MICRURGICAL TECHNIQUES FOR NEUROSPORA

WILSON, JAMES F. (Hartnell Coll., Salinas, Calif.) Micrurgical techniques for Neurospora. Amer. Jour. Bot. 48(1): 46-51. Illus. 1961.-Procedures for carrying out micrurgical operations on the mold, Neurospora crassa, are described. These procedures include interstrain transfer of protoplasm, micro-injection of fluids, excision and culture of injected cells. The special microtools and culture methods for these micrurgical procedures are discussed. SINCE the time Barber developed his micro-injection apparatus (Barber, 1911a), only a few investigators have made micro-injections into plant cells (Barber, 1911b; Chambers and Kerr, 1932; Kerr, 1933; Hofmeister, 1940), and apparently only Barber (1911b) has successfully injected fungi. The present paper is a description of techniques developed specifically for use with the mold Neurospora crassa-techniques which reduce micro-injecting to a routine performance in which 60-80 injections can be made in 1 or 2 hr. The techniques needed for micro-injecting Neurospora are considerably different from those used on animal cells. The morphological and physiological characteristics of Neurospora and their variability under different environmental conditions make the application of standard micrurgical techniques extremely difficult. Instead of individual cells in hanging drops, coverslip microcultures growing on agar films are used. To penetrate the tough cell wall, a relatively large micropipette with a tip like a hypodermic needle is necessary. The microcultures are placed in a hypertonic solution to reduce the internal pressure, and the cells are injected indirectly through the septal pores. The microtools, culture methods, and micrurgical procedures used are described in the following sections. EQUIPMENT.-A microforge is necessary to make most of the tools needed. For the present work, a microforge was constructed by mounting an electrically heated platinum filament on a Taylor micromanipulator (Taylor, 1925). The filament holder was made from a platinum electrode (Harvard Apparatus Co.) by removing the terminals and replacing them with heavy copper wires cut off so that they projected 1 cm. from the holder. A piece of 28-gauge platinum wire, 18 mm. long, was soldered to the copper wires to form a V-shaped filament. The filament was heated with AC current stepped down and controlled with a Powerstat Vari1 Received for publication April 13, 1960. This paper is a portion of a dissertation submitted to the Department of Biological Sciences and the Committee on Graduate Study of Stanford University in partial fulfillment of the requirements for the degree of Doctor of Philosophy. The work was done during the tenure of a National Science Foundation pre-doctoral fellowship. The author wishes to thank Dr. E. L. Tatum for his advice and help in developing this technique, and for his critical reading of the paper able Transformer (Superior Electric Co.). The voltage potential across the leads of the filament holder varied from 700 to 900 millivolts at minimum and maximum operating temperatures of the platinum loop. The filament holder was mounted in one instrument holder of the Taylor micromanipulator, and the microtools either in the other instrument holder or in a flat-jawed clamp mounted in the holder. For observing the microforge operations, a triple objective dissecting microscope with a substage and 15X oculars was used in a tilted position. The most commonly used objectives were 1.3X and 6X. The substage mirror and glass stage were removed and the operations illuminated directly by a small twin-tubefluorescent lamp. The micrurgical operations were carried out with a de Fonbrune micromanipulator and a standard binocular microscope equipped with 15X wide field oculars, and lOX and 43X objectives. MICROTOOLS.-Although micropipettes useful for many purposes can be made by hand, when employed with Neurospora, such pipettes plug almost immediately with coagulated protoplasm. Kopac (1955) has recommended coating pipettes with silicones and filling them with silicone oil to lessen the tendency of protoplasm to coagulate, but filling hand-drawn pipettes with oil is impractical without special equipment. However, micropipettes made on a microforge can be filled with silicone oil, and can be made with sharp tips and larger apertures; these reduce difficulties in penetration of the cell wall and coagulation of protoplasm. These larger pipettes are made from 1-mm. capillary tubing (Aloe Co.) The tubing is cleaned thoroughly and coated with Desicote (Beckman Instrument Co.). The tubes are then pulled down by hand to 0.2-0.3 mm. outside diameter, while preserving as large a lumen as possible, and broken off in suitable lengths. For this operation, a microburner such as described by Chambers and Kopac (1950) is used. The pulled-out end is bent into a hook, and the capillary tubing is mounted in a de Fonbrunie pipette holder equipped with a chuck, or in a plain pipette holder with DeKhotinsky cement. The pipette holder and capillary are filled with DC 200 silicone oil, 50 centistokes viscosity (Dow Corning Co.) from a glass syringe contain-