Protocol for Making Apple Juice Plates for Fly Laying for Cell Culture

Steps

  • Make a 1/2 liter recipe for ~20 plates
  • Get a new 1000mL erlenmeyer flask
  • Weight 7.5g of fly agarose (by the hood) and add to the dry flask
  • Measure 375mL of distilled water in a graduated cylinder
  • Pour the water into the flask
  • Add a stir bar to the flask
  • Stir the flask on the plate stirrer for a few minutes
  • Autoclave the flask on cycle 3 to dissolve the the agarose: foil the top of the flask and place in a small autoclave bucket with a layer of water at the bottom
  • While the autoclave has ~10 min left, make the apple juice mix
  • Get a small 250mL erlenmeyer flask
  • Measure 12.5g of sugar (on the shelf) and pour into the small flask
  • Measure 125mL of apple juice in a graduated cylinder and pour into the small flask
  • Swirl and microwave for 2.5min on power 7
  • Take out and swirl (the amount of sugar is reaching the saturation point so it may be hard to get into solution)
  • Microwave again for 3 minutes then leave out on the counter
  • Get the autoclaved agar mix and place the flask on the stir plate spinning with no heat
  • Measure 0.75g TEGOSEPT (anti-fungal) and pour into a small beaker
  • Squirt in a little 95% ethanol to the beaker to dissolve the TEGOSEPT
  • Wait until the apple juice is at 55 degrees C before pouring in the TEGOSEPT mix (at higher temps, the TEGOSEPT will inactivate)
  • Swirl the apple juice to mix after adding the TEGOSEPT
  • Wait until the agar mix is at 55 degrees C, spinning on the mixer (here you want to be careful that the AJ doesn’t cool too much in the meantime, they both need to be warm to not solidify the agar but not de-activate the TEGOSEPT)
  • Add the apple juice flask to the big flask by pouring down the side of the flask to minimize bubbles
  • Mix with the stir bar until completely mixed
  • Open the plate sleeve by cutting open only one side
  • Take out the plates and leave them in a stack with the lids up
  • Pout plates in the stack by lifting the stack from the lid of the bottom most plate and pouring that one, then place the lid/stack down and do the next plate above. This way you don’t move the plates after they’re poured
  • Pour ~20mL into each plate, pour until the liquid is almost filling the plate then stop, it will continue to fill up the plate
  • Pour quickly and carefully so the agar doesn’t solidify
  • Let the plates sit 20-40min until they are solid
  • Clean the flask immediately when you’re finished with it so the agar doesn’t solidify: soap and water, then distilled water. Same with the other flask and the cylinders
  • For the TEGOSEPT beaker: needs to be rinsed with 95% ethanol to remove any TEGOSEPT residue, then water and distilled water
  • Plate the plates back into the sleeve and tape up the sleeve
  • Place the plates in the 4 degree fridge with the agar side up to avoid condensation on the agar