PROCEDURE – COOKING

1. On a cart bring empty trays for bottles, boxes of vials, ice, and any additions necessary to “Fly Kitchen” (5-150 Hogan Hall).  Bring the keys attached to the gray plastic card, the kitchen key has a green sticker on it.
2. Open boxes of vials and remove plastic prior to cooking food. Also put 16 bottles in each empty box.
3. Measure out all ingredients except Nipagen, propionic acid, and ethanol.
4. Find the metal knob and place it in the kettle’s spout. Make sure the black rubber ring is pulled tight on the end. Screw the spout closed (. Make sure plastic bin is underneath spout. In plastic bin, place a plastic liner to catch water that leaks.
5. Pour 7L for single, 14L for double, or 21L for triple batch of water in kettle
6. Add in agar while turning on the stirrer and heat. The stirrer turns on via a knob on the back of the green apparatus. The heat is turned on by making the yellow handle parallel to the pipe; turn this handle all the way down.
7. Hydrate the soy flour and yeast in 3L of water for single, 6 for double, and 9 for triple in bucket
8. Hydrate corn meal in 3L of water for single, 6L for double, or 9L for triple in bucket
9. Let agar mixture heat for 6 minutes. Make sure the temperature is well above 60C or 140F. Release the pressure from the kettle (lift the small switch in the back, left of the kettle). Allow agar and water to come to a boil, then quickly add soy flour/yeast mix to boiling agar
10. Add corn meal mix (mix up the mixture first and pour slowly and make sure the cornmeal and water come out together, otherwise it will make the food clumpy)
11. Allow to boil for 1 minute.
12. Add corn syrup and molasses and boil for ten minutes
13. Measure out nipagen and dissolve in ethanol. Measure propionic acid.
14. Turn off heat, add three scoops of ice. (Use styrofoam container on shelf to get ice from second floor by the Cook/Hogan bridge ice machine.) Let cool for another 3 minutes.
15. Add acids and allow them to mix into the food.
16. Open spout and empty food into container until it looks well mixed. Pour these contents back into the kettle.
17. Turn off stirrer.
18. Pour the bottles and vials
    1. Pour 50mL of food into each bottle
    2. Pour between 800 and 1000 mL of food for each tray of vials.
    3. For EtOH or RU486 food, pour 800 mL of food, add 1 mL of blue dye and 8 mL of EtOH or RU486 and mix before pouring.
19. Put finished vials and bottles onto drying racks and cover with plastic.
20. Wash and return to appropriate locations everything that was used
21. Clean kettle (including the spout)

PROCEDURE – BUNGING

1. The next morning after food has been made, place a cotton ball in each vial. Do not stuff it too deep.
2. Styrofoam plugs are used for bottles
3. Make sure the food has been assigned and dated before putting it into the cold room for storage.
4. Stack each person’s food near their piles in the cold room.

PRO TIPS

• Occasionally use spatula to mix the food in the kettle. Do not leave the stirrer on, this will cause the food to congeal faster.
• Use the plastic from the cotton balls to cover the plastic bin under the kettle, the table that you pour vials on. (It makes clean-up easier!)
• Use a empty box to pour corn syrup and molasses over. (Again save yourself the mess)
• Wash dishes as you go.
• Do not turn the stirrer past 50%, it will cause the machine to drop and rub against the kettle.
• Place new food behind old food in the cold room. This helps encourage the grad students and postdocs to use all the food they have before it goes bad.

UNLOADING BOARDS AND CLEANING TUBES

1. Remove the tubes from the boards
   1. For small boards, pull tubes out so that black caps come off (leaving the yellow on).  This makes fly disposal easier.
   2. For large boards, use the metal spatula to remove them all by placing the spatula behind the tubes and running it down the board.
2. Return boards to Pancoe ASAP.
3. Remove yellow caps and pour flies into morgues.
4. Place empty tubes in a 600mL beaker with agar food at the top.
5. Add water and a splash of bleach to the beakers, up to 450mL, and microwave for 4 minutes.
6. Rinse out 2-3 times and check that all the tubes are now clear. If not, repeat step 5. If there’s only a few that still have agar or stubborn flies, pull them out with the blue tweezers and clean manually.
7. Fill beakers with half bleach, half water until it just covers the tubes. Let sit for no more than 30 minutes
8. Rinse out bleach and fill again to 500mL. Microwave for 4 minutes.
9. Rinse 2-3 times again.
10. Cover with aluminum foil and place a small piece of autoclave tape. Autoclave on the “uncovered 45 minute” cycle.

