Effects of Various Stabilizers on the Formation and Stabilities of Protoplasts from Aspergillus flavus

Protoplasts of Aspergillus flavus were successfully obtained by treating the young mycelia with snail gut juice. Effective formation and stabilization of the protoplast were achieved by using 1.0M MgSO_4 as a stabilizer. The method for formation of the protoplasts was as follows : 1. Aspergillus flavus was cultivated for 5 days on malt extract agar to allow conidia to form. 2. The conidia were collected by centrifugation and inoculated (10^7 conidia/ml) into Sabouraud's glucose medium supplemented with 0.5% yeast extract (Difco) and cultivated at 30℃. 3. After 16-24 hours, resulting mycelia were collected, washed on a filter paper and resuspended in 0.005M citrate-phosphate buffer solution (CPBS, pH 6.0) containing 10% snail gut juice and 1.0M MgSO_4 as a stabilizer. 4. After incubation for 4∿5 hours with gentle shaking at 30℃, the reaction mixture was subjected to filtration through a filter paper (Toyo Roshi No.1, Toyo Roshi Ltd., Tokyo) for separation of protoplasts from intact mycelia or debris.