Fling, M.

Horowitz, N. H.

MacLeod, H.

Fungal Genetics Reports

English

M. Fling

N. H. Horowitz

H. MacLeod

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Large scale culture of Neurospora

Kansas State University

Fungal Genetics Reports Volume 3 Article 7 Large scale culture of Neurospora M. Fling N. H. Horowitz H. MacLeod Follow this and additional works at: https://newprairiepress.org/fgr This work is licensed under a Creative Commons Attribution-Share Alike 4.0 License. Recommended Citation Fling, M., N.H. Horowitz, and H. MacLeod (1963) "Large scale culture of Neurospora," Fungal Genetics Reports: Vol. 3, Article 7. https://doi.org/10.4148/1941-4765.2152 This Technical Note is brought to you for free and open access by New Prairie Press. It has been accepted for inclusion in Fungal Genetics Reports by an authorized administrator of New Prairie Press. For more information, please contact cads@k-state.edu. Large scale culture of Neurospora Abstract Large scale culture of Neurospora This technical note is available in Fungal Genetics Reports: https://newprairiepress.org/fgr/vol3/iss1/7 to IO hours for the duration of the experiment. Under no circumstances, however, ore the conidio per- mitted to germinate sufficiently to form o mycelium which con fragment. Filtration is accomplished by pouring the contents of the incubated flasks through 4 layers of cheese cloth (grade 60) into an empty sterile 500 ml. flask. The cheese cloth, held in place by o rubber band, forms o bag hanging into the mouth of the empty flask. The filter is covered with aluminum foil or paper during autoclaving and storage. To aid the germination of chemical-or ultraviolet-treated conidia,one- third of the original volume of fresh Fries minimal medium (e.g., 100 ml. ) is added to the incubation flask after the first 24 hours. Both chemical and ultraviolet treatments appear to cause delayed erratic germination of conidia. An initial washing of the treated conidia prior to placement in the filtration flasks does not seem to eliminate this effect. The addition of the new medium, however, appears to help overcome this delayed germination phenomenon. Plating: The suspension is plated on o Fries-sorbose minimal medium with added supplementation for the type of mutant desired. Under the experimental conditions described the first sample from the filtration flasks con be plated after 40 hours. At this time the sample should be clear with no obvious germination. Mutant recovery should be adequate if in o treated sample of 0.5 ml. plated at 40 hours there ore no more than 50 colonies present. At 40 hours the number of colonies from o control sample should be no more than 5 to IO/O.5 ml. It is advisable to begin plating os early (IS feasible if the survival of the mutant type which is desired is not optimal under starvation conditions. ---Biology Department, Yale University, New Haven, Connecticut, U.S.A. Fling, M., N. H. Horowitz, and H. In order to obtain the large quantities of mycelium Macleod. Large stole culture of Neurospom. necessary for enzyme isolation, we are now growing Neurospora in o converted washing machine. The machine is o top-loading automatic washer of the type which spins the water out through o single row of holes around the to of the inner tub (Westinghouse model LUC 27). The holes have been covered with o strip of stainless 30 wire mesh and the top of the inner tub fitted with o removable plexiglass cover con- ! !  taining two one-inch ports, one for an air line and one for introducing medium. The blades on the agitator have been cut down so that the center spindle is smooth and the blades at the bottom ore about on inch high. Aeration is through o ring of heavy rubber tubing ( 15/16” OD, 3/8” ID) in which sixteen 0.07” holes have been drilled along the inner side. The ring rests on the bottom of the tub and fits closely to the sides of the tub. It is connected with two plastic T-tubes to two vertical lengths of rubber tubing which are secured to fittings on the underside of the plexiglass lid. This support enables the aerator to go through the spin cycle of the machine without damage. Aeration is through one of the vertical lengths of tubing which passes through one of the ports in the lid. (The air line is disconnected before spinning!) The other vertical length of tubing ends blindly. Sterilization is accomplished by filling the tub half-full (g. 25 liters) with hot (60’) top-water and adding 1600 ml. of 5.25% sodium hypochlorite solution (ordinary household bleach), plus 100 ml. con- centrated HCI. This brings the pH to about 7.5 and liberates chlorine gas. The solution is left in the machine for 30 minutes, with agitation. It is then spun out, the tube is rinsed once with 8 liters of sterile water and is then filled with 40 liters of sterile medium. (The medium is outoclaved in corboys and siphoned into the tub). Both oemtion and agitation ore used during growth. The machine’s pro- grammer is disconnected and is replaced by o timer which opemtes the agitator approximately half of the time (8 minutes on, 12 minutes off), thus preventing overheating of the bearings. A fan blowing across the motor prevents transfer of heat to the medium. Under these conditions, the temperature of the medium has been 22-25’. (The machine is not located in o constant-temperature room). We have found the modified machine particularly useful for inducing tyrosinase synthesis in Neurosporo. After 3 days’ growth, we spin off the medium, wash the mycelium twice with sterile water and then re- suspend it in sterile phosphate buffer. Yield of mycelium after induction has been 300-500 grams wet weight. (The mycelium loses about 25% of its weight during induction). This work was supported by the National Science Foundation. --- Biology Division, California Institute of Technology, Pawdena, Cali- fornia, U.S.A. 

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Large scale culture of Neurospora

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