fly culture manual by Jessica

Fly culture general instructions

Monday: stock culture

Current stocks:

·       29 inbred lines

·       20 vials FM (yellow-bodied and bar-eyed)

·       20 vials LHm-bw (brown-eyed)

·       60 vials LHm (wildtype)

·       6 vials Andro (male colour pattern selection line)

Yeast new vials.  Use high yeast for inbred lines, low yeast for all other stocks.  Combine multiple vials from the same stock to avoid genetic drift between vials.  If you are confident in your ability to handle the flies on the gas plate quickly, do this using a collector bottle.  If not, mix 2 vials on the plate.  Make sure to mix between bundles in both cases (i.e. using collector or mixing individual vials).  When using collectors, place 1/3 to 1/2 of the flies on the plate at one time—more than this and you’ll have trouble handling them quick enough.  The longer the flies are kept in the collectors, the stickier they will become, so it’s best to only fill one or two collectors at a time.

Remember not to leave the flies on the gas plate for longer than 4 minutes!

Put 16 pairs of flies (i.e. 16 males and 16 females) in each new vial.  Discard excess flies.  Wipe off gas plate every time you switch to a new stock.  Remember to wipe when switching between different inbred lines as well.  Using damp paper towel reduces static electricity on the plate somewhat.

For the inbred lines, the vials are ordered from left to right in the following way: 2 vials of old backups from two culture cycles ago, 2 vials of old backups from the last culture cycle, and 2 stock vials from the last culture cycle (with foam plugs).  Use the stock vials for culturing, but if you have too few flies then it’s OK to get extras from the backups.

Use fresh plugs for the new vials.  Put old vials in the freezer and wash plugs later.

Label with white tape and current date.  Make sure to add a piece of brown tape to the LHm-bw vials so that it’s easy to distinguish between them and the regular LHm stock.

To wash plugs, fill a white plastic container with hot water and a bit of washing liquid.  Let the vials soak for at least 15 minutes.  Then scrub gently between your hands and rinse well.  Let dry in another white plastic container.  The empty frozen vials from which you removed the plugs get thrown away in the dumpster marked “brännbart” behind the Ecology Building.

Wednesday: flips

Flip flies into fresh vials on Wednesday afternoon.  The inbred lines take longer to lay than the other stocks, so they can be flipped earlier (anytime after 13.00 is fine).  The LHm and LHm-bw stocks in particular are very fecund, so it’s best to flip them later in the day.  Anytime after 15.00 is fine, but you’ll have less trimming to do if you wait until 16.00.

Make sure to transfer the foam plugs to the new vials, and plug the old (backup) vials with rayon (人造丝)balls.  Leave vials on their side while flies are laying eggs.

Once you’ve done the flips you can throw away the old backups in the incubator.  For the inbred lines, throw away the 2 vials on the left (backups from two culture cycles ago), and move the other vials two steps to the left, so that you have (from left to right): 2 vials with backups from the previous culture cycle, 2 vials with the new backups, and 2 empty slots for the new stock vials (which we leave lying down until after clearing).

Thursday: clearing and trimming

Clear (discard) flies from egg-laying vials at 9:00 on Thursday morning.  Make sure to check that the inbred lines have laid enough eggs before discarding them.  If the number of eggs looks too low, check them again later in the day (how much approx. is too few?).  Too few eggs is unlikely to be a problem for the other stocks (except maybe the FMs).

Trim excess eggs by scraping them off the food.  Target number of eggs is 100-150 for all stocks except the inbred lines.  For the inbred stocks anything from 50-150 is OK—here the acceptable range is wider because you don’t have to worry about the stocks adapting to changes in density. For vials where the flies will be used in an experiment, make sure to count fairly carefully.  For all other vials it’s ok to count around 10 vials to calibrate yourself, then estimate for the rest.

Clearing needs to be done in the morning, otherwise the flies will lay too many eggs and you’ll have to do a lot of work to trim them.  Trimming can wait until later in the day if necessary, although you still need to do it on Thursday.

After trimming the vials, make sure to stand them upright in the incubator.