Sean Lauber:Mouse Endotracheal Intubation: Difference between revisions
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Revision as of 22:41, 10 July 2013
JANE ANN is the person to train you. She has all the equipment and is probably the nicest person in the world. She won't laugh at you if you fail at your first attempt. Note that you will fail your first attempt.
Arrange with her before you plan to intubate. She has a calendar and you need to mark down when you plan on using her equipment. Remember that it is HER equipment and you must show respect for it. When you're done using the equipment, clean it with vitrol in the CAF, dry the canulas with the pressurized air, and put the stuff back where you found it. If you killed one of the two batteries for the ottoscope, make sure you arrange to have it charged (unscrew the ottoscope and plug it in overnight). Once the battery is charged, write the date that it was charged on a piece of tape and place it in the spare battery box. Please remmeber to charge the battery. It is horrible if you're downstairs in the CAF and you've run out of battery power, because at this point you cannot continue your experiment.
- Collect the necessary components for the intubation:
The rack The ottoscope The ottoscope fittings The canulas The spare battery Masking tape PBS Virus Calculations for virus (if you're making the virus in the CAF) Ependorffs 2x P200 pipettes Filter tips Regular tips Forceps Isoflurane
- Prepare your virus
- Prepare the rack and accessories (place folded tape over the rack, set the rack at the furthest notch, assemble the ottoscope, remove the canula from its box)
- Prepare your pipette for confirming entry in trachea (load a p200 with a non-filter tip and set it to 150 ul, draw up about 75 ul of PBS and draw air the rest of the way)
- Load 50 ul into the other pipette with a filtered tip
- Place 1 mouse in the isoflurane chamber (at the 5th setting)
- Once the mouse starts showing hunched breathing (about 2 minutes) wait another 30 seconds
- Remove the mouse from the chamber by the base of the tail (for better stabilty in arranging the head) and place it on the rack, moving its head to hang it from its teeth using the thinner of the two strings
- Secure the tape on around the chest of the mouse
- Turn the ottoscope on and pick up the forceps
- Lift the mouse's tongue with the forceps and enter the mouse's mouth with the attached ottoscope piece (don't jab it in too far or you'll injure the mouse)
- While looking through the magnifier on the ottoscope, and with a lifting (of the tongue) and pulling motion, draw back until you see the opening to the trachea (the vocal cords should open and close as the mouse breathes)
- Once the vocal cords are located, grab the canula and insert it into the trachea (easier said then done) - again, do not put it too deep down the trachea
- Hold the canula with one hand as you turn off and place the ottoscope down
- Remove the insert in the canula and get the pipette to confirm you've gained access. Make sure the pipette tip is forming a good seal so you can note the breathing. If the mouse stops breathing you can press on its chest (sometimes works) or remove the pipette tip and wait for it to start breathing again, and then put the tip back. Occasionally the PBS in the tip will splash around and will not move up and down with the mouse's breathing. Simply repace the tip and get new PBS.
- Once you've confirmed you're in the trachea you can trade for the virus pipette and eject the liquid into the canula. The mouse should inhale it almost instantly. Watch the opening in the canula to make sure it's all gone before removing it. The mouse should inhale it deeply and there shouldn't be any trouble. You shouldn't have to force the liquid into the trachea, although I have done this in extreme situations. The mouse should be in a state of anasthesia that it breathes deeply and inhales the liquid in one or two breaths. Make sure you get this right!
- Move the mouse to a recovery cage until you can move it back to its home. Warm the mouse if you see it's struggling.
- Clean up everything with vitrol, especially the canula (then rinse with water). Once upstairs dry the canula with the pressurized air and place everything back
- If the battery needs charging, do it now. If it's Friday, leave yourself a note to remind you to do it Monday.
When you get really good you should be able to do about 10 mice in 30 minutes. I'm talking really good. The main thing is practice. I went through probabl 100 mice before I was able to do it without mistakes.
If you screw a mouse up (it will happen), ti's okay to put the mouse back in the chamber for 30-60 seconds and then try again. The worst I've done is repeat a mouse about 5 times. I didn't expect him to take me out for drinks after (his trachea did not look good) but if this is what it takes then so be it. Once you get good you can do multiple mice in a chamber and be able to pump them out every 1-2 minutes or so. It can go very quickly if you have the timing right, but can be a huge disaster if you start messing them up. If you have multiple mice in a chamber and you have to keep repeating one, start alternating. You don't want one mouse to be in the chamber for too long or they will be so deep that they won't inhale the virus effectively. This is why you need to start off slow, develop your skills, and then try to be more efficient. You must learn how to do each mouse in about 30 seconds. It sounds crazy but this is the only way to efficiently do things (if that's your goal).