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[Drosophila] Culture problems

Mark Thorson via dros%40net.bio.net (by nospam from sonic.net)
Wed Jul 4 19:48:37 EST 2012

I'm having inconsistent results my cultures.
Sometimes every culture is good, but more
often there will be a few poor performers.

Unlike the more standard methods, I don't
use any methyl paraben and I don't autoclave.
I make a new batch every day, so autoclaving
would be a real burden, adding an hour or more
to a procedure that is already about an hour.

My culture vessels are large plastic cups with
dome lids, like what are used for bubble tea.
I use small coffee filters between the cups and
lids to allow for air exchange.  I usually make
a dozen cultures every day, sometimes two dozen.

My medium consists of chunks of bananas (six
bananas per dozen cups), four teaspoons of
cream of tartar, one packet of active dry yeast,
and a couple ounces of water.  I cut each banana
into discs about half an inch thick, then quarter
each disc.  I don't mash them up because I want
to slow down their decomposition by the yeast.
I mix the cream of tartar, yeast, and water,
then use the mixture to coat the banana chunks,
like I was making potato salad.

While mixing, it appears that a significant
amount of liquid is coming out of the bananas.
Within minutes, there is foaming as the yeast
grows.  Once I place the mix in the cups, the
foam rises for several hours, then declines.
All of the liquid gets absorbed within 24 hours,
then it reappears 24 hours after that and
persists for the rest of the life of the culture.

I suspect my variable results are due to
bacterial growth.  I have had no problems with
mold.  In my early experiments working out the
recipe, bacteria was a terrible problem, some
cultures being completely dead.  It usually looks
like a layer of melted cheese on top.  Sometimes
it smelled like limburger cheese, but other times
it had no odor.

Since adopting the current recipe, I seem to have
the bacterial growth greatly reduced, but I still
notice a difference in odor among the cultures,
even cultures in the same batch.  Some smell
strongly of vinegar, alcohol, or both, while other
cultures have very little odor.  Also, I sometimes
notice a "bite" in the odor, which I suspect is
carbon dioxide.

I recently noticed in a batch that was three or
four days old that a few of the cups had lots of
dead adults -- sometimes all of them.  I usually
start a culture with 30-50 adults.  I'm beginning
to suspect that the variability in my cultures
is due to early death of the adults due to carbon
dioxide poisoning, though there didn't seem to
be a correlation between strong odor or "bite"
and dead adults.  (I could be wrong about that.)

Another factor I've considered is whether the age
of the adults might be contributing to variability.
Somtimes I use adults that are freshly emerged,
while other times they will be a few days old.
I've started marking the young adult batches to
see if it correlates with good yield.

I'm wondering what experienced Drosophilists would
attribute the variability to?  Does it sound
plausible that the yeast could generate enough
carbon dioxide to kill the adults?  Does the age
of the adults make a big difference?  I'm still
suspecting that bacterial growth is the factor
behind the variabililty.  I'm considering seeding
new cultures with a bit of the culture medium from
old cultures that were good performers.  I already
tried this with several batches and it didn't solve
the problem, but maybe I didn't try hard enough.
The last batch I made that way was very good, so
I'm thinking of trying that again.

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