More training on Infections with Serratia on D. innubila and D. innubila
My previous attempt at infections was not completely informational based on the mortality. I could have either poked too lightly or that innubila are more resistant to serratia infections - both could be true. This time I am trying different ODs of serratia, and also adding melanogaster flies.
I had previously separated out 8 vials of innubila, each with 22 males and 10 females. These had aged 7-10 days in the incubator. There was some loss of fly life with these. Melanogaster flies from Kistie were a little older, probably 12-14 days but they would do for a test.
20220904 Making Sugar Agar Vials and Growing Serratia Overnight
Sugar Agar
- Made up 30 vials
- Recipe:
- 33.2g sugar
- 8g drosophila agar
- 400mL DI water
- Combined these into a 1L flask and microwaved until boiling
- This was basically impossible to not boil over
- When it had boiled and was clear, let sit on the counter until it was 60C
- Measured 0.8g tegosept and dissolved it in 95% ethanol
- At 60C, added the tegosept and mixed on the stir plate
- Used serological pipettes to add 10mL of sugar agar to each vial
- There was a little left over
- Covered the vials in a pillow case and let cure overnight
- Next day stored in the fridge with press-n-seal covering
Growing Serratia
- Using the Unkless lab strain # 93
- Made up 3 liquid culture LB tubes
- Picked 1 colony from the plate and put into the culture tubes
- Placed in the 37C incubator overnight
20220905 Infections
Spectrophotometer
- Let bulb warm up for 10 minutes before use
- Plan was to make 0.1, 1, and 4 ODs
- I titrated drops of culture and LB, and used a concentrated bacterial culture to get the correct OD readings. This took me about 30 minutes. I felt like I was running short on time so I ended up not making an OD of 4, but just getting it to ~3 and using that one
- Exact ODs were: 0.099, 1.004, and 3.153
- Each one was pipetted into a 0.6mL tube, and an LB tube was made as well
- Test tubes were bleached and the area was cleaned with 70% ethanol
Serratia Infections
- Plan was to do 30 LB innubila, 40 0.1 OD innubila, 40 1 OD innubila, and 40 3 OD innubila. I also got some melanogaster c564 flies from Kistie to also try infecting. I wanted to do 20 LB dmel, 20 0.1 OD dmel, and 20 1 OD dmel
- Process for infecting flies:
- Got a new tube of 95% ethanol
- Cleaned a probe and an infection needle
- Prepared/labeled 2 sugar agar vials per vial of innubila or 2 molasses vials per vial of c564
- Dumped the fly vial on the C02 and recorded the time on the vial
- Sexed flies and split number of males in half. Half would get 1 treatment, and the other half would get a different one. The order of what treatment/poke they got I varied throughout
- I infected half the flies, dipping the needle in the infection liquid after each fly, then put the flies in their new vial, then disinfected the probe and the needle. Then I moved on to the other half of the flies
- I tried hard this time to be more consistent with my poking, and I tried to poke more below the wing joint which is softer
- Set up for innubila:
| vial # | infected with | N# | time put on CO2 | Extra time on CO2? |
|---|---|---|---|---|
| 1 | LB | 9 | 3:40pm | no |
| 2 | 0.1 | 9 | 3:40pm | 11 min on CO2 |
| 3 | 1 | 9 | 3:53pm | no |
| 4 | 3 | 9 | 3:53pm | no |
| 5 | 1 | 8 | 4:03pm | no |
| 6 | 0.1 | 8 | 4:03pm | no |
| 7 | 3 | 7 | 4:13pm | no |
| 8 | LB | 7 | 4:13pm | no |
| 9 | LB | 9 | 4:21pm | no |
| 10 | 1 | 9 | 4:21pm | no |
| 11 | 0.1 | 7 | 4:30pm | no |
| 12 | 3 | 7 | 4:30pm | no |
| 13 | 3 | 9 | 4:39pm | no |
| 14 | 1 | 9 | 4:39pm | no |
| 15 | 0.1 | 10 | 4:47pm | no |
- Set up for c564 melanogaster
| vial # | infected with | N# | time put on CO2 | Extra time on CO2? |
|---|---|---|---|---|
| 1 | LB | 10 | 4:54pm | no |
| 2 | 0.1 | 10 | 4:54pm | no |
| 3 | 1 | 10 | 5:04pm | no |
| 4 | 0.1 | 10 | 5:04pm | no |
| 5 | LB | 10 | 5:13pm | no |
| 6 | 1 | 10 | 5:13pm | no |