I need to clear some rice roots, preserved in 70% ethanol, I am using 10% KOH overnight soaking and then heating at 90 degree for 15 to 20 minutes. I am using 10% sheaffer black ink for staining. But in some case the thin roots are degreded but not cleared and the thick roots are not cleared as well. Could anyone suggest me anything which might be helpful?
Cytological studies on the infection of rice root by Ustilaginoidea virens
January 2018Microscopy Research and Technique 81(2)
DOI: 10.1002/jemt.22990
Mingli YongYijia LiuTianqi ChenShow all 6 authorsDongwei Hu
Try to boil the roots for 2min, more or less, in lactophenol cotton blue under a hood. Preparation of lactophenol: 200g phenol, 400ml glycerol, 200ml lactic acid, 200ml deionized water. Concentration of cotton blue: 0,05% or less in order to have a good contrast of the blue stained fungal structures amongst the cleared plant tissues.
Many thanks Henri, I should try the protocol. Just need a thing to clear from you,
After clearing with lactophenol by boiling should i go for the staining at 0.05% cotton blue, and do i need to destaining after that for removal of excess ink?
Hello Monira,
I advise to boil in lactophenol containing cotton blue and to keep the roots later on in clear lactophenol awaiting examination under a stereo- or high magnification microscope. This allows some destaining. Depending on the roots, bowling time and cotton blue concentration may need to be adjusted. If the root tissues are stained too strongly, try bowling in 0,01 or 0,005% cotton blue by dilutions of the colored lactophenol with clear one. Thus prepare first a stock solution of lactophenol and add cotton blue to 100ml for trials.
All the best,
Henri
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