TITLE: Isoflurane Anesthesia Protocol
AUTHORS: Michaela Selby, Alicia Avelar

[DESCRIPTION]
protocol describes how to use anesthesia machine with isoflurane to anesthetize a rat. 

[BEFORE_START]
Handle rat beforehand to reduce stress
Bring rat from vivarium (or wherever it is kept) to surgery room in a clean cage 

[GUIDELINES]
Ensure the anesthesia box that contains the rat has a fully closed and locked lid.  Ensure that the nose cone used to maintain anesthesia is tightly around the rats nose.  Perform procedure in a hood for safety and wear lab coat and gloves.  Turn off the gas flow as soon as you are done using it for anesthesia. 

[STEPS]
SECTION: Setup
1. Make sure you have a surgery room booked 
SECTION: Setup
1.1. Some sort of table or surgical workbench to transfer rat to when fully anesthetized 
SECTION: Setup
2. Grab isoflurane cart and/or supplies and bring it to the surgery room
SECTION: Setup
3. Place paper towels in the plastic container 
SECTION: Setup
4. Make sure the tubing is tightly sealed together (if tape is needed add tape)
SECTION: Setup
4.1. Make sure all of the tubing is in the correct place and connected
SECTION: Setup
4.2. Make sure the valves are parallel with the tubing (the pink and orange tubing)
SECTION: Setup
5. Use larger thicker tape to secure white tubing down onto the surgery table (where you want it on the workbench)
SECTION: Setup
6. Snip open a little hole in a glove and place it over the tubing for the rat's mouth as a nose cone for anesthesia during surgical procedures
SECTION: Setup
7. Check isoflurane level (make sure it is between the minimum and maximum fill lines) 
SECTION: Setup
7.1. If the isoflurane level is low then unscrew the cap and slowly add in the isoflurane 
Set isoflurane regulator on top-adjust dial as appropriate
SECTION: Setup
8. Use the black wrench to open the oxygen tank 
SECTION: Setup
8.1. Turn to the right to open
SECTION: Setup
9. Look at the oxygen regulator and turn the green knob so the silver ball is in the middle of the 1 on the regulator.
SECTION: Setup
10. Place the rat in the plastic box and close the lid
SECTION: Setup
11. Adjust isoflurane dial as needed
SECTION: Setup
11.1. Start at 4 (if female)
SECTION: Setup
11.2. Start at 5 (if male), heavier animals usually need more isoflurane to become anesthetized
SECTION: Setup
12. Keep rat under for a few minutes or until the rat’s breathing slows down
SECTION: Setup
13. Take the rat out of the plastic container and place it onto the surgery table
SECTION: Setup
14. Do a toe pinch using forceps to make sure the animal does not feel any pain
SECTION: Setup
14.1. If the rat has a pain reflex or a jerk motion of the limb then place back into the plastic container and continue anesthesia
SECTION: Setup
14.2. If the rat has no pain reflex or jerk then continue
SECTION: Setup
15. Put the tubing that has the glove with the slit over the rat's mouth and nose to maintain anesthesia
SECTION: Setup
16. Then when the rat is fully anesthetized you can proceed with your surgery
SECTION: Turning off machine
17. Turn off the isoflurane dial (back to 0)
SECTION: Turning off machine
18. Take the black wrench to close the oxygen tank
SECTION: Turning off machine
18.1. Turn to the left to close
SECTION: Troubleshooting
19. Check to make sure there is no tubing loose/disconnected
SECTION: Troubleshooting
19.1. In case there is a leak
SECTION: Troubleshooting
19.2. Or if something is not working
SECTION: Troubleshooting
20. Adjust dial 
SECTION: Troubleshooting
20.1. More or less isoflurane depending on how the rat is doing 
SECTION: Cleanup and Transport
21. Press the green oxygen flush button
SECTION: Cleanup and Transport
21.1. This clears out oxygen from the tubing  
SECTION: Cleanup and Transport
22. Remove the paper towels and dispose of them
SECTION: Cleanup and Transport
23. Clean out the plastic box with paper towels and water only
SECTION: Cleanup and Transport
24. Return isoflurane machine and supplies to its storage place
SECTION: Cleanup and Transport
25. Place cage where the dirty cages go to be cleaned