Using 16Cq DiNV For Infections with D. innubila im New Protocol to Maximize Sterility
Previous infections have brought up questions about sterility and the keeping of control flies not infected with virus. I am trying here a small infection with new safety measures to hopefully keep things sterile.
I want to use 10 flies, control and infected, to freeze on day 0. And 32 flies to keep going in the experiment. I only plan to have the infection go for 7 days because this is mostly just to see if I can do this steriley.
To move flies I have autoclaved toothpicks and autoclaved two forceps. One forceps will be for control flies, the other for infected flies.
I have also bleach cleaned the CO2 pad. The pad was soaked in 10% bleach for 10-20 minutes, then rinsed with DI water at least 10 times, then dried with air running through it, and finally dried with 95% ethanol and air. The pad was left to dry overnight before use.
All sterile poke flies were done first, and all flies were frozen right after being poked. I wore gloves throughout the infection, and I put on new gloves when switching to the DiNV infection. Everything was set up before I touched the DiNV tube. Between moving each fly to a tube for freezing, the tweezers were dipped in fresh 95% ethanol.
| vial | Species | Treatment | sex | Day_emerged | Day_Infected | time_infected | fly_age | Original_N_number |
|---|---|---|---|---|---|---|---|---|
| 1 | innubila | SP | M | 7/19-07/21 | 20230724 | 2:57 | 5-7 days | 8 |
| 2 | innubila | SP | M | 7/19-07/21 | 20230724 | 3:04 | 5-7 days | 8 |
| 3 | innubila | DiNV | M | 7/19-07/21 | 20230724 | 3:21 | 5-7 days | 8 |
| 4 | innubila | DiNV | M | 7/19-07/21 | 20230724 | 3:29 | 5-7 days | 8 |
Info on the infected flies can be found here
When transferring flies on the 3rd day to new food, a freshly bleached CO2 pad was used, and the autoclaved toothpicks were used to move the flies into the new vials. Sterile poked flies were done first, then infected ones. No flies had died at this time.
When freezing flies, flies were put on a freshly bleached CO2 pad (sterile poked first), then transferred to vials with sterilized forceps (bleach, DI water, and 95% ethanol). There was 1 forceps for sterile poked flies, one for DiNV poked flies. Information on the frozen flies is found here
| sample_ID | vial_from | sex | treatment | day_frozen | alive_when_frozen? |
|---|---|---|---|---|---|
| 1 | NA | M | SP | day0 | yes |
| 2 | NA | M | SP | day0 | yes |
| 3 | NA | M | SP | day0 | yes |
| 4 | NA | M | SP | day0 | yes |
| 5 | NA | M | SP | day0 | yes |
| 6 | NA | M | DiNV | day0 | yes |
| 7 | NA | M | DiNV | day0 | yes |
| 8 | NA | M | DiNV | day0 | yes |
| 9 | NA | M | DiNV | day0 | yes |
| 10 | NA | M | DiNV | day0 | yes |
| 11 | 1 | M | SP | day7 | yes |
| 12 | 1 | M | SP | day7 | yes |
| 13 | 1 | M | SP | day7 | yes |
| 14 | 1 | M | SP | day7 | yes |
| 15 | 1 | M | SP | day7 | yes |
| 16 | 1 | M | SP | day7 | yes |
| 17 | 1 | M | SP | day7 | yes |
| 18 | 1 | M | SP | day7 | yes |
| 19 | 2 | M | SP | day7 | yes |
| 20 | 2 | M | SP | day7 | yes |
| 21 | 2 | M | SP | day7 | yes |
| 22 | 2 | M | SP | day7 | yes |
| 23 | 2 | M | SP | day7 | yes |
| 24 | 2 | M | SP | day7 | yes |
| 25 | 2 | M | SP | day7 | yes |
| 26 | 3 | M | DiNV | day7 | no |
| 27 | 3 | M | DiNV | day7 | yes |
| 28 | 3 | M | DiNV | day7 | yes |
| 29 | 3 | M | DiNV | day7 | yes |
| 30 | 3 | M | DiNV | day7 | yes |
| 31 | 3 | M | DiNV | day7 | yes |
| 32 | 3 | M | DiNV | day7 | yes |
| 33 | 3 | M | DiNV | day7 | yes |
| 34 | 3 | M | DiNV | day7 | yes |
| 35 | 4 | M | DiNV | day7 | yes |
| 36 | 4 | M | DiNV | day7 | yes |
| 37 | 4 | M | DiNV | day7 | yes |
| 38 | 4 | M | DiNV | day7 | yes |
| 39 | 4 | M | DiNV | day7 | yes |
| 40 | 4 | M | DiNV | day7 | yes |
| 41 | 4 | M | DiNV | day7 | yes |