| Popis: |
Publisher Summary This chapter describes the basic procedures for handling echinoderm oocytes, with particular emphasis on sea urchin oocytes. Other echinoderm oocytes, especially starfish, have received a great deal of attention and such references will be provided along with a brief description of handling procedures. Oocytes are far more vulnerable to mechanical, chemical, and bacterial demise than the resultant eggs or embryos. Sea urchin oocytes removed from their ovarian environment are viable in vitro and can be cultured for up to several days. These cells are fragile and are best used for experiments right away, with the least handling possible. The most important consideration for culture of sea urchin oocytes is a sterile environment. Oocytes are not equipped to counter microbial invaders and are easily overtaken by bacteria and other microorganisms in the seawater. To prevent contamination of the oocyte culture, it is essential to use filter-sterilized artificial sea water (ASW), supplemented with 100 ug/mL ampicillin. For prolonged culture, it is critical to change the oocyte incubation medium once every 12 h. The best containers for in vitro culture are either silicon-coated glass Pyrex multiwell spot plates, or Falcon plastic 96-well microtiter plates. These containers need to be stored in a humid chamber to prevent excessive evaporation of seawater. |
