Shake the cells for 5 min, set in the hood 1h with occasional swirling. Pellet cells, wash two times with dH20. Resuspend cells in an eppendorf tube. Incubate cells at 37 C for 30-45 min. Check spheroplasting under the phase microscope.
Unspheroplasted cells are phase bright, spheroplasted cells are phase dark.
Do this while cells are spheroplasting. Inc. 5 min at room temp. Remove excess polylysine. Let slides dry. Wash extensively by running distilled water over slide.