Chapter 9 



TECHNIQUES FOR MICRODISSECTION AND PRESERVATION 



OF CULTURES 



THE DISSECTING NEEDLE 



We use a solid glass needle with a flat tip about 20 microns in 

 diameter for the isolation of single ascospores. The needle can be 

 made by hand as follows: A pyrex rod is drawn out in a flame to 

 make a small rod about a millimeter in diameter. This is broken 

 up into six-inch lengths. Two burners are used to make the tip: one 

 is the pilot light of an ordinary Bunsen burner and the other is a 26- 

 gauge hypodermic needle. The hyT)odermic needle and the Bunsen 

 burner are connected to the same gas jet by a T-tube; the flame of 

 the hypodermic needle is controlled by a screw clamp on the tubing. 

 The hypodermic needle is mounted by clamping it into the small jaw 

 of a burette holder. The room must be without drafts, otherwise the 

 small flame will blow out. A small guard to shield the flame from 

 drafts may be necessary. The six-inch pyrex rod, one millimeter 

 thick, is thinned out to about one tenth its thickness by melting and 

 drawing out in the pilot flame. The next step involves thinning out 

 the small end again down to a 20 -micron tip and bending at the same 

 time. This is done by bringing the small end of the pyrex rod over 

 the flame from the hypodermic needle while maintaining a slight 

 tension so that as soon as the glass softens it pulls out of the flame. 

 This produces a thin rod and a right angle bend at the same time. 

 The needle can be cut off using small manicure scissors. Several 

 trials may be required before a needle the right size with an even 

 end is produced. 



The technique of using flat-tipped needles for microdissection 

 was originally developed by Dickinson. With such a needle it is pos- 

 sible to move cells around over an agar or a glass surface, or to 

 remove them from it by the capillary water that adheres to the flat 

 needle surface. 



The needles can also be made by the de Fonbnme microforge 

 (fig. 9-1). In this apparatus an Incandescent platinum wire, in which 

 the heat can be controlled, is mounted before a microscope. The 

 operator can move the glass needle and the incandescent platinum 

 wire by a number of knurled knobs and perfect control of the melted 

 glass with continuous observation through the microscope is pos- 

 sible, (fig. 9-2). 



9-1 



