Diluting Titered DiNV Stock to Specific FFU and Injecting in Male and Female D. innubila
Making Dilutions
Previously I planned a way I would like to do dilutions on DiNV for injection infections. The fluid to use DiNV passage 4 from Kent. He showed me where it is kept in the -80 and gave me a ~1mL tube. From that previous post, I planned on infecting with a total of 6.375FFU (undiluted), 6, 3, 1, 0.1, and 0.01 FFU. I only have to dilute to 6FFU, and then I can use that solution to make all the others.
The tube of virus solution was thawed on ice and inverted multiple times before using (cannot spin down the cyrotubes, also they have cell debris in them that you won’t want to spin out of solution probably).
To make 80ul of 6FFU (I probably didn’t need to make this much but that’s what I chose), I calculated how many total FFU I’d need at that volume. To deliver 6FFU per injection, that is 0.217FFU/nl solution, or 217FFU/ul. 80ul * 217FFU/ul = 17,360 FFU total. The undiluted solution is at 238.7FFU/ul. 17,360FFU/238.7FFU/ul = 72.73ul. I will be using cell culture medium (including mushroom extract) as the diluent and control for these. From that tube I will make the other dilutions, cutting it by half for the 3FFU, then that by 1/3 for the 1FFU and so on (see table below).
All samples were made on ice in 0.65ml tubes:
| tube # | volume virus solution | virus from tube | volume Cell culture medium |
|---|---|---|---|
| 1 | 80ul | undiluted stock | NA |
| 2 | 80ul | undiluted stock | NA |
| 3 | 80ul | undiluted stock | NA |
| 4 | 80ul | undiluted stock | NA |
| 5 | 72.73ul | undiluted stock | 7.27ul |
| 6 | 50ul | tube 5 | 40ul |
| 7 | 13.3ul | tube 6 | 16.7ul |
| 8 | 3ul | tube 7 | 27ul |
| 9 | 3ul | tube 8 | 27ul |
tubes 11-29 are 30ul of undiluted stock. I forgot to do a tube 10.
Injections
Flies used in this are slightly older than I would have liked but I did not have any flies in the 5-7 day range. I think this age range though guarantees that the females are mated because this is when they are the most fertile. 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.
Surprisingly I did not break a needle, so every fly got the same needle, although I did cut it too thick at first, and had to redo the needle at the beginning. I think someone is changing the settings in the needle puller on the 5th floor and it is making the needles slightly different.
I made 16 new vials of mushroom food for this. Kent asked me to do 5 males and 5 females injected with the undiluted fluid so that he can sample them when they die. I will monitor them along with mine and maybe add in their data. He wants to fix the flies immediately when they die if possible.
The set up of the injection was the same as previous injections
Of note all the viral dilutions were kept on ice until use, and pipette mixed with 15ul before drawing up the fluid into the needle.
| vial | species | sex | mated status | days_emerged | day_infected | age_infected | tube | treatment | volume | time | time on CO2 | original_N_number |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 1 | D. innubila | male | mated | 20231025-20231026 | 20231103 | 8-10 days | CCM | CCM | 27.6nl | 2:22 | 10 min | 10 |
| 2 | D. innubila | female | mated | 20231025-20231026 | 20231103 | 8-10 days | CCM | CCM | 27.6nl | 2:28 | 6 min | 10 |
| 3 | D. innubila | male | mated | 20231025-20231026 | 20231103 | 8-10 days | 9 | 0.01 FFU | 27.6nl | 2:37 | 5 min | 10 |
| 4 | D. innubila | female | mated | 20231025-20231026 | 20231103 | 8-10 days | 9 | 0.01 FFU | 27.6nl | 2:43 | 5 min | 10 |
| 5 | D. innubila | male | mated | 20231025-20231026 | 20231103 | 8-10 days | 8 | 0.1 FFU | 27.6nl | 2:53 | 6 min | 10 |
| 6 | D. innubila | female | mated | 20231025-20231026 | 20231103 | 8-10 days | 8 | 0.1 FFU | 27.6nl | 2:59 | 5 min | 10 |
| 7 | D. innubila | male | mated | 20231025-20231026 | 20231103 | 8-10 days | 7 | 1 FFU | 27.6nl | 3:09 | 5 min | 10 |
| 8 | D. innubila | female | mated | 20231025-20231026 | 20231103 | 8-10 days | 7 | 1 FFU | 27.6nl | 3:13 | 6 min | 10 |
| 9 | D. innubila | male | mated | 20231025-20231026 | 20231103 | 8-10 days | 6 | 3 FFU | 27.6nl | 3:28 | 6 min | 10 |
| 10 | D. innubila | female | mated | 20231025-20231026 | 20231103 | 8-10 days | 6 | 3 FFU | 27.6nl | 3:34 | 5 min | 10 |
| 11 | D. innubila | male | mated | 20231025-20231026 | 20231103 | 8-10 days | 5 | 6 FFU | 27.6nl | 3:43 | 6 min | 10 |
| 12 | D. innubila | female | mated | 20231025-20231026 | 20231103 | 8-10 days | 5 | 6 FFU | 27.6nl | 3:49 | 6 min | 10 |
| 13 | D. innubila | male | mated | 20231025-20231026 | 20231103 | 8-10 days | 1 | undluted DiNV | 27.6nl | 4:00 | 6 min | 10 |
| 14 | D. innubila | female | mated | 20231025-20231026 | 20231103 | 8-10 days | 1 | undluted DiNV | 27.6nl | 4:06 | 6 min | 10 |
| 15 | D. innubila | male | mated | 20231025-20231026 | 20231103 | 8-10 days | 1 | undluted DiNV | 27.6nl | 4:12 | 4 min | 5 |
| 16 | D. innubila | female | mated | 20231025-20231026 | 20231103 | 8-10 days | 1 | undluted DiNV | 27.6nl | 4:14 | 6 min | 5 |
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