2] Micromanipulation and dissection of asci

Abstract

Publisher Summary This chapter describes the micromanipulation and dissection of asci. Separation of the four ascospores from individual asci by micromanipulation is required for meiotic genetic analyses and for the construction of strains with specific markers. In addition, micromanipulation is used to separate zygotes from mass-mating mixtures and, less routinely, for positioning of vegetative cells and spores for mating purposes and for single-cell analyses. Micromanipulation can be implemented directly on the surfaces of ordinary petridishes filled with nutrient medium or in special chambers on thin agar slabs. The petridish is positioned so that the inoculum is in the microscope field over the microneedle. Examination of the streak should reveal the presence of the desired four-spored clusters as well as smaller clusters and vegetative cells. The relocation and transfer of ascospores, zygotes, and vegetative cells are almost exclusively carded out on agar surfaces with a fine glass microneedle mounted in the path of a microscope objective and controlled by a micromanipulator. Although specialized equipment and some experience are required to carry out these procedures, most workers can acquire proficiency with a few days of practice.