Diluting Titered DiNV Stock to Specific FFU and Injecting in Male and Female D. innubila Replicate 3
Making Dilutions
Previously I had done a first replciate of this infection, and I had planned how to dilute the virus. I had done a second replicate as well. This is the third replicate of that experiment, I diluted the virus new this day, and I did not include an undiluted injection because it was so similar to the 6 FFU injection. Virus stock tube # 3 was used to make these dilutions.
| tube # | volume virus solution | virus from tube | volume Cell culture medium |
|---|---|---|---|
| 1 | 72.73ul | undiluted stock | 7.27ul |
| 2 | 50ul | tube 5 | 40ul |
| 3 | 13.3ul | tube 6 | 16.7ul |
| 4 | 3ul | tube 7 | 27ul |
| 5 | 3ul | tube 8 | 27ul |
I also used a tube of cell culture medium for the controls.
Injections
I kept the fly age on the older side (8-9 days) to go along with the first and second replicate, this also insures females are mated. I made sure that I ordered the injections so that I did the highest dilutions first (least amount of virus) and move up to the higher concentrations. This is so just in case there was any extra viral fluid in the needle, it did not make the solution more concentrated than I would want (although it could make it less concentrated). However when switching out the solution in the needle, I made sure to eject all of the fluid until a few drops of mineral oil came out each time. I also used a new tooth pick each new dilution I used to move the flies around.
The same needle was used for every vial.
I also did 5 flies per sex per dilution that were injected and put into 1.5mL tubes for day 0. These were done after the flies for the keep vials, but before moving on to the next dilution.
I made 14 new vials of mushroom food for this. Kent asked me to do 10 males and 10 females injected with the virus and also control so that he can sample them. I will monitor them along with mine and maybe add in their data.
The set up of the injection was the same as a previous injections
Of note all the viral dilutions were kept on ice until use, and pipette mixed with 20ul before drawing up the fluid into the needle.
| vial | species | sex | mated status | days_emerged | day_infected | age_infected | original_vial_from | tube | treatment | volume | time | time on CO2 | original_N_number |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 1 | D. innubila | male | yes | 11/26-11/27 | 20231205 | 8-9 days | 3 | CCM | CCM | 27.6nl | 2:55 | 5 min | 10 |
| 2 | D. innubila | female | yes | 11/26-11/27 | 20231205 | 8-9 days | 4 | CCM | CCM | 27.6nl | 3:01 | 5 min | 10 |
| 3 | D. innubila | male | yes | 11/26-11/27 | 20231205 | 8-9 days | 6 | CCM | CCM | 27.6nl | 3:06 | 6 min | 10 |
| 4 | D. innubila | female | yes | 11/26-11/27 | 20231205 | 8-9 days | 1 | CCM | CCM | 27.6nl | 3:12 | 7 min | 10 |
| 5 | D. innubila | male | yes | 11/26-11/27 | 20231205 | 8-9 days | 2 | 5 | 0.01FFU | 27.6nl | 3:28 | 7 min | 10 |
| 6 | D. innubila | female | yes | 11/26-11/27 | 20231205 | 8-9 days | 3 | 5 | 0.01FFU | 27.6nl | 3:37 | 5 min | 10 |
| 7 | D. innubila | male | yes | 11/26-11/27 | 20231205 | 8-9 days | 3 | 4 | 0.1FFU | 27.6nl | 3:51 | 5 min | 10 |
| 8 | D. innubila | female | yes | 11/26-11/27 | 20231205 | 8-9 days | 2 | 4 | 0.1FFU | 27.6nl | 3:56 | 5 min | 10 |
| 9 | D. innubila | male | yes | 11/26-11/27 | 20231205 | 8-9 days | 1 | 3 | 1 FFU | 27.6nl | 4:11 | 6 min | 10 |
| 10 | D. innubila | female | yes | 11/26-11/27 | 20231205 | 8-9 days | 5 | 3 | 1FFU | 27.6nl | 4:17 | 5 min | 10 |
| 11 | D. innubila | male | yes | 11/26-11/27 | 20231205 | 8-9 days | 2 | 2 | 3 FFU | 27.6nl | 4:32 | 6 min | 10 |
| 12 | D. innubila | female | yes | 11/26-11/27 | 20231205 | 8-9 days | 3 | 2 | 3 FFU | 27.6nl | 4:38 | 5 min | 10 |
| 13 | D. innubila | male | yes | 11/26-11/27 | 20231205 | 8-9 days | 4 | 1 | 6 FFU | 27.6nl | 4:53 | 5 min | 10 |
| 14 | D. innubila | female | yes | 11/26-11/27 | 20231205 | 8-9 days | 6 | 1 | 6 FFU | 27.6nl | 4:57 | 5 min | 10 |
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
For transferring flies and freezing dead ones, this is the process that is followed:
- Make new food vials the day you need them
- Use the sterilized CO2 pad, toothpicks, and forceps for this
- Place flies on CO2 to transfer, count the number when adding the flies to the vial
- Use new 1.5mL tubes from the molec lab only (less likely to be contaminated)
- Use clean forceps to move a dead fly to the 1.5mL tube
- Between each fly move, put the forceps in 10% bleach, then DI water, then 95% ethanol (these are in tubes that are for dipping in)
- The CO2 pad will be sterilized afterwards, along with the forceps
- The toothpicks will be soaked in ethanol before disposing in the glass disposal box