18/7/2014 Culturing Drosophila - Bloomington

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Basic Methods of Culturing Drosophila

Updated March 30, 2007

Stockkeeping
Mechanics
Balanced stocks and balancers
Culture contaminants
Mites
Fungi and Bacteria
Experimental populations
Matings
Virgins

Stockkeeping

1. Mechanics
Most stocks can be successfully cultured by periodic mass transfer of adults to fresh
food. Bottles or vials are tapped on the pounding pad to shake flies away from the
plug, the plug is rapidly removed and the old culture inverted over a fresh bottle or
vial. Flies are tapped into the new vessel, or some shaken back into the old one, as
necessary, and the two are rapidly separated and replugged. Good tossing technique
combined with plugs that are easily removed and replaced result in very few escapees.
You will learn from experience which stocks require a medium or large inoculation of
adults and which do better with only a few.

The frequency with which new subcultures need to be established depends on the
health and fecundity of the genotype, the temperature at which it is raised, and the
density of the cultures. Temperature has a large effect on the rate of Drosophila
development. Generation time (from egg to adult) is approximately: 7 days at 29°C, 9
days at 25°C, 11 days at 22°C, 19 days at 18°C. For most purposes stocks are maintained
by live culture, transferring adults to fresh medium every few generations. Stocks kept
at room temperature should be transferred to fresh food every 20 to 30 days. Mites and
mold are more likely to be a problem in older stocks, so it is good policy to set 30 days
as an absolute upper limit for room temperature stocks. This period can be extended by
keeping stocks at lower temperature. 18°C buys more time than 22°C, for example, but
a significant number of genotypes fail to thrive at 18°C, and mold can be a serious
problem. It is wise to keep a room temperature backup of stocks to be maintained at
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low temperature for the first two or three transfers in case the stocks do poorly. If the
quality of your fly food is unreliable it is wise to have at least two cultures for each
stock, staggered to assure the use of different batches of medium (at least until you
find a new cook).
Cryopreservation of ovaries (see Ashburner, 1989) or embryos (Cole et al., 1993;
Steponkus and Caldwell, 1993) are viable alternatives to continuous culture for some
purposes. Genotypes that are unstable due to reversion, breakdown, or accumulation of
modifiers, especially those with non-visible phenotypes that are time consuming to
select, are good candidates for freezing. Also, if you are generating hundreds of stocks
that will not be in use but must be kept for many years it might be cost-effective to
maintain these as frozen stocks. For most routine stockkeeping purposes, however, live
culture remains the preferred route.
Identify stocks with tags showing the complete genotype of the stock, sans shorthand.
Writing the genotype on the vial or bottle at each transfer invites transcription errors
and takes longer than moving a tag. Don't use a stock center stock number or other
potentially cryptic symbol as the only identifier of a stock. Stocks are often kept for
many years and what is obvious to you now may be meaningless even to you in a few
years, and is easily misinterpreted by someone inheriting your stocks. Unless you are
careful to maintain complete stock data elsewhere, record all relevant information on
the tag.
2. Balanced stocks and balancers
Mutations that are homozygous viable and fertile are most easily kept as homozygous
stocks. Lethal or sterile mutations must be maintained in a heterozygous state. A
balanced stock is one that regenerates the same set of heterozygotes each generation
so the stock can be maintained by mass transfer of adults instead of by mating specific
genotypes each generation. A simple balanced lethal stock carries different recessive
lethals on each of the two homologues, allowing only heterozygotes to survive.
Dominant male or female steriles (Ms or Fs) can be maintained in stock without
selection by double balancing - one Ms, one Fs and one recessive lethal. Fs/lethal male
and Ms/lethal females will be the only fertile genotypes present in the stock each
generation.

