lipids, are rich in peptidoglycan and are cross-linked to form a dense grid structure. When treated with ethanol, the peptidoglycan mesh shrinks, which can block the crystal violet-iodine complex. It remains in the cell wall, giving it the purple color of crystal violet. Gram-negative bacteria, on the other hand, have thin cell walls, low peptidoglycan content and loose cross-linking. When exposed to ethanol, the lipid-rich outer membrane dissolves, a large gap appears in the cell wall, and the crystal violet-iodine complex flows out, so the cells become colorless after ethanol decolorization. At this point by safranin counterstaining, Gram-negative bacteria will be stained red.

Gram staining solution contains four components, namely ammonium oxalate crystal violet staining solution, 1% iodine solution, destaining solution and fuchsin staining solution. After staining, gram-positive bacteria appear purple to blue-purple, and gram-negative bacteria appear red.

Storage and Handling Conditions

Store and transportation at room temperature; valid for 12 months.

Component

Assay Protocol / Procedures

1. Dewax the paraffin section to water; dry the bacterial smear naturally or warm it with an alcohol lamp to dry and fix it.

2. Initial dyeing: Add crystal violet staining solution dropwise to cover the sample for 10-30 s, wash with water, and spin dry.

3. Mordant dyeing: Add 1% iodine solution dropwise to cover the sample for 1-1.5 min, wash with water, and spin dry.

4. Decolorization: dropwise add decolorization solution to rinse the tissue from one end of the slide for decolorization, until the decolorization solution that flows down no longer appears purple, and immediately wash with water to remove the decolorization solution. Take care to control the destaining time.

5. Counterstaining: add Fuchsin staining solution dropwise for 20-60 s, then wash with water. Blot the edges with filter paper.

6. Bacterial smears can be observed directly by microscopy. After drying at 60 °C, the tissue sections were rapidly dehydrated in absolute ethanol for three times, 1 s, 3 s, and 5 s, respectively. Then, it was transparentized with xylene for 5 min, and finally sealed with neutral gum for microscopic examination.

Note

1. Tighten the cap in time after using the reagent to prevent the solvent from volatilizing.

2. The bacterial smear is too thick, the crystal violet staining time is too long, and the decolorization is insufficient, which may lead to false positive results. It is recommended to do both negative and positive bacterial control groups.

3. Each set of staining solution can be used to stain approximately 30 sections (drop staining).

4. Please wear lab coat and disposable gloves during operation.

For Research Use Only!

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