● Answer the questions in Part A of the Lab Report.
• Inject 1-2 mL of isotonic KCl (0.53M = 3.9%) through the perisomal membrane into the perivisceral cavity.
• Be sure to penetrate the cavity, but not much farther.
• This will cause smooth muscles of gonads to contract and spawn their gametes.
• After2-5 minutes, repeat injection to achieve a heavier spawning.
• Spawning should begin a few minutes after injection.
• As soon as spawning begins check the color of your gametes to tell the sex of the animals.
–For males the spawn will be creamy white.
–For females the eggs will be yellow, pink or dark red depending on the species.
–If you have a male - immediately pour off first sperm to get rid of perivisceral fluid. This will interfere with the sperm ability to fertilize. Then allow the animals to shed into the watch glass or petri dish without diluting the sperm (called dry sperm). Dry sperm are good for 6-10 days at room temperature and even longer in the fridge.
–If you have a female– allow the female to shed eggs into seawater by placing her, inverted on top of a beaker of seawater. The beaker should be small enough that she doesn’t fall in and must be full enough that the seawater touches her aboral side. As the animals sheds, the eggs will drift down through the seawater and settle in the bottom of the beaker. After shedding is complete, decant off the seawater and add fresh seawater. Repeat 2X. This washes the eggs of perivisceral fluid which interferes with fertilization.
•Place a drop of sea urchin eggs, diluted with sea water on a depression slide. DO NOT add a coverslip yet and be careful not to get objective lenses wet.
• Inducing spawning with KCl will cause the release of both mature and immature eggs.
• Answer the questions 5 & 6 in Part B of the Lab Report.
• Observe freshly spawned eggs.
• Diagram an immature egg and a mature egg.
• Answer the remaining questions in Part B of the Lab Report.
NOTE - if no Sea Urchins are available your instructor will provide you with images to complete this part of the lab.
• To fertilize the eggs first make a standard sperm suspension of 1 drop of dry sperm in 10 ml seawater.
–You must use this solution within 20 minutes. Diluted sperm become active and quickly use up their energy stores.
–Note the time you made your standard sperm solution.
• With the low power (10x) objective still in focus on your eggs move objective lens slightly to one side and add 1 drop of living sperm cells and add a coverslip.
• Re-align the low power objective lens and immediately observe the cells
• Note the time it takes for fertilization envelope to rise.
• Observe a fertilized eggs (look for thin membrane “halo” surrounding the egg).
• Answer the question 10 in Part C of the Lab Report.
• Diagram a fertilized egg and an unfertilized egg.
• Label the parts of the fertilized egg listed in the Lab Report.
• Answer the remaining questions in Part C of the Lab Report.
NOTE - if no Sea Urchins are available your instructor will provide you with images to complete this part of the lab.
• Repeat the steps above but use pH 5 seawater instead of the regular seawater to observe fertilization in pH 5 seawater
• Answer questions 22-28 in Part D of the Lab Report.
• Repeat the steps above using sodium free water to observe fertilization in sodium free water.
• Answer questions 29-34 in Part D of the Lab Report.
• Try to answer the bonus question #35 in Part D of the Lab Report.
NOTE - if no Sea Urchins are available your instructor will provide you with images to complete this part of the lab.
• Look at eggs that were fertilized this morning.
• Look at prepared slides.
• Answer the question 36 in Part E of the Lab Report.
• Diagram and label a blastula stage embryo.
• Observe Prepared slides of sea urchin development and make notes and diagrams in the space provided in Part E of the Lab Report.