Tetrad disection of yeast

1. Preparation of spores:
   Starting from a liquid sporulation culture, mix 60 µl of sporulation culture with 30 µl of O-glycosidase (Boehringer; stored at 4°C)
   Incubate at 37°C for 10 min
2. Preparation of petri plates:
   15 ml of medium per plate
   ! Plates have to be dry
   ! (pour plates at least four days before dissection and leave them at room temperature)
   
   Mark inoculum area on the dish with a lab pen using the tempate
   Inoculate with O-glycosidase-treated spore suspension with a single stroke from an inoculation loop
3. Switch on apparatus
   - Plug in power cord
   - Monitor on
   - Console on
4. Wait until you hear a beep (takes a few seconds)
5. Push right button
6. Lift microscope until it latches firmly
   Turn the micromanipulator knob anticlockwise to lower the needle
   Load petri dish in holder in good orientation (inoculum at the left)
   Confirm fixation of petri dish
   Microscope down (pull handle toward you)
7. Push twice on right button (‘SEARCH’ mode will be displayed)
8. Focus on inoculum
9. Go completely to the left to clean the needle in the agar
10. Move back to the inoculum and search for a suitable tetrad (use joystick to move the petri dish; red button allows making fine movements)
11. Once a tetrad is on the needle, ensure the needle is well lowered and then press the right button
    The needle will go to position A1 on the matrix
    Deposit one spore
    Use the joystick to move to B1 ...(can be verified on the display !)
    After complete dissection, go back to position A1 and make a reference hole in the centre at A1.
    Push right button to go back to inoculum area
12. After having picked up a second tetrad, go back to A1 (by pressing right button)
    Refocus
    Use joystick to move to A2
    Continue dissection (10 tetrads can be dissected at the top and 10 at the bottom)
13. To get from A10 to F1 (or any other matrix point), key F1 followed by =.

1. Before switching off, return needle to A1
2. When finished,
   - key ?,
   - then # (to centre the needle),
   - then E
   before switching off.
3. Mark the dish for easy relocation
4. Raise the microscope and remove the plate
5. Load an empty petri dish in the holder

Swich off the monitor and console
Power cord out