Infecting Male D. innubila with Middle Band DiNV to Check Titering Accuracy
I planned on diluting the stock virus to concentrations to deliver 0.01 FFU, 0.05 FFU, 1 FFU, and 3 FFU in a 27.6nL volume to see if the mortality curves between the P4 stocK I have already done and the newly titered stocks are similar.
I decided to use the middle band stock that had been titered to 527,500 FFU/mL. (this is also 527.5 FFU/uL) I also decided to calculate out a 3 FFU dilution and then make further dilutions from that tube.
- For 3FFU delivery in 27.6nL solution, we’d need a 0.1086 FFU/nl solution
- If I wanted to make 60ul of 0.1086 FFU/nl solution, we multiply 60ul * 108.6 FFU/uL = 6,516 FFU total needed
- 6,516 FFU / 527.5 FFU/uL = 12.35 ul of stock MB virus
- 60 - 12.35 = 47.65uL of cell culture medium to make the dilution
- The other solutions were made from the 3 FFU delivery solution, these solutions were made and kept on ice until use. All solutions were pipette mixed and kept bubble free:
| tube | dilution for delivery | ul of virus solution | virus solution tube | ul of medium |
|---|---|---|---|---|
| 1 | 3 FFU | 12.35ul | stock tube 42 | 47.65ul |
| 2 | 1 FFU | 15ul | tube 1 | 30ul |
| 3 | 0.1 FFU | 5ul | tube 2 | 45ul |
| 4 | 0.05 FFU | 20ul | tube 3 | 20ul |
| 5 | 0.01 FFU | 5ul | tube 3 | 45ul |
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note that tube 3 was not used in the infections, just for further dilutions
- 11 new mushroom food vials were made
- Males had been separated out the day prior, and were 5-7 days post emergence
- Infections followed the nanoinjection protocol
| vial | species | sex | mated status | days_emerged | day_infected | age_infected | tube | treatment | virus | volume | time | time on CO2 | original_N_number |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 1 | D. innubila | male | yes | 2/25-2/26 | 20240302 | 6-7 days | CCM | CCM | middle band | 27.6nl | 2:22 | 6 min | 9 |
| 2 | D. innubila | male | yes | 2/25-2/26 | 20240302 | 6-7 days | CCM | CCM | middle band | 27.6nl | 2:27 | 6 min | 10 |
| 3 | D. innubila | male | yes | 2/25-2/26 | 20240302 | 6-7 days | CCM | CCM | middle band | 27.6nl | 3:33 | 5 min | 9 |
| 4 | D. innubila | male | yes | 2/25-2/26 | 20240302 | 6-7 days | 5 | 0.01 FFU | middle band | 27.6nl | 3:39 | 5min | 10 |
| 5 | D. innubila | male | yes | 2/25-2/26 | 20240302 | 6-7 days | 5 | 0.01 FFU | middle band | 27.6nl | 3:44 | 6 min | 10 |
| 6 | D. innubila | male | yes | 2/25-2/26 | 20240302 | 6-7 days | 4 | 0.05 FFU | middle band | 27.6nl | 3:52 | 5 min | 10 |
| 7 | D. innubila | male | yes | 2/25-2/26 | 20240302 | 6-7 days | 4 | 0.05 FFU | middle band | 27.6nl | 3:57 | 5 min | 10 |
| 8 | D. innubila | male | yes | 2/25-2/26 | 20240302 | 6-7 days | 2 | 1 FFU | middle band | 27.6nl | 4:04 | 5 min | 10 |
| 9 | D. innubila | male | yes | 2/25-2/26 | 20240302 | 6-7 days | 2 | 1 FFU | middle band | 27.6nl | 4:10 | 5 min | 10 |
| 10 | D. innubila | male | yes | 2/25-2/26 | 20240302 | 6-7 days | 1 | 3 FFU | middle band | 27.6nl | 4:17 | 5 min | 10 |
| 11 | D. innubila | male | yes | 2/25-2/26 | 20240302 | 6-7 days | 1 | 3 FFU | middle band | 27.6nl | 4:23 | 5 min | 10 |
Infection monitoring can be found here, note that the first vial was removed from the experiment, and that the last 3 were from another experiment that ended up not being used
Frozen fly information can be found here
Analysis can be found here, note that this is replicates combined