Using 4 Different Laying Plate Types to See What D. innubila Lays on Best

20220110 Made Fresh Apple Juice Plates and Mushroom Agar Plates

  • Made new apple juice agar plates, and poured them into the small petri dishes as well as the large ones
  • Also made mushroom agar (Lab recipe, but used frozen mushrooms that were then autoclaved) and poured into small petri dishes
  • Made fresh yeast paste and autoclaved twice

20220111 Added Yeast to Plates

  • Trying plain plates for both mushroom and apple juice, and also plates with a little yeast on them
  • Experimental plates:

20220112 Setting up Flies

  • Used the 8 vials of flies that had been saved from the week before’s innubila cell culture attempt
  • Used 2 vials per cage, and had 4 cages set up at a time. One mushroom plate, one AJ plate, one mushroom plate with yeast, and one AJ plate with yeast
  • Did not knock out flies when adding to the cages so some flies were lost but happiness of flies should have remained
  • Noted that the mushroom plates were kind of wet

20220113 Cell Culture Day 1

  • Had Rob show me how to pick out eggs from the plates with a tooth pick
  • Transferred flies to new plates to lay again
  • Many of the flies on the mushroom plates had gotten stuck and died, mostly because the plates were too wet
  • Imaged each plate to count eggs x
  • Picked out eggs and put them on a yellow 100um filter
  • Returned to 4012 and mostly followed the Prepping Cell Culture Protocol with some modifications:
    • Used 10% FBS Schneider’s medium without gentamicin or 0% FBS Schneider’s medium without gentamicin made fresh that day
    • Did not use trypsin to minimize the number of washes needed to prevent loss of cells/eggs
    • Instead, I used 0% FBS medium to wash out the beach solution, 3 washes, then homogenized the eggs in 2mL of 10% medium
    • There was no waiting period after homogenization, and the homogenate was added to a flask with 3mL of 10% medium and put into the incubator at 23 degrees

20220114 Cell Culture Day 2

  • Transferred flies to food vials
  • Imaged plates to count eggs x
  • There were a lot more eggs on the mushrooom plates today, but the darkness/opaqueness of the plates made it very hard to see in the images
  • Picked out eggs and put them in a beaker with some eggs wash buffer
  • Returned to 4012 and mostly followed the Prepping Cell Culture Protocol using the same modifications from above
  • Tried to remove as much of the agar pieces or at least break them up by swishing a paintbrush on the filter while washing it with egg wash
  • Imaged flasks from both days. There was a lot of debris but there was also visible cells and cell clumps!
  • 20220113 flask:
  • 20220114 flask:

20220121 Imaging Flasks Again and Fluid Addition

  • Added 1mL of 20% Schneider’s medium to the 20220114 flask
  • Imaged flasks, there is still debris of course, but the cells look big and still alive, some are even contracting