In order to observe the pathogens inside and outside the diseased leaves as well as other tissue sections, the entire tissue can be subjected to clearing treatment, which allows for clear visualization of both epiphytic pathogens and those residing within the tissue. Common methods for whole tissue clearing include the use of lactophenol, lactophenol cotton blue, chloral hydrate, lactophenol-chloral hydrate, chloral hydrate-phenol, lactic acid, pyridine, etc. Among these, the chloral hydrate method is the most frequently used.
Aladdin Lactic Acid Clearing Solution is mainly composed of lactic acid and water, and it is acidic. This product requires a relatively long time to achieve tissue clearing. This reagent is for research use only and is not intended for clinical diagnosis or any other purposes.
Self-prepared Materials:
1. Distilled water, lactophenol cotton blue staining solution, glycerol mounting medium
2. Gradient ethanol, xylene or environment-friendly dewaxing clearing solution, Canada balsam
3. Tweezers, microscope, glass slides
Operating Procedures (For Reference Only):
1. Rinse small pieces of diseased leaves thoroughly with water.
2. Use tweezers to immerse the diseased leaves in lactic acid clearing solution and soak for 2–5 days to achieve complete clearing.
3. Take out the tissues with tweezers, rinse them with distilled water, and place them on glass slides.
4. Pigmented mycelia and fruiting bodies in the tissues can be observed directly; for non-pigmented and transparent tissues, staining with lactophenol cotton blue followed by dehydration and mounting is required before observation.
5. Add a drop of glycerol mounting medium, cover with a coverslip, and proceed with microscopic observation.
6. If permanent slides are to be prepared, perform dehydration with gradient ethanol, treat with xylene or dewaxing clearing solution, and seal the slides with Canada balsam.
Precautions:
1. Lactic acid clearing solution is acidic; avoid direct contact with the skin.
2. Lactic acid requires a relatively long time to clear diseased tissues, so ensure that the clearing process is thorough.
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 prevent compromising subsequent experimental results.