In most cases, balanced lethal schemes work only if one of the lethal chromosomes is
itself a balancer chromosome. Recombination between lethal (or sterile) mutations on
different homologues can produce one homologue with both mutations and one wild-
type homologue. The wild-type chromosome will rapidly predominate or become fixed
in the stock. Balancers are structurally rearranged chromosomes that prevent
recombination between homologues in females (meiotic recombination is absent in D.
melanogaster males and in the tiny 4th chromosome in females). This is accomplished
in part by reducing recombination directly and in part by preventing transmission of
recombinant chromosomes. The most commonly used balancers carry overlapping sets
of inversions and prevent recombination throughout most of the length of the
chromosome. Some special purpose balancers work well only for specific regions of a
chromosome. Suppression of recombination is less effective when balancers for two or
more heterologues are present in a stock.

Culture contaminants

Drosophila is relatively pestilence-free, but mites, fungi and bacteria can be problems
in laboratory cultures. It is good practice to clean your bench top and fly pushing
equipment regularly. This is particularly important if a problem is evident. Clean the
bench top and all equipment that comes into contact with potentially contaminated
stocks with 10% bleach, 70% ethanol or soap and water after use. Sharing pounding
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pads, CO2 pads, fly pushers and sorting plates can aid the spread of contaminants. If
sharing is unavoidable, the need for cleanliness should be understood by all and
enforced.
1. Mites
"I have mites" is not an admission you want to have to make to your fly colleagues (but
you must make it, if true). The most dangerous species are egg predators, but even
those that simply feed on the medium can out-compete weak genotypes and
compromise experimental observations. Frequent stock transfer, tight plugs, and zero
mite-tolerance by all the fly workers in a building are the best defenses. In general,
cultures grown at 24-25°C should never be kept for more than 30 days. If mites are
known to be a problem in your lab or building, cultures should be checked and
discarded after 18 to 20 days. Lining stock trays with benzyl benzoate-treated cheese
cloth (soak cloth in 10% benzyl benzoate in 95% ethanol, air dry; replace the cloths
every 6 months) may help prevent infestation. Some kinds of plastic are dissolved by
benzyl benzoate, so test first if you use plastic vials or trays (the cloth will fuse with
the plastic within 24 hours) and protect paper items such as stock tags from direct
contact with the cloths.
To prevent the importation of mites from outside sources all stocks new to your lab
should be quarantined for at least two generations. Never open a foreign bottle or vial
at your fly bench (or your neighbor's) without first inspecting the culture for mites.
Using a microscope, examine the surface of the medium and the walls of the container,
especially around pupae or pupal cases. If no mites are evident, replace foam or paper
stoppers with tight cotton plugs and isolate cultures in a quarantine tray. As an added
precaution, cultures can be wrapped in the mite cloths described above. Keep the
original bottle or vial for about 20 days, even though you have established fresh
cultures, rechecking for mites every 5 to 10 days. We check the new cultures too, just
to be safe, but we have never found mites in a subculture when the parental vial was
mite free.
Any culture found to contain mites should be frozen or autoclaved immediately if it can
be replaced from a mite-free source. If replacement is not possible, use one of the
methods described in Ashburner (1989) to disinfect the culture, such as daily transfer of
adults for about a week, using only the final transfer to establish a new and mite-free,
it is hoped, culture. Keep infected cultures wrapped in mite cloths until they have been
mite free for three generations.

2. Fungi and Bacteria

If mold is a problem in isolated cultures it can usually be eliminated by daily transfer of
adults for 7-10 days. Visually inspect cultures from the later transfers for hyphae (look
around the pupal cases) and use one that appears to be free of fungal growth for further
subculture. In extreme cases this process may need to be repeated for an additional
generation. If fungal contamination is a wide spread problem be sure that fungal
inhibitor (p-hydroxy-benzoic acid methyl ester) is being added to the medium after it
is cooked (boiling destroys the inhibitor), add a small amount of live baker's yeast to
every culture (the yeast tends to inhibit the growth of unwanted fungi) and check for
sources of infection in the lab, such as incubator fan housings. Clean any contaminated
or suspicious areas with disinfectant.
A variety of bacterial contaminants can occur in fly cultures. The most common
problems are caused by mucus producing bacteria. Although not directly toxic, larvae,
and to some extent adults, become trapped in the heavy layer of mucus that coats the
surface of the food. Large numbers of larvae overcome the effects of the bacteria in a
healthy stock, but weak stocks or pair matings can be seriously compromised. A
widespread bacterial problem may indicate that the pH of your medium is too high; try
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lowering the pH to about 5. Individual stocks can be treated with antibiotics for one
generation. A quick approach that often works: add 100 µl of penicillin-streptomycin
solution (10,000 u/ml and 10,000 µg/ml, respectively) to the surface of the medium in a
vial and allow it be absorbed. Add a small amount of yeast and transfer flies to the
treated medium. Discard adults before progeny eclose; subculture progeny on untreated
medium. Other antibiotics may be tried if the contaminant proves to be resistant to
penicillin and streptomycin.
Alternatively, clean cultures can be established from embryos dechorionated with 5.25%
sodium hypochlorite (liquid household bleach, full strength). A convenient method is to
transfer eggs to a bleach soaked wedge of filter paper, wick away bleach after
chorions have dissolved (3-5 minutes), wash eggs several times with water and transfer
to a fresh piece of filter paper (small enough to sit on the surface of the medium in a
vial) moistened with water. Place the filter paper with eggs into a fresh vial of food
and place a larger strip of filter paper along the wall of the vial. Wet this strip of paper
to maintain high humidity in the vial until the eggs hatch.

Experimental populations

1. Matings
While mass transfer of adults works well for most stockkeeping purposes, it often results
in overcrowded cultures. Overcrowding can effect the outcome of crosses and
experimental procedures. Development time is slowed, different genotypes may be
disproportionately affected by competition for food and pupation sites, and many
pupae and adults will drown in the soup of larvae and liquified medium. The best yield
of healthy adults is obtained from cultures established with an optimum number of
animals. Expect 50-100 adults from a vigorous 8 dram vial culture, 300-600 from a
comparable half-pint bottle culture. For most genotypes the optimum number of
females will range from 1 to 3 per vial and 5 to 20 per bottle. Set up a few test bottles
or vials to determine this number empirically for the genotypes involved; control the
age of the food, the age of the females, and the number of days the females are left in
the vials. One or two males are usually sufficient to rapidly inseminate several females,
but some genotypes will require an equal or excess number of males to insure rapid
mating. If necessary, the effects of overcrowding can be reduced mid-culture by adding
baker's yeast and a tissue (such as a Kimwipe®) to provide additional nutrition and
pupation sites, respectively, for the excess larvae. Extremely crowded cultures are best
dealt with by distributing larvae (scoop them out with a spatula) to several fresh
bottles or vials.
If you cannot distinguish parent from progeny by phenotype, parents should be
discarded before the progeny begin to hatch. Experimental crosses maintained at 25°C
should be discarded after 18 days to prevent recovery of second generation progeny.
An effective schedule is to establish crosses on day 0 (start on a Friday if you want to
begin virgin collection on a Monday), discard adults and add yeast and papers (optional)
on day 7, collect virgins or score progeny on days 10 through 18.
Some mutant phenotypes are affected by temperature or genetic background. Before
setting up a large scale effort such as a screen, make a test cross of the relevant
genotypes under the conditions to be used and confirm that all phenotypes are
scorable. It is also prudent to assure the absence of 'background' lethals in a stock to be
used for mutagenesis by isogenizing a chromosome for use in a screen. To isogenize a ri
e chromosome, for example, cross to an appropriate balancer stock, recover 10-20
progeny heterozygous for ri e and the balancer, backcross them individually to the
balancer strain, cross sibling ri e/balancer progeny, and then recover ri e homozygotes
from one of the lines and establish a stock. Only lines carrying lethal-free chromosomes
will produce homozygotes among the progeny of the sib matings.
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2. Virgins
Most experimental schemes require virgin females. D. melanogaster adults do not mate
for about 10 hours after eclosion, allowing virgins to be collected within 8-12 hours
after the culture has been cleared of adults. The timing of this virgin window appears
to be genotype dependent. Collect females within 8 hours to be safe, or determine
empirically for a given strain how long you can wait and still recover virgins. Ashburner
(1989) describes a variety of environmental and genetic tricks to facilitate the
