produced in the following way mycelium from a flask was suspended in 15 ml of a freshly prepared and sterile filtered solution of the lytic enzyme mixture novozym 234 3 mg ml novo nordisk bagsvaerd denmark in omt medium in a sterile erlenmeyer flask the mycelium resuspended in the enzyme solution was incubated at 30 u00b0 c on a shaking incubator infors at a low speed 80 rpm for 1 to 3 h during the incubation the formation of the protoplasts was observed under the microscope freely moving protoplasts were normally to be seen after 1 h the end point of the protoplasting was determined by visual inspection under the microscope and the protoplasts were separated from the remaining mycelium by filtration through glass wool in a glass filter the glass wool was carefully washed with ice cold omt medium protoplasts were isolated by centrifuging the suspension in a sterile sample vessel 2 000 rpm 2 500 u00d7 g 4 u00b0 c 10 min further processing of the cells took place at 4 u00b0 c the protoplast pellet was washed by suspension in omt medium and was reisolated by centrifugation the washing step was repeated if required the concentration of protoplasts was determined in a counting chamber under the microscope the protoplast suspension was.