technical specifications

Tips:

Clearing: A good clearing of the root with KOH is essential to obtain good staining results. You have to adapt the clearing time (with KOH) to the roots you are working with. After clearing, roots should be transparent. Thick, old roots need more clearing time than, thin, young roots. If you clear roots too long, they will disintegrate and you will find only single root cells with the central cylinder of the root left.
We experienced that it is best to work with a water bath and not to heat the KOH directly in a tube.

) Ink:
(i) Check in the literature whether the type of ink you want to use has been already reported to stain the fungus, as some inks do not stain.
(ii) Alternatively, check first whether the ink you want to use really stains the fungus (stain a root piece where you know that it is colonized by a AM-fungi).
(iii) Once you publish your study give the precise description of the ink you used (unfortunately I observed that people only mention e.g,….black ink… but give no further details).
(iv) We obtained best results with a Shaeffer JetBlack ink.
(v) Instead of the 5% ink-vinegar solution (e.g., 5 ml ink in 95 ml vinegar) we tried as well a 4% ink-vinegar solution (e.g. 4 ml ink in 96 ml vinegar). It worked fine.

Observations: With any microscope you should clearly see the stained fungal structures. With a stereo-microscope structures are much easier to see when using a dark field illumination (unfortunately not all stereo microscopes do have a dark field illumination).

Storage of stained roots: We observed that when storing the stained roots in tap water for more than a week at room temperature, a fungus starts to grow, which after a time makes it impossible to clearly see the stained fungal structures in the root. If you want to keep your samples for a longer time before watching, keep them in the fridge (max. 2 weeks otherwise again you also will have a fungus growing) or replace the tap water for storage by ETOH (min 50%). In ETOH you can store your samples for a long time.

Destaining of roots: Probably you want to stain something else in the root after having quantified the AM-Fungi. You can destain the roots by putting the roots shortly back into KOH. You will see that the dye (ink) redisolves immediately in the KOH.

By the way, the same thing happens when the pH of your tap water is too high. Roots destain quickly and you might think that there is no fungal root colonization. For this reason (to lower the pH) we add some drops of vinegar to the tap water used for rinsing the stained roots.

Staining of other fungi: The described technique can also be used for the staining of other fungi such as Rhizoctonia solani. It seems to us that the chitin in the cell wall is the substance which is stained e.g. Phytophtora spp. is not stained (no chitin in the cell wall).