Testing Nanoject II Infections with 16Cq DiNV on D. innubila For a Second Replicate
The first round of infections seemed to go ok, so I decided to do a second replicate of the infections. Rob also wanted me to expand my day 0 samples, so I did 10 day 0 males with the injection for DiNV, and I also included 10 day 0 males poked with the needle dipped into the DiNV solution. Additionally, Kent asked me to infect 10 males and 10 females, and control inject 4 males and 4 females. So those were added in as well. I did my best to make sure the infection went sterilely with the new method. Two days before I separated out flies into easy vials to use for infections: - 10 vials of 10 males - 1 vial of 10 females - 1 vial of 5 males - 1 vial with 5 males and 5 females - These flies had emerged 9/1-9/4
Set up area
- Thawed 16Cq virus on ice and changed gloves after touching
- Made 11 new vials of mushroom food
- Wiped down fly room bench with 95% ethanol before using/putting things on it
- Things taken to the fly room
- tube rack with 10 tubes for day 0
- mineral oil
- sterile Co2 pad
- pulled needles
- sterile forceps (2)
- two sets of gloves
- 95% ethanol
- Nanoject
- autoclaved toothpicks
- Notebook
- Virus and control medium on ice
- Used one of the forceps to clip off the tip of one of the pulled needles
- Used the metal needle to backfill the pulled needle with mineral oil, avoiding any bubbles
- Placed the pulled needle on the machine
- Slide the collet onto the needle
- Pushed the 2nd o-ring onto the needle
- Slid the needle onto the machine
- Tightened the collet down
- (this method is easier than pushing the needle through the ring on the machine)
- Ejected the mineral oil until the beep
- Filled the needle with control medium (10% FBS, 4% mushroom Schneider’s medium with antibiotics)
- Tested needle to make sure fluid ejects when pressing inject
Injecting flies
- Placed 1st vial of 5 flies on CO2
- Separated out males with toothpick
- Injected each fly with 27.6nl control medium
- Placed each fly in 1.5mL tubes with forceps, dipped in 95% ethanol between each fly
- Placed 1st vial of 10 flies on CO2
- Separated out males with toothpick
- Injected each fly with 27.6nl control medium
- Used toothpick to place flies in new vial
- Repeated steps for 2 more vials for all the control flies
- Repeated steps for the vial of 4 males 4 females for Kent
- This time I was able to eject the rest of the medium, and refill the needle with the 16Cq virus fluid. I think some bubbles got into the needle at this time but I proceeded anyways (this might not have been the correct choice)
- For virus infected flies, infections went the same way:
- Placed vial of 10 flies on CO2
- Separated out males with a newtoothpick
- Injected each fly with 27.6nl 16Cq virus
- Placed each fly in 1.5mL tubes with forceps, dipped in 95% ethanol between each fly (separate pair of forceps from the control flies)
- Placed next vial of 10 flies on CO2
- Separated out males with the virus toothpick
- Poked them with a needle dipped in the 16Cq virus solution, dipping in after each fly
- Used virus toothpick to place flies in new vial
- Placed next vial of 10 flies on CO2
- Separated out males with the virus toothpick
- Injected each fly with 27.6nl 16Cq virus
- Used virus toothpick to place flies in new vial
- Repeated those steps for 3 more vials for me and the vials of males and females for Kent
The time of infection and duration on CO2 was recorded for each fly:
| vial | species | sex | days_emerged | day_infected | age_infected | treatment | volume | original_N_number |
|---|---|---|---|---|---|---|---|---|
| 3 | D. innubila | male | 9/1-9/4 | 20230908 | 4-7 days | cell culture medium | 27.6nl | 9 |
| 4 | D. innubila | male | 9/1-9/4 | 20230908 | 4-7 days | cell culture medium | 27.6nl | 10 |
| 5 | D. innubila | male | 9/1-9/4 | 20230908 | 4-7 days | cell culture medium | 27.6nl | 10 |
| 6 | D. innubila | male | 9/1-9/4 | 20230908 | 4-7 days | 16Cq DiNV | 27.6nl | 10 |
| 7 | D. innubila | male | 9/1-9/4 | 20230908 | 4-7 days | 16Cq DiNV | 27.6nl | 10 |
| 8 | D. innubila | male | 9/1-9/4 | 20230908 | 4-7 days | 16Cq DiNV | 27.6nl | 10 |
| 9 | D. innubila | male | 9/1-9/4 | 20230908 | 4-7 days | 16Cq DiNV | 27.6nl | 10 |
The flies that were frozen for day 1:
| tube number | vial from | day frozen | treatment | dead when frozen? |
|---|---|---|---|---|
| 1 | NA | day0 | cell culture medium | no |
| 2 | NA | day0 | cell culture medium | no |
| 3 | NA | day0 | cell culture medium | no |
| 4 | NA | day0 | cell culture medium | no |
| 5 | NA | day0 | cell culture medium | no |
| 6 | NA | day0 | 16Cq DiNV needle poke | no |
| 7 | NA | day0 | 16Cq DiNV needle poke | no |
| 8 | NA | day0 | 16Cq DiNV needle poke | no |
| 9 | NA | day0 | 16Cq DiNV needle poke | no |
| 10 | NA | day0 | 16Cq DiNV needle poke | no |
| 11 | NA | day0 | 16Cq DiNV needle poke | no |
| 12 | NA | day0 | 16Cq DiNV needle poke | no |
| 13 | NA | day0 | 16Cq DiNV needle poke | no |
| 14 | NA | day0 | 16Cq DiNV needle poke | no |
| 15 | NA | day0 | 16Cq DiNV needle poke | no |
| 16 | NA | day0 | 16Cq DiNV injection | no |
| 17 | NA | day0 | 16Cq DiNV injection | no |
| 18 | NA | day0 | 16Cq DiNV injection | no |
| 19 | NA | day0 | 16Cq DiNV injection | no |
| 20 | NA | day0 | 16Cq DiNV injection | no |
| 21 | NA | day0 | 16Cq DiNV injection | no |
| 22 | NA | day0 | 16Cq DiNV injection | no |
| 23 | NA | day0 | 16Cq DiNV injection | no |
| 24 | NA | day0 | 16Cq DiNV injection | no |
| 25 | NA | day0 | 16Cq DiNV injection | no |
Mortality is assessed every day, and flies are transferred on CO2 every 3 days. When transferred, dead flies are frozen. Mortality information can be found here and frozen fly information can be found here