CLEANING CAPS

1. Keep yellow and black caps separate.
2. Soak in bleach and water (approximately 1:10 ratio) for 30 minutes.
3. Rinse thoroughly and place in a tray with paper towel lining the bottom. Place in drying oven downstairs.
4. Return all yellow caps to the fly room in Pancoe by Friday morning. Black caps go in the drawer labeled “black caps” by the lab bench.

PRO TIPS

• Keep tubes separate depending on if they have black, plastic normal food, or plastic blue food caps. This makes the job go faster and means that you only need gloves when handling the blue food rather than all of them.
• Autoclave beakers with the dishes from Pancoe if you can to save time and space in the autoclave.

POURING BEAKERS

1. Remove enough tubes from center of the beaker (using blue tweezers) to insert a funnel.
2. Heat tube food:
   1. Take off cap
   2. Start with two or three minutes, depending on how full the bottle is. Then continue with 30 second bouts (so it doesn’t boil over) until heated through.
   3. Make sure there are no chunks
3. Pour food SLOWLY and a little at a time. If you pour over the stem of the funnel, it will make bubbles in the tubes.
4. Shake the beaker as you go (using your wrist as if you were jiggling a door knob) in order to pour evenly.
5. Fill to the 100mL line, replace tubes you removed earlier, and replace aluminum foil cover.
6. Let sit on the counter until they harden naturally. Then refrigerate for AT LEAST 20 minutes to allow food to set.
7. Store in +4 degree fridge.

WIPING AND CAPPING

1. Use a metal spatula to separate agar from the sides of the beakers and dump tubes from the beaker
2. Wipe excess agar off from the outside of the tubes
3. Place five sets of three rubber bands on the tubes. Place back into the beaker the opposite direction, food down.
4. Using tool, push into all of the tubes to push the agar to the correct length. Then take the tubes out of the beaker, flip them again, and use the edge of the tool to cut off the exposed ends of agar. Remove rubber bands.
5. Clean each individual tube.
6. Place black cap on end of the tube (make sure there are no air bubbles) and place tubes in a clean cylinder . Refrigerate immediately.
7. Label with date and mark tally on the board

PRO TIPS

• Get more than one person working on a beaker to make the job go faster. Assembly lines work well with this job.
• Reprocess tubes that are older than three weeks.
• Relabel the tubes from previous weeks so they look like they were made this week. This encouraged grad students and postdocs to use them. Use tallies on the side of the tape to keep track of how old they are, however.
• Place cylinders that are older in the front of the fridge so the graduates and postdocs grab them first.

PROCEDURE

1. Let food sit at room temp for at least 10 minutes.
2. Transfer at least 8 to 10 flies into the new vial (make sure there are males and females)
   1. DO NOT use vials if they are too short/too tall.
   2. If there are not enough flies to transfer, find the duplicate and place both vials in a box labeled hospital. Write the date on a piece of tape and tape it to the vials.
   3. Rewrite label if ripped or falling off.
3. Discard old vials into biohazard bin.
4. Take biohazard bag to Panco freezer in hallway. Put the date on a piece of tape and put it on the bag before leaving it in the freezer for 24 hours.
5. Remove bags from freezer and put in biohazard bins on the storage room (Silverman 2-589). Make sure to fill out the purple sticker and fill out the form on the clipboard.

Clean/Organize lab bench   Clean out morgues – weekly

1. Place a Kimwipe over a sieve and filter out flies
2. Discard Flies in biohazard bin
3. Rinse out bottle and refill with water and dish soap

Clean Pancoe Incubator

1. Remove flies on the shelf and arrange them on a counter in the same way. DO NOT MIX UP WHERE THEY GO.
2. Wipe each shelf/walls with bleach solution, then ethanol.