Propidium Iodide Staining on Agar Plates

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This protocol is for staining fruiting bodies on agar/agarose plates with propidium iodide (PI).

1. Put on gloves to prevent the PI stain from contacting your skin.

2. Remove an aliquot of PI stain (one of the PCR tubes of purple PI stain) from the -20C freezer.

3. Once thawed, dilute the PI stain 1:100 in TPM (2uL into 198uL for example). You need 20ul of diluted PI solution per spot that you want to stain, so make sure to calculate enough.

4. Mix the PI stain thoroughly and drop a 20uL droplet of PI stain over each spot of fruiting bodies, trying carefully to cover the entire spot but do not touch the agar or cells with the pipet tip.

5. DO NOT move the plates and leave uncovered to incubate at room temp by the bunsen burner for 10-15min, or until the PI spot has completely dried. Cover.

6. Capture images in brightfield and using the dsRED fluorescent filter on the Zeiss dissecting microscope. Typically about 400ms exposure at 25% intensity is enough to capture good images of the PI stain.