Diluting DiNV and Injecting D. innubila to For Measuring Viral Load During Infection

I wanted to do infections with virus dilutions of 0.01 FFU, 0.1 FFU, and 3 FFU and measure viral load on the flies at day 1, day 3, and day 5 after injection. Note that I originally made the 0.01 dilution incorrectly and it was actually 0.1, so those flies went first in the order. I then diluted that by 1:10 and made the 0.01 dilution. I make sure to remove all of the previous fluid from the needle when changing out liquids so I am not super concerned, but I may need to do this again.

Stock virus is 238.7 FFU/ul, the same passage 4 DiNV I have been using for recent infections.

tube dilution vol virus vol CCM
1 3 FFU 18.2ul stock 21.8ul
2 1 FFU (not used) 10ul tube 1 20ul
3 0.1 FFU 3ul tube 2 27ul
4 0.01 FFU 3ul tube 3 27ul

I also did 10-8 flies per sex per dilution that I injected and put into individual tubes for day 0 samples. Flies had emerged the 4th and 5th of December and should be 7-8 days old. Flies were separated out the day before in ~10 flies per sex in vials.

18 new vials of food were prepared.

The set up of the injection was the same as a previous injection except that males and females were done at the same time because they were kept in the same tubes.

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 tube treatment volume time time on CO2 original_N_number
1 D. innubila male yes 12/4-12/5 20231212 7-8 days 3 0.1 FFU 27.6nl 2:34 10 min 10
2 D. innubila female yes 12/4-12/5 20231212 7-8 days 3 0.1 FFU 27.6nl 2:34 10 min 10
3 D. innubila male yes 12/4-12/5 20231212 7-8 days 3 0.1 FFU 27.6nl 2:45 10 min 10
4 D. innubila female yes 12/4-12/5 20231212 7-8 days 3 0.1 FFU 27.6nl 2:45 10 min 10
5 D. innubila male yes 12/4-12/5 20231212 7-8 days 3 0.1 FFU 27.6nl 2:54 10 min 10
6 D. innubila female yes 12/4-12/5 20231212 7-8 days 3 0.1 FFU 27.6nl 2:54 10 min 10
7 D. innubila male yes 12/4-12/5 20231212 7-8 days 4 0.01 FFU 27.6nl 3:43 10 min 10
8 D. innubila female yes 12/4-12/5 20231212 7-8 days 4 0.01 FFU 27.6nl 3:43 10 min 9
9 D. innubila male yes 12/4-12/5 20231212 7-8 days 4 0.01 FFU 27.6nl 3:52 10 min 10
10 D. innubila female yes 12/4-12/5 20231212 7-8 days 4 0.01 FFU 27.6nl 3:52 10 min 10
11 D. innubila male yes 12/4-12/5 20231212 7-8 days 4 0.01 FFU 27.6nl 4:02 10 min 10
12 D. innubila female yes 12/4-12/5 20231212 7-8 days 4 0.01 FFU 27.6nl 4:02 10 min 10
13 D. innubila male yes 12/4-12/5 20231212 7-8 days 1 3 FFU 27.6nl 4:25 10 min 8
14 D. innubila female yes 12/4-12/5 20231212 7-8 days 1 3 FFU 27.6nl 4:25 10 min 8
15 D. innubila male yes 12/4-12/5 20231212 7-8 days 1 3 FFU 27.6nl 4:33 10 min 9
16 D. innubila female yes 12/4-12/5 20231212 7-8 days 1 3 FFU 27.6nl 4:33 10 min 8
17 D. innubila male yes 12/4-12/5 20231212 7-8 days 1 3 FFU 27.6nl 4:41 10 min 10
18 D. innubila female yes 12/4-12/5 20231212 7-8 days 1 3 FFU 27.6nl 4:41 10 min 10

Mortality daily is not measured, but when taking the day samples, their information is here. The vials for day 5 will be transferred to new food on day 3:

For transferring flies and freezing, 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 if transferring
  • Use new 1.5mL tubes from the molec lab only (less likely to be contaminated)
  • Use clean forceps to move a fly to the 1.5mL tube if freezing
  • 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