TECHNIQUES ^ *' 



course adjustment screw in the nose-piece permits an initial up- 

 ward-downward needle movement prior to operating the instrument. 

 A particular advantage in the de Fonbrune instrument is the pro- 

 vision of a collar around the vertical piston cylinder. An adjust- 

 ment of this collar regulates the working distance of the pistons and, 

 consequently, the latitude of movement of the dissecting needle in 

 the field of the microscope. Such an adjustment is especially de- 

 sirable since, lifting asci from a mass of cells in a water droplet 

 one requires a rapid movement of the dissecting needle (effected 

 by increasing the latitude of needle movement), while the actual dis- 

 section of the ascus and lifting of the ascospores needs to be done 

 within a narrower range of needle movements in order to avoid in- 

 jury to the spores during manipulation. 



THE MOIST CHAMBER 



An essential device used in micro-dissection is a rectang^ar 

 open-top moist chamber made of plastic cemented onto a 3 by 2 

 inch glass slide and placed on the mechanical stage of the micro- 

 scope. The outside measurements of the chamber are 2 by 1 inch 

 and the walls are about 3 mm. thick and 12 mm. high. It supports 

 two one inch No. 2 coverslips placed close to each other. One end 

 of the chamber has a short wall (8 mm. high) so that the dissecting 

 needle can be inserted to work on the bottom of the coverslip. This 

 is sufficient to reduce air currents within the chamber and lessens 

 the chances of contaminations while the latter is in actual use. 

 When in use, a humid atmosphere is maintained in the chamber to 

 prevent drying up of the droplet bearing the sporulated cells, and 

 the dessication of the agar droplets. The top lens of the condenser 

 is removed to increase the working distance of the condenser and 

 accurate focusing of the light source is obtained by laying a piece 

 of lens paper on the cover glass covering the moist chamber and 

 focusing the condenser until the maximum intensity of light appears 

 on the lens paper on top of the moist chamber. Control of the light 

 intensity by a rheostat makes it possible to use different magnifi- 

 cations without too much eye -strain. A small bottle of agar is kept 

 melted, using a 50 cc. beaker, as a water bath, over the pilot light 

 of a Bunsen burner (fig. 9-3). The sides of the moist chamber are 

 lined with filter paper kept moist by adding water from an eyedrop- 

 per. 



A very small quantity of sporulated yeast cells is collected from 

 a gjrpsimi block by means of a sterile inoculation needle and quickly 

 transferred to a drop of clean water on a sterile cover glass. The 

 cover glass is lifted with a pair of sterile forceps and inverted, 

 sporulated cell suspension downwards, over the rim of the dissec- 

 tion chamber. Next, another cover glass is flamed while held with 

 forceps, and four droplets of melted sterile agar are quickly de- 



