Lab Protocol: Preparing for Mouse Intestine Dissection

Transcription

1 Lab Protocol: Preparing for Mouse Intestine Dissection A Guide for Peter Hudson Lab Undergraduate Researchers Whether you are new to the lab or simply need a refresher in lab protocols, this guide is designed to lead you through the steps of pre-dissection preparation. This guide provides instructions only for the preparation prior to intestine dissection, not for the dissection and parasite identification itself. Before You Begin: Time and Requirements You must be trained by Justin or another senior lab member before you dissect independently. Designate at least 2 hours for dissection [preparation should only take about 5 mins]. All tools are provided in the lab, but you should have all tools laid out ahead of retrieving a mouse in order to limit exposure to air. Materials Needed All of these materials are available in lab in either in the labeled drawers or on the counter adjacent to the sink. The water, ethanol and scissors may on occasion already be laid out in the fume hood. Note: Gloves should be worn at all time in the lab. Before retrieving your mouse, gather the listed items below and place in the fume hood. These materials include those to be used later for dissection since it is best to set up everything at once. Materials: o Latex gloves o 2 large petri dishes with lids o 2 small petri dishes with lids o Dissection scissors o 2 angled probes o Forceps o Spray bottle of 70% ethanol o Bottle of distilled (DI) water o Labeling tape and marker 1

2 IMPORTANT: Mouse fur and droppings can contain Hanta virus. It is easily aerosolized from dry specimens so be sure to keep carcass away from your face, spray carcass with ethanol to kill virus and decrease aerosolization, and wear gloves so as not to contaminate your clothes or belongings on departure. Always put carcass back into its bag and back in the freezer as soon as possible. FIG 1. Gloves Procedure: 1.Sign up. Make sure you have signed up in the weekly lab spreadsheet in your . You should give yourself at least two hours. Only two people can work in the lab at a time so sign-up is very important. 2.Put on Gloves. Once you ve arrived at the lab, immediately put on gloves before touching surfaces, mice or tools to prevent possible transmission of Hanta virus. 3. Gather supplies. Petri dishes, forceps, probes, scissors, DI water tand ethanol should be collected and transported to your primary tworkspace, the fume hood. All supplies will be located either on the tcounter next to the sink or already be in place in the hood. FIG2.-80 Freezer 4.Retrieve mouse from freezer. Next you will need to get your mouse from the -80 C freezer in the hallway. FIG C Freezer DO NOT remove mouse from bag unless in the fume hood. The mice you will be working with will be located on the shelf labeled Mice w/o organs need guts dissected. Do not use mice labeled for Viral Discovery or Non-Dissected. FIG3. Freezer label 2

3 FIG4. Dissection record binder 5. Record mouse in binder. Back in the lab, spray the baggy with ethanol and record your initials next to your mouse s ID# (found on top of bag) in the dissection binder. This is the binder on top of the chest freezer next to the fume hood. This is also where you will be recording your dissection findings later so it may be helpful to mark your page. Put bagged mouse in fume hood. Label your petri dishes using the labeling tape with your mouse s ID#. NOTE: The shield on the fume hood should be as low as possible to work with. This is to protect you from possibly inhaling Hanta virus from the carcass. 6. Unbag mouse. Take your mouse out of bag and spray, place on dissection tray (already in the hood) and spray mouse and bag with ethanol. 7.Pin mouse down. Using pins from tray, secure the mouse to the tray at the shoulders and hips. There is a flap of skin from the removal of the other organs. Pull up this flap of skin to expose intestines. FIG 5. Mouse position and anatomy. Stomach and gut outlined. 8. Remove digestive tract. The gut will already be cut at the end of the large intestine, just prior to the anus so only the stomach will need to be cut. Just above the stomach cut the esophagus and lift the tract into a large petri dish. Some membranes may need to be cut but at this point you should be able to gently lift the entire tract and place in the large petri dish. 3

4 9. Separate the intestine. Use the angled probe push the contents of the gut away from the stomach so the gut is translucent. Cut to separate from the stomach. Return the stomach to the mouse and cover intestine with just enough water to cover the bottom of the dish. 10. Return mouse. Unpin mouse and return to bag. Seal and spray bag with ethanol before putting it into the freezer on top of the refrigerator in the lab. FIG6. Lab refrigerator, freezer on top 11. Cut the intestine into segments. The intestine is usually dissected in roughly 5-6 cm segments. Using the angled probes and forceps, gently unravel the intestines and choose which end you will begin your dissection from. Parasites are more common in the small intestine so it is common to start there but is not necessary. Using the angled probes, press the contents of the gut away from where you intend to cut until intestine is translucent so that no worms are cut when the intestine is. Cut with scissors and using forceps move to small petri dish trying not to spill the contents. 12.Ready for dissection. Cover segment with just enough water to cover the bottom of the dish. Put lid labeled with mouse ID# on large petri dish containing the remainder of the tract. Take probes and small dish with gut segment to microscope for dissection. After this you will be ready for dissection. Troubleshooting: - If your gut seems too frozen to unravel enough to cut for dissection, spray with a little more water. It will take no more than a minute or two for the gut to thaw and separate. - If you have any questions or are unsure about these procedures, Kurt or Justin will be nearby or in their offices, Office 239A and the adjacent cubicle, to help you or look over your work. 4

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