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 a timely and general understanding of mycorrhizal development, and can save plant materials. Its disadvantage is that the results are not very accurate. To accurately examine mycorrhizal development and determine the colonization rate, the staining method must be adopted, which generally involves the processes of root clearing, staining, and differentiation.
Aladdin Lactate Trypan Blue Staining Solution (0.05%) is mainly composed of lactic acid, glycerol, trypan blue 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, lactate trypan blue staining and destaining 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 10–20 minutes.
2. Discard the solution and rinse the roots thoroughly under running water. Add alkaline hydrogen peroxide, leave it at room temperature for 15 minutes, then discard the solution and rinse again under running water.
3. Add 2% HCl solution to submerge the roots, incubate at room temperature for 5–20 minutes, discard the solution and rinse under running water.
4. Add 0.05% lactate trypan blue staining solution, and stain either in a water bath at 90 °C for 30–60 minutes or overnight at room temperature.
5. Discard the staining solution and rinse under running water. Add lactate trypan blue destaining solution and destain at room temperature for 1–2 days until only the AMF in the mycorrhizae shows blue coloration, with no excess color remaining in the root tissues.
6. Place a drop of glycerol on a glass slide, mount the root segments, seal the slide and observe under a microscope.
Staining Results:
Plant cells remain unstained (sometimes the stele is stained), while fungal tissues are stained blue.
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 0.05% lactate trypan blue 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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