Male D. innubila Injections with 0.01, 0.05, and 0.005 FFU DiNV
Rob suggested doing some in-between FFU concentrations for injections and just trying them out to see what they are like. I Separated out ~20 males for each treatment on the 30th, and they had emerged on the 24th and 25th, making them 7-8 days old for injection. I injected 10 control and 10 undiluted flies for Kent as well. No day 0 flies were made, but flies were frozen after death every 3 days, and the experiment ends in 2 weeks.
Making dilutions:
I planned the dilution calculations similar to here, and here. I planned to dilute the stock virus to give 0. FFU with the injection, and dilute it 1:5 and 1:10 for the other dilutions.
If I want to give 0.05FFU in a 25.6nl injection: 0.05/27.6 = 0.00181FFU/nl is needed. If I want to make 50ul of 0.00181FFU/nl (or 1.81FFU/ul), I need 90.5FFU total in that 50ul. Stock virus is 238.7FFU/ul. 90.5/238.7 = 0.379ul of stock in 50ul. I did not want to pipette that, so I did a 1:10 dilution of the stock, and decided to use that to make the 0.05FFU:
- Stock tube 26 used, thawed on ice
- Diluted 1:10 in tube “2” 3ul of stock in 27ul of CCM
- Tube 3 is for 0.05FFU: 3.79ul of tube 2 in 46.21ul of CCM
- Tube 4 is for 0.01FFU: 10ul of tube 3 in 40ul of CCM
- Tube 5 is for 0.005FFU: 5ul of tube 3 in 45ul of CCM
- Tubes were mixed after add additions and before use with pipetting
10 new vials of mushroom food were made fresh for the infection.
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. The needle did not break so only one needle was used for every fly.
| vial | species | sex | days_emerged | day_infected | age_infected | tube | treatment | volume | time | time on CO2 | original_N_number |
|---|---|---|---|---|---|---|---|---|---|---|---|
| 1 | D. innubila | male | 1/24 - 1/25 | 20240131 | 7-8 days | CCM | CCM | 27.6nl | 3:42 | 9 min | 10 |
| 2 | D. innubila | male | 1/24 - 1/25 | 20240131 | 7-8 days | CCM | CCM | 27.6nl | 3:52 | 6 min | 10 |
| 3 | D. innubila | male | 1/24 - 1/25 | 20240131 | 7-8 days | 5 | 0.005 FFU | 27.6nl | 4:03 | 7 min | 10 |
| 4 | D. innubila | male | 1/24 - 1/25 | 20240131 | 7-8 days | 5 | 0.005 FFU | 27.6nl | 4:10 | 7 min | 10 |
| 5 | D. innubila | male | 1/24 - 1/25 | 20240131 | 7-8 days | 5 | 0.005 FFU | 27.6nl | 4:17 | 6 min | 10 |
| 6 | D. innubila | male | 1/24 - 1/25 | 20240131 | 7-8 days | 4 | 0.01 FFU | 27.6nl | 4:27 | 6 min | 10 |
| 7 | D. innubila | male | 1/24 - 1/25 | 20240131 | 7-8 days | 3 | 0.05 FFU | 27.6nl | 4:37 | 6 min | 10 |
| 8 | D. innubila | male | 1/24 - 1/25 | 20240131 | 7-8 days | 3 | 0.05 FFU | 27.6nl | 4:43 | 6 min | 9 |
| 9 | D. innubila | male | 1/24 - 1/25 | 20240131 | 7-8 days | undil | 6.2 FFU | 27.6nl | 4:56 | 6 min | 10 |
| 10 | D. innubila | male | 1/24 - 1/25 | 20240131 | 7-8 days | 4 | 0.005 FFU | 27.6nl | 5:04 | 6 min | 11 |
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