Note: Roots can be stored indefinitely in 70% ethanol at room temperature prior to staining. For long term storage, make sure they are in a well-sealed vessel where the ethanol can’t evaporate.

Materials:

Reagents

• Potassium hydroxide pellets
• “Fine pore” tissue casettes
• Black fountain pen ink such as “Pelikan Brilliant Black 4001”
• White vinegar or glacial acetic acid
• Glycerol (for storage)

Note on selection of inks: Pelikan black ink or shaeffer black ink both give good contrast. (The latter has become somewhat difficult to source). Blue inks are pretty and stain fungal structures effectively but tend to have poorer contrast.

Preparation of 5% acetic acid (“white vinegar”) from glacial acetic acid

• Prepare 5% white vinegar from glacial acetic acid (e.g., by adding 50 ml glacial acetic acid to 950 ml DI water). Glacial acetic acid should only be opened in the hood.

Preparation of 10% (w/v) potassium hydroxide

• Weigh 100 g of potassium hydroxide.
• Add 800 ml DI water to a 1 L vessel.
• Slowly a Add potassium hydroxide to water while mixing.
• Top off vessel to 1 L with DI water.

Preparation of storage solution (30% glycerol in vinegar)

• Mix glycerol with 5% acetic acid (white vinegar) in a 3:7 ratio. For example, to prepare 1 L, add 700 ml vinegar to 300 ml glycerol.

Preparation of (5%) ink solution for staining

• Add 12.5 ml of ink to a 250 ml vessel.
• Fill to 250 ml with 5% acetic acid.

Staining protocol

1. Place roots into “fine pore” tissue casettes.
2. Clear roots by boiling in 10% KOH for 3-5 minutes
3. Rinse roots with tap water
4. Stain roots for 3 minutes by boiling in 5% ink solution in white vinegar
5. Rinse roots with acidified tap water for 20 minutes (minimum).

To achieve greater contrast, roots can be optionally destained in “ClearSee” solution as follows:

1. Rinse roots with DI water to remove all traces of vinegar.
2. Place in clearsee solution and for 1-3 minutes
3. Rinse with DI water to remove all traces of clearsee.

Caution: Roots must be thoroughly rinsed with DI water before/after exposure to clearsee solution because Clearsee constituents precipitate under acidic conditions. Once precipitated, Clearsee can be very difficult to remove from roots.

1. After destaining, roots can be stored in the 30% glycerol storage solution.

Note: 24 well microplates are a convenient format for storing stained roots. To prevent evaporation, sealing film can be placed over the wells. For longer-term storage, roots can be refrigerated.

References

Vierheilig, H., Coughlan, A.P., Wyss, U. & Piché, Y. (1998). Ink and Vinegar, a Simple Staining Technique for Arbuscular-Mycorrhizal Fungi. Appl. Environ. Microbiol., 64, 5004–5007.

Kurihara, D., Mizuta, Y., Sato, Y. & Higashiyama, T. (2015). ClearSee: a rapid optical clearing reagent for whole-plant fluorescence imaging. Development, 142, 4168–4179.