Root Staining

Yesterday, we began staining our MIP roots we had collected and washed over the last few weeks. Luckily for us, the new arbuscular ink and vinegar procedure is really simple compared to old fungi staining techniques. At its core, the procedure is only two steps. The first being boiling the roots (in tissue cassettes) in 10% potassium hydroxide (KOH) for 3-5 minutes. This clears the root cells of their contents, leaving the cell walls and fungi unharmed.

Boiling the roots in the 10% KOH solution.

The second major step is boiling the roots in a vinegar and ink solution for 3 minutes. This stains the fungal tissue within the cells, making it possible to see the hyphae (regular fungal body tissue) and vesicles (reproductive capsules).

10% KOH on the left hotplate and vinegar/ink on the right.

Intermediate steps include removing the cassettes from the hot solutions and rinsing them with water several times. Washing after the first step rinses the remaining KOH from the roots. Washing after the second step initially rinses the excess ink which will leach from the roots.

Susanna removing the cassettes from the solution into water.

Because we are boiling the solutions, the beakers and hot plates obviously get really hot. We got the chance to open and use our brand new thermal safety gloves. They are thick, orange, and terry cloth. Embarrassingly, this is the first pair of elbow length gloves I've ever worn.

Me, looking snazzy and being safe.

Our final product is a beaker full of stained roots, all ready to be mounted on slides. We are temporarily storing them in a beaker of water with a splash of vinegar in the fridge.

Purple haze, all in my roots.