Archived Protocol

<HTML>   <HEAD> <TITLE>Sea Urchin gamete collection</TITLE> <META NAME=GENERATOR CONTENT="Claris Home Page 3.0"> <X-CLARIS-WINDOW TOP=140 BOTTOM=630 LEFT=65 RIGHT=811> <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=90 HEIGHT=1> <SPACER TYPE=block WIDTH=90 HEIGHT=1> </TD> <TD WIDTH=564 HEIGHT=1> <SPACER TYPE=block WIDTH=528 HEIGHT=1> </TD> <TD WIDTH=174 HEIGHT=1> <SPACER TYPE=block WIDTH=174 HEIGHT=1> </TD> </TR> <TR> <TD WIDTH=1 HEIGHT=86> <SPACER TYPE=block WIDTH=1 HEIGHT=86> </TD> <TD WIDTH=90 HEIGHT=86> </TD> <TD WIDTH=564 HEIGHT=86> <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=174 HEIGHT=86> </TD> </TR> <TR> <TD WIDTH=1 HEIGHT=526> <SPACER TYPE=block WIDTH=1 HEIGHT=526> </TD> <TD VALIGN=top WIDTH=90 HEIGHT=526> <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=91 content csheight=197 WIDTH=564 HEIGHT=526> <H3><A NAME=Heading1></A>Sea Urchin Gamete collection</H3> <SPACER TYPE=vertical SIZE=2> Spawning can be induced in adult sea urchins<SPACER TYPE=horizontal SIZE=6>by injecting 1 ml of a<SPACER TYPE=horizontal SIZE=6>0.5M KCl solution into <SPACER TYPE=vertical SIZE=5> several sites in the soft membrane around the mouth. Within minutes, the gametes should <SPACER TYPE=vertical SIZE=5> appear: the sperm are off-white, the eggs are tan to orange. Collect the sperm by inverting the <SPACER TYPE=vertical SIZE=5> male onto a small, dry petri dish. After several minutes, cover the sperm and put on ice. Collect <SPACER TYPE=vertical SIZE=5> the eggs by inverting the female onto a small beaker filled with <A HREF="urchin_solutions.html">ASW</A> (room temperature for <SPACER TYPE=vertical SIZE=5>L. variegatus, and below 14ºC for S.purpuratus and L.pictus ). Keep urchins from falling in <SPACER TYPE=vertical SIZE=5>with aluminum foil supports. Allow eggs to settle, pour off ASW and replace with fresh ASW <SPACER TYPE=vertical SIZE=5>(at same temperature). <EMBED SRC="movies/Sea_urchin_spawn.mov" PLUGINSPAGE="http://quicktime.apple.com" WIDTH=240 HEIGHT=196 CONTROLLER=true LOOP=false AUTOPLAY=false> Colletion of sperm (left) and eggs (right) from L. variegatus The sperm can be transfered to a small tube and stored at 4° C for up to 2 weeks. Eggs should normally be used on the same day as they are collected. Consult the <A HREF="http://www.stanford.edu/group/Urchin">Sea Urchin Embryology</A> web site for a protocol for collecting the eggs with antibiotics, which allows them to be stored for several weeks before use. Check to see if the population contains a large percentage of immature eggs; they will not fertilize. <IMG SRC="img_urchin/mature%26immature.jpg" WIDTH=576 HEIGHT=324 X-CLARIS-USEIMAGEWIDTH X-CLARIS-USEIMAGEHEIGHT ALIGN=bottom> <A HREF="urchin_prep_sheet.html">Instructor's prep sheet</A> Proceed to <A HREF="Urchin_fert.html">Fertilization</A> </TD> <TD WIDTH=174 HEIGHT=526> </TD> </TR> </TABLE> <CENTER>© 2001 Cebra-Thomas Last Modified: 29 August, 2001 <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>