The observation of mycorrhizal colonization and the determination of colonization rate can be conducted using the vital microscopic examination method and the staining microscopic examination method. The advantage of the vital microscopic examination method is that it allows repeated continuous observation of living samples, enables timely and general understanding of mycorrhizal development status, and helps save plant materials. Its disadvantage is that the results are not very accurate. To accurately examine mycorrhizal development and determine colonization rate, the staining method should be adopted, which generally involves the processes of root clearing, staining and differentiation.
Aladdin Lactofuchsin Staining Solution (0.01%) is mainly composed of lactic acid, glycerol, acid fuchsin and water. It is used for the staining and observation of molds, and also applicable to the staining research of arbuscular mycorrhizae. This reagent is for research use only and is not intended for clinical diagnosis or any other purposes.
Self-prepared Materials:
1. Distilled water, FAA fixative, 5%–10% KOH solution, 2% HCl solution
2. Microscope, water bath
Operating Procedures (For Reference Only):
1. Cut root segments or roots fixed with FAA into small pieces of 0.5–1.0 cm in length, place them into a test tube or other staining container, add 5%–10% KOH solution, and incubate in a water bath at 90 °C for 20–60 minutes.
2. Discard the alkaline solution, then rinse the roots gently with tap water for 3 times, and subsequently soak them in 2% HCl solution for acidification for 5 minutes.
3. Discard the acid solution, add lactofuchsin staining solution (0.01%), and incubate again in a water bath at 90 °C for 20–60 minutes or overnight at room temperature.
4. Add lactic acid for differentiation and then proceed with microscopic examination.
Staining Results:
Plant cells remain unstained (sometimes the stele is stained), while fungal tissues are stained red.
Precautions:
1. Generally, the clearing time required for the tender roots of herbaceous plants is relatively short, whereas that for the roots of woody plants is longer.
2. The 0.01% lactofuchsin staining solution can be recovered and reused.
3. For your safety and health, please wear a lab coat and disposable gloves during the operation.
4. Please use the reagent as soon as possible after opening to avoid affecting the results of subsequent experiments.
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