Testing Nanoject II Infections with 16Cq DiNV on D. innubila
Because the first test injections with control fluid on innubila seemed to go ok, I decided to test doing real infections with DiNV to see if this method would give me a robust infection. I decided to use the 27.6nl I had tested previously because it is the middle volume the machine can give, so I can adjust up or down in the future if needed. I used the 16Cq virus cell culture fluid because it has given me good infections in the past.
I did my best to make sure the infection went sterilely with the new method. The day before, 2 vials of 5 males and 2 females, and 7 vials of 11 males and 4 females were made up. These will be the males I will infect
Set up area
- Thawed 16Cq virus on ice and changed gloves after touching
- Made 7 new vials of mushroom food
- Wiped down fly room bench with 95% ethanol before using/putting things on it
- Things taken to the fly room
- tube rack with 10 tubes for day 0
- mineral oil
- sterile Co2 pad
- pulled needles
- sterile forceps (2)
- two sets of gloves
- 95% ethanol
- Nanoject
- autoclaved toothpicks
- Notebook
- Virus and control medium on ice
- Used one of the forceps to clip off the tip of one of the pulled needles
- Used the metal needle to backfill the pulled needle with mineral oil, avoiding any bubbles
- Placed the pulled needle on the machine
- Slide the collet onto the needle
- Pushed the 2nd o-ring onto the needle
- Slid the needle onto the machine
- Tightened the collet down
- (this method is easier than pushing the needle through the ring on the machine)
- Ejected the mineral oil until the beep
- Filled the needle with control medium (10% FBS, 4% mushroom Schneider’s medium with antibiotics)
- Tested needle to make sure fluid ejects when pressing inject
Injecting flies
- Placed 1st vial of 5 flies on CO2
- Separated out males with toothpick
- Injected each fly with 27.6nl control medium
- Placed each fly in 1.5mL tubes with forceps, dipped in 95% ethanol between each fly
- Placed 1st vial of 10 flies on CO2
- Separated out males with toothpick
- Injected each fly with 27.6nl control medium
- Used toothpick to place flies in new vial
- Repeated steps for 2 more vials for all the control flies
- Note here I tried to remove all the control medium from the needle by pressing eject and then tried to fill it with virus fluid but I broke the needle. So I had to get another needle, break off the tip, backfill it, and fill it with virus for the infection treatment flies. So two needles were used.
- For virus infected flies, infections went the same way:
- Placed 2nd vial of 5 flies on CO2
- Separated out males with a newtoothpick
- Injected each fly with 27.6nl 16Cq virus
- Placed each fly in 1.5mL tubes with forceps, dipped in 95% ethanol between each fly (separate pair of forceps from the control flies)
- Placed next vial of 10 flies on CO2
- Separated out males with the virus toothpick
- Injected each fly with 27.6nl 16Cq virus
- Used virus toothpick to place flies in new vial
- Repeated those steps for 3 more vials
The time of infection and duration on CO2 was recorded for each fly:
| vial | treatment | volume | time | time on CO2 | n# |
|---|---|---|---|---|---|
| 1 | control medium | 27.6nl | 3:34pm | 6 min | 9 |
| 2 | control medium | 27.6nl | 3:41pm | 6 min | 10 |
| 3 | control medium | 27.6nl | 3:48pm | 7 min | 10 |
| 4 | 16Cq DiNV | 27.6nl | 4:39pm | 6m min | 10 |
| 5 | 16Cq DiNV | 27.6nl | 4:45pm | 7 min | 9 |
| 6 | 16Cq DiNV | 27.6nl | 4:53pm | 6 min | 10 |
| 7 | 16Cq DiNV | 27.6nl | 4:59pm | 6 min | 10 |
The flies that were frozen for day 1:
| tube# | treatment |
| 1 | CCM |
| 2 | CCM |
| 3 | CCM |
| 4 | CCM |
| 5 | CCM |
| 6 | DiNV |
| 7 | DiNV |
| 8 | DiNV |
| 9 | DiNV |
| 10 | DiNV |
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