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<HTML>   <HEAD> <TITLE>Sea Urchin fertilization</TITLE> <META NAME=GENERATOR CONTENT="Claris Home Page 3.0"> <X-CLARIS-WINDOW TOP=220 BOTTOM=658 LEFT=199 RIGHT=844> <X-CLARIS-TAGVIEW MODE=minimal> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 3.2//EN"> </HEAD> <BODY BGCOLOR="#FFFFFF" VLINK="#660099"> <TABLE BORDER=0 CELLSPACING=0 CELLPADDING=3 WIDTH=793 HEIGHT=613 cool gridx=16 showgridx gridy=16 showgridy> <TR> <TD WIDTH=1 HEIGHT=1> </TD> <TD WIDTH=104 HEIGHT=1> <SPACER TYPE=block WIDTH=108 HEIGHT=1> </TD> <TD WIDTH=475 HEIGHT=1> <SPACER TYPE=block WIDTH=475 HEIGHT=1> </TD> <TD WIDTH=209 HEIGHT=1> <SPACER TYPE=block WIDTH=209 HEIGHT=1> </TD> </TR> <TR> <TD WIDTH=1 HEIGHT=72> <SPACER TYPE=block WIDTH=1 HEIGHT=72> </TD> <TD WIDTH=104 HEIGHT=72> </TD> <TD WIDTH=475 HEIGHT=72> <MAP NAME=map1> <AREA SHAPE=rect COORDS="329,10,387,49" HREF="../Mouse/mouse_protocols.html"> <AREA SHAPE=rect COORDS="276,7,325,51" HREF="../Chick/chick_protocols.html"> <AREA SHAPE=rect COORDS="184,6,274,54" HREF="../Frog/frog_protocols.html"> <AREA SHAPE=rect COORDS="137,3,178,57" HREF="../Fish/fish_protocols.html"> <AREA SHAPE=rect COORDS="1,3,75,60" HREF="../DB_lab.html"> </MAP><IMG USEMAP="#map1" SRC="imgs/top_bar.gif" WIDTH=396 HEIGHT=63 X-CLARIS-USEIMAGEWIDTH X-CLARIS-USEIMAGEHEIGHT ALIGN=bottom> </TD> <TD WIDTH=209 HEIGHT=72> </TD> </TR> <TR> <TD WIDTH=1 HEIGHT=540> <SPACER TYPE=block WIDTH=1 HEIGHT=540> </TD> <TD VALIGN=top WIDTH=104 HEIGHT=540> <MAP NAME=map2> <AREA SHAPE=rect COORDS="0,125,79,153" HREF="urchin_adv.html"> <AREA SHAPE=rect COORDS="2,52,76,79" HREF="urchin_notes.html"> <AREA SHAPE=rect COORDS="1,88,57,115" HREF="urchin_stage.html"> <AREA SHAPE=rect COORDS="0,15,69,45" HREF="urchin_basic.html"> </MAP><IMG USEMAP="#map2" SRC="imgs/side_bar.gif" WIDTH=81 HEIGHT=211 X-CLARIS-USEIMAGEWIDTH X-CLARIS-USEIMAGEHEIGHT ALIGN=bottom> </TD> <TD VALIGN=top xpos=109 content csheight=193 WIDTH=475 HEIGHT=540> Sea Urchin Fertilization Gametes are collected as <A HREF="Urchin_gamete.html">described</A>. To remove some of their jelly coat,<SPACER TYPE=horizontal SIZE=6>wash eggs several times in ASW by allowing them to settle and gently pouring off excess liquid. Transfer enough eggs to barely cover the bottom to 10 ml ASW in a 50 ml beaker. <SPACER TYPE=horizontal SIZE=9>Sperm will only remain viable for a short time in water. Collect them in a dry petri dish. Before use, dilute 50 ul of sperm in 5 ml ASW (diluted sperm). Use 0.1 ml of diluted sperm to fertilize eggs in 10 ml of ASW. Check for the presence of fertilization membranes after 5 and 10 minutes. Discard the ASW containing sperm and replace with 40 ml fresh ASW. For long term cultures, scale up 4 fold and transfer to shallow finger bowl after washing fertilized embryos. <TABLE BORDER=0 CELLPADDING=4> <TR> <TD> <IMG SRC="img_urchin/sperm%26egg.JPG" WIDTH=216 HEIGHT=206 X-CLARIS-USEIMAGEWIDTH X-CLARIS-USEIMAGEHEIGHT ALIGN=bottom> Sea urchin egg surrounded by sperm burrowing through jelly coat </TD> <TD VALIGN=top> <IMG SRC="img_urchin/zygote.jpg" WIDTH=216 HEIGHT=186 X-CLARIS-USEIMAGEWIDTH X-CLARIS-USEIMAGEHEIGHT ALIGN=top> Zygote with fertilization membrane </TD> </TR> </TABLE> <A HREF="urchin_prep_sheet.html">Instructor's prep sheet</A> </TD> <TD WIDTH=209 HEIGHT=540> </TD> </TR> </TABLE> <CENTER>© Cebra-Thomas, 2003 Last Modified: 23 January, 2003 <A HREF="Desktop%20Folder/research.html"> <HR ALIGN=left> </A>[<A HREF="../DB_protocols.html">Lab Protocols</A> | <A HREF="../DB_student.html">Students</A> | <A HREF="../CebraThomas.html">Cebra-Thomas</A> | <A HREF="../Bio_306.html">Course</A> | <A HREF="../links.html">Links</A><A HREF="Desktop%20Folder/research.html"> </A>]</CENTER> <A HREF="Desktop%20Folder/research.html"> <HR ALIGN=left> </A> </BODY> </HTML>