Rhodamine B Assay for Estimating Activity of Killer Toxins Permeabilizing Cytoplasmic Membranes

Killer yeasts secrete proteins (killer toxins, zymocins) that kill sensitive yeast strains (1 ,2 ). The killer strain is immune (R⁺ ) to the effects or its own toxin, but it can be sensitive to the toxins of another immunity group (2 ). The killing ability of strains (K⁺ ) is routinely screened (1 ) using buffered rich agar media containing methylene blue (the pH range usually from 4.2 to 4.8) thinly spread with potentially sensitive tester strain. Potential killers are replica plated or heavily streaked across the background lawn. Killer colonies are identified by clear zones usually fringed with blue-stained dead cells. The same assay can be used for distinguishing the sensitivity of selected strains to various killers. A well test (1 ) based on measuring the diameter of inhibition zones was evolved that enables killer toxin activity to be assayed in liquid media added to wells. Also the fraction of colony-forming cells after killer toxin treatment can be considered the measure of killer toxin activity. However, the assays based on the formation of colonies or inhibition zones last usually at least 2 d.