Training on Infections with Serratia on D. innubila and D. falleni
20220825 Growing Serratia Overnight
- Took out the serratia plate from the fridge (Kistie had grown up the Unckless lab strain (stock number 93) that month)
- Turned on the bunsen burner
- Got 2 LB media tubes and labeled serratia, name, and date
- Picked 1 colony from the plate with a pipette tip and dropped into the LB liquid for both tubes, trying to keep sterile, things open for a quick amount of time and close to the flame
- Placed the test tubes in the 37 degree incubator shaking overnight
20220826 OD Spectrophotometer
When learning the spec, I made 0.1, 1, and 10 ODs. I only ended up using the 0.1.
- Note, this takes ~30 minutes of time to titrate the desired OD
- Note, need to turn on the spec 10 minute before use so the bulb can warm up
- Note, you want your reading to be within 0.02 of the number you want
- Note, make sure to not contaminate transfer pipettes, it’s ok to get new ones
- Once warmed up, clicked OD600
- Got 2 LB test tubes
- Got a fresh cuvette
- Used transfer pipette to fill cuvette to line with LB
- Put in spec to read as blank
- Pressed arrow to go on to samples
- For making the 0.1 OD
- Basically it was titrating drops of bacteria and LB to get the concentration within 0.02 of the desired number of 0.1
- Started by adding 1 drop of bacteria from the overnight culture into the LB blank
- This was about 0.01
- Increased the drops to get to 0.1, this took multiple tries of doing 2 drops, then reading it
- After every drop before reading, the solution was pipette mixed with the transfer pipette
- Note, make sure to use the appropriate transfer pipette for mixing, you don’t want to add in extra bacteria or LB thats on the sides of the pipette
- Once i got the right OD, I got a 0.6mL tube and used a transfer pipette to fill it all the way to the top of the tube (easy to put the needle in)
- Making OD of 1
- Based off how many drops it took to get to 0.1, it would be tons of drops to get it to 1
- To concentrate the bacterial culture, I put the second culture tube in a 1.5mL tube and centrifuged for ~1 min to make a bacterial pellet
- Then sucking off the supernatant to start of the taper of the tube where 500ul is)
- Then resuspending the bacterial pellet in the liquid
- Used 1 drip of that into the previous cuvette and reading it on the spec
- This happened to be ~1, but if it hadn’t, I would have used either LB or the less concentrated bacteria to get it to 1
- This OD bacteria was also put in a 0.6mL tube, but I ended up not infecting with it
- Making OD of 10
- The spec only reads to 4, so you can’t directly read an OD of 10
- Get a new cuvette if needed, if you get a new cuvette, it needs to be blanked
- Did a 1:10 dilution for the spec: 540ul of LB and 60ul of the concentrated bacteria
- This was about 1, which means the stock was 10
- Make a 0.6mL tube of pure LB (made sure to use a new transfer pipette)
- Cleaned everything up afterwards:
- Cuvettes, tips, pipettes into the orange trash
- Wiped bench with ethanol
- Wiped pipettes and tip boxes with ethanol
- Bleached test tubes
- Placed the infection tubes on a rack and took to the fly room
innubila and falleni 0.1 OD Serratia Infections
- Note: I have previously made up ~20 vials of mushroom food for the infected flies to live in
- Added a 1.5mL tube of 95% ethanol to my tube rack
- Set up at a microscope, put 2 kim wipes in front of the scope area
- Dipped new infection needed and probe in the ethanol and laid on the kim wipes to dry
- Notes about the process:
- Main thing to note is that you don’t want flies to be on the CO2 for more than 10 minutes
- Best to switch up the order of LB and serratia, so you don’t always do LB first and potentially bias
- The infection point between two suture points below where the wing attaches (this picture is from melanogaster but this spot is also in innubila and falleni)
- Kistie suggests to try to poke the fly with the needle close to verticle
- Want to poke the fly until the taper of the needle goes in, or about 1/3 of the depth of the fly (this is different for different species)
- Hold the probe and the vial of infection material in the left hand, for manipulating the fly’s position, and the needle in the right hand
- Infect only males because mating or not mating doesn’t effect their immune system
- Process for infecting 1 vial of flies:
- Prepared 2 new vials for the poked flies from the fly vial, one labeled for LB and one for serratia
- Dumped the fly vial on the CO2 pad and recorded the time of dumping on the 2 new vials set up
- Lined up the flies on the CO2 and sex them quickly by pushing the females off the line
- Then I counted up the number of males and divided them in half, where one half would get LB poke and the other half would get serratia
- Then I’d get the probe, liquid tube, and needle into my hands
- Between each fly, dip the needle into the liquid, position to easily access the infection point, then poke, pick up the fly with the needle (they should stick on the needle) and push them gently off with the probe on the side of the CO2 pad
- Once done with a set of flies, dip the needle and probe into ethanol and let dry on the kimwipe
- Brush the poked flies in their respective vial and write the n number on the vial
- Keep the vial with the poked flies horizontal for a long time to let the poked flies recover and wake up (at least 10 minutes)
- All infected flies should be kept in the incubator for the duration of their infections (they should have been aged in the incubator too)
- Notes for these infections, the first innubila vial was LB and the flies were on the CO2 for about 15 minutes, all other vials were 10 minutes or less
- falleni are a lot smaller flies than innubila, especially the males. I tried to not poke them as hard as the innubila, but it was hard to get consistent with the poking. I would say I was not very consistent with the poking, either depth or angle of poke
- I checked the flies ~24 hours later, then left them for the weekend, then checked them again on Monday, the rest of the days of the week until it had been 7 days
- On Monday I transferred the flies to new food, trying not to loose any flies (but some of them did get lost, see big table below)
innubila infection info:
| tube # | treatment | time of infection | n# |
|---|---|---|---|
| 1 | LB | 2:50pm | 9 |
| 2 | serratia 0.1OD | 3:07pm | 7 |
| 3 | LB | 3:07pm | 8 |
| 4 | LB | 3:19pm | 5 |
| 5 | serratia 0.1OD | 3:19pm | 7 |
| 6 | serratia 0.1OD | 3:28pm | 8 |
| 7 | LB | 3:37pm | 9 |
| 8 | serratia 0.1OD | 3:45pm | 8 |
falleni infection info:
| tube # | treatment | time of infection | n# |
|---|---|---|---|
| 9 | LB | 3:52pm | 9 |
| 10 | serratia 0.1OD | 3:52pm | 9 |
| 11 | serratia 0.1OD | 4:03pm | 9 |
| 12 | LB | 4:03pm | 9 |
| 13 | LB | 4:14pm | 11 |
| 14 | serratia 0.1OD | 4:14pm | 11 |
Infection/survival spreadsheet:
| Vial | Species | Treatment | Replicate | Day_emerged | Day_Infected | time_infected | fly_age | OD600 | Number_of_flies_per_vial | Day 1 (thur 8/26) | Day 2 (sat 8/27) | Day 3 (sun 8/28) | Day 4 (mon 8/29) | Day 5 (tues 8/30) | Day 6 (wed 8/31) | Day 7 (thur 9/01) | Notes |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 1 | innubila | LB | 1 | 08/18-08/21 | 08/25 | 2:50 | 4-7 days | NA | 9 | 9 | NA | NA | 9 | lost 1 fly in food transfer | |||
| 2 | innubila | serratia | 1 | 08/18-08/21 | 08/25 | 3:07 | 4-7 days | 0.1 | 7 | 4 | NA | NA | 3 | ||||
| 3 | innubila | LB | 2 | 08/18-08/21 | 08/25 | 3:07 | 4-7 days | NA | 8 | 8 | NA | NA | 8 | ||||
| 4 | innubila | LB | 3 | 08/18-08/21 | 08/25 | 3:19 | 4-7 days | NA | 5 | 5 | NA | NA | 5 | ||||
| 5 | innubila | serratia | 2 | 08/18-08/21 | 08/25 | 3:19 | 4-7 days | 0.1 | 7 | 4 | NA | NA | 4 | ||||
| 6 | innubila | serratia | 3 | 08/18-08/21 | 08/25 | 3:28 | 4-7 days | 0.1 | 8 | 4 | NA | NA | 4 | ||||
| 7 | innubila | LB | 4 | 08/18-08/21 | 08/25 | 3:37 | 4-7 days | NA | 10 | 10 | NA | NA | 9 | ||||
| 8 | innubila | serratia | 4 | 08/18-08/21 | 08/25 | 3:45 | 4-7 days | 0.1 | 8 | 4 | NA | NA | 3 | ||||
| 9 | falleni | LB | 1 | 8/15-8/18 | 08/25 | 3:52 | 7-9 days | NA | 9 | 9 | NA | NA | 9 | ||||
| 10 | falleni | serratia | 1 | 8/15-8/18 | 08/25 | 3:52 | 7-9 days | 0.1 | 9 | 8 | NA | NA | 8 | lost 1 fly in food transfer | |||
| 11 | falleni | serratia | 2 | 8/15-8/18 | 08/25 | 4:03 | 7-9 days | 0.1 | 9 | 6 | NA | NA | 6 | ||||
| 12 | falleni | LB | 2 | 8/15-8/18 | 08/25 | 4:03 | 7-9 days | NA | 9 | 9 | NA | NA | 9 | ||||
| 13 | falleni | LB | 3 | 8/15-8/18 | 08/25 | 4:14 | 7-9 days | NA | 11 | 11 | NA | NA | 11 | lost 1 fly in food transfer | |||
| 14 | falleni | serratia | 3 | 8/15-8/18 | 08/25 | 4:14 | 7-9 days | 0.1 | 11 | 10 | NA | NA | 7 | lost 1 fly in food transfer |