This is about a set of stocks that had a bacterial infections in 2016 in the lab (we think Acinetobacter spp.)
the infection is most likely to due to the presence of some acinetobacter species which contain stable antibiotic resistance for at least streptomycin and penicillin.
It also seems to be persistent, and can not be controlled using rapid transfers of lines to fresh vials (i.e. transferring the same flies to fresh vials each hour over a period of 3-4 hours, followed by fresh vials each day for the next 4-5 days). Normally this latter approach works for fungal infections.
It also appears that decontaminating eggs via 3-4 minutes in bleach (which dechorionates the eggs) is only partially successful. This is quite surprising.
We also had poor luck switching the sugar source to dextrose. While this reduced the mucous (apparently a dextrin), we still had larval lethality.
My most recent trials have used a combination of tetracycline (final concentration of 50 micrG/mL of food) and chloramphenicol (20 microG/mL) that seem to be working well. At least in the first generation, I see no sign of the slimy bacteria. However, there are a few things to note. First, the antibiotics are a bit harsh and it does kill some flies. So for populations with lots of natural variation, this may not be optimal. Second of all, it is well known that tetracycline kills wolbachia bacteria, which can have huge effects on sexual behaviours (and sex specific survival). So be cautious when using it. I describe how to make the antibiotic solution for the food below.
The other approach that seems to be keeping the effects of the bacteria minimized for the moment is the use of the new yeast (red star). If you have a relatively healthy number of flies, grow them in a vial or bottle well seeded with live yeast from this strain. Not only will this stimulate rapid egg laying in the females, but it seems to inhibit the growth of the bacteria enough such that the larvae seem viable. The only issue with this, is you can run into an issue with the yeast growing too much (especially for a culture with a small number of flies). You can tell this when the surface of the food looks uniformly tan (like the colour of the yeast particles).
If you do have a weaker culture, and want to use the red star yeast, I suggest the following. Seed a vial with lots of yeast (red star) on the food surface. Place the flies in this new vial. Repeat with a fresh vial (with fresh yeast) for each of the next two days (i.e. daily transfers). On the 4th day, transfer to a fresh vial without any new yeast seeded. HOPEFULLY, most of the problematic bacteria will have been replaced by the fresh yeast, and there is enough live yeast on the surface of the flies to start fresh community, but not too much too cause overgrowth.
Much of the effects of yeast is likely in part due to its effect on lowering the pH of the fly media. Indeed Bloomington stock center suggests this is a common explanation for persistant bacterial infection. they suggest lowering the food to around pH of 5, and this can help a lot.
I have made up two stock solution. 16.5mg/mL tetracycline in 70% ethanol and 20 mg/mL chloramphenicol in 95% ethanol.
To get final concentrations in a standard food vial (~8mL of food) you want to add the following to each vial (make a solution of the following):
| reagent | for one vial | for 20 vials |
|---|---|---|
| 16.5 mg/mL Tet | 24.2 microL | 484 microL |
| 20 mg/mL Chlor | 10 microL | 200 microL |
| water | 15.8 microL | 316 microL |
Add water, tet and chlor together into a stock solution. With a sterile spatula or forceps, poke holes in fly food (to allow solution to penetrate). Add 50 microL of stock solution to each vial, taking care to cover entire surface. Allow dry for at least 4 hours (I leave it overnight). Then you can use the vials. Mark each vial with antibiotics with a black dot on top (so you know it has the antibiotics). After the next generation of flies emerges from that food, label the flug with a "P" on top (post-antibiotic).