9-6 THE YEAST CELL 



posited on this cover glass (fig. 9-4) and the latter placed, drop- 

 lets downward, over the rim of the moist chamber close to the cov- 

 erslip bearing the droplet of sporulated yeast (fig. 9-5). 



DISSECTION 



When the coverslips have been placed in position, the microma- 

 nipulator is ready for use. The tip of the dissecting needle is cen- 

 tered in the field of the microscope with the aid of the low power 

 lens. Spores and needle point are now centered under the 16 -milli- 

 meter lens. Once this has been achieved, needle and cells are cen- 

 tered under a 4 -millimeter, 0.65 N.A., objective. An 0.85 objective 

 has too short a working distance for this purpose. Using the course 

 adjustment screw on the manipulator nosepiece, the needle is brought 

 to within approximately the same plane of focus as the cells in the 

 water droplet. A large four-spored ascus from the fringe of the 

 water droplet is next pulled out deftly and brought to the dry part of 

 the cover glass. This is done with a rapid movement of the needle. 

 Increased latitude (and therefore rapidity) of the needle movement 

 can be secured by adjusting the collar attached to the vertical piston 

 cylinder. It is particularly essential that one employs a quick move- 

 ment to separate asci from the water droplet since forces of sur- 

 face tension tend to prevent a single cell from being pulled out from 

 the fringe of the water drop onto the dry part of the cover slip. The 

 tip of the dissecting needle is then brought directly beneath the iso- 

 lated ascus and with a careful movement the ascus is lifted from 

 the coverslip and the needle lowered clear of the lower surface of 

 the coverslip. The ascus is now on the needle and ready to be de- 

 posited on the second coverslip which bears the agar droplets. To 

 do this, the moist chamber is moved with the mechanical stage un- 

 til the agar droplets are within the field of the lowest power ob- 

 jective. The edge of one of the agar drops is then brought within 

 the focus of the high power objective and the ascus is deposited very 

 close to it. With sufficient practice it is always possible to locate 

 the exact site of deposition of the ascus even after it has moved out 

 of the field of the microscope during the succeeding operations. It 

 is a good plan always to deposit an ascus at a fixed border of any 

 one of the four agar droplets, as this makes it easier to return to 

 the same site after separation and deposition of each spore onto an 

 agar drop. 



Dissection of an ascus must be performed very delicately; else 

 injury to the ascus will result and cause the collapse of one or more 

 spores. In order to rupture the ascus wall and liberate the contained 

 ascospores, direct pressure is never exerted on the ascus. Rather, 

 the latter is gently rolled about until the ascus wall collapses. 

 Freshly sporulated cells are more amenable to this operation for 

 young spores withstand manipulation to a greater degree than do those 



