Iodine staining (based on Bresch et al 1968)
After meiosis each newly formed haploid will become a spore, of which four together form an ascus. These spores contain starch which the spore uses during germination and which we can use to detect spores at the macroscopic level by staining for starch using Iodine vapour.
- On a plate to induce mating EMM-lowN or EMM-N
- Patch the strain to be tested
- Plate out a population or mix in a low dilution to obtain single cell colonies
- Incubate the plate for 2 to 7 days at 25°C (longer incubation gives clearer result)
Make sure you do the rest of the protocol in the fumehood!!. Iodine sublimates readily and the gasses are slightly toxic due to their oxidative character.
- pour into an empty petridish some iodine beads
- place the to be tested plate over it (upside down). Make sure the beads don't get onto your probably slightly electrostatic plate.
- Wait for 1 to 2 minutes; the longer you wait the stronger the effect, but the more likely the toxic Iodine fumes will have killed your cells.
- If a patch or colony does not stain completely yellow, it is homothallic, or was a mix of cells of two opposite mating types. Not yellow for the standard labstrain means that the colony turns black when stained long enough, but for many natural isolates and mutants, this can be anything from purple at the edges, faintly black/purple, black with yellow spots etc.
Iodine/ Potassium iodide (IKI) staining
To stain cell walls of pombe ascospores for microscopic and flow cytometric studies use a 2% IKI solution
- distribute some cells in liquid on a glass slide
- Add a drop of equal volume IKI to the cells
- The spores should become darkly stained and can be more easily be visually distinguished from other cells