Day 1 – Pushing the Flies:
Hatched flies of all test groups (e.g. wild type controls, experimental groups) are disposed of, in case their age is not controlled.
Day 2 – Collecting the Flies:
All newly hatched flies from all vials of each test group are collected into one new ‘experimental’ vial each (“Exp.”). The flies are now 0 to 1 day old.
Day 3 – Wing Clipping:
The collected flies from the day before become anesthetized with CO2 and their wings are shortened. It is important to keep the flies anesthetized for no longer than 5 minutes. Therefore, it is helpful to portion the flies: They get transferred into an empty vial; from this vial smaller groups of flies can be transferred into another empty vial (via tube or funnel).
The single fly portions then get anesthetized by holding the vial upside down, inserting the CO2 pistol and fill in the CO2 for a few seconds (stop when all flies fall down onto the plug). Afterwards, the vial can be opened and the flies can be placed down the activated CO2 pad.
Flies need to be sorted in case the experiment is asking for distinct gender or stocks are balanced. The selected flies can be held by the forceps at the very tip of the wings. The wings need to be shortened to one third of their original length and straight through the crossing line of both wings. Too long wings can cause the fly to jump more often; cutting the wings too shortly can injure the fly. If the wings of a fly are spread into different directions it is still possible to cut them straightly, even if hitting the crossing line is more difficult.
After cutting the wings the still anesthetized flies get transferred carefully to the now empty experimental vial by using a brush; following portions of wing clipped flies can be added the same way, as long as the work is done quickly enough.
The vial with the freshly wing shortened flies needs to be set aside as long as flies recovered from anesthesia; sleeping flies fall down into the food and stick to it. The completely prepared flies are put back into the incubator.
Day 4 – Experiment:
- darken the room (close blinds and curtains, switch off lights)
- switch on the lights of the Buridan arena
- assemble the bottom of the arena such that the platform perfectly fits into the designated hole (the grommet must seal the hole perfectly)
- fit the bottom into the arena and make sure that the platform is straight in all directions (use water-level)
- check whether the camera is in a straight position above the arena (water level)
- start the computer
- start the program (Buritrack)
- choose “Main”
- choose “New Video Capture”
- choose the camera (if there is only one camera linked to the computer, leave the default camera number “0”)
- adjust the camera such that the platform is clearly visible and the edge of the platform is not too dark but clearly visible as well, make sure that the “Auto Focus” is switched off and focus the platform manually in the web cam soft ware user surface.
- use the function “Set Camera ROI” to check whether the dark stripes are in the exact north and south position of the platform: Therefore click left on “Set Camero ROI”; click left in the middle of the platform (if you misplaced the cursor, click right and choose “Set Camera ROI” again); move the cursor up to the north stripe and down to the south stripe (the edge of the square has to cross the stripe in the middle); move the stripes if necessary
- fill in water (approx. 2,5 big fly vials per arena); the platform is supposed to be surrounded by a “wall” of water so that the fly cannot jump out of the platform
- clean the platform with 70% Ethanol
Experiment: Choose a tight area around the platform which shall be recorded (“Set Camera ROI”); the stripes or the edge of the arena are not supposed to be recorded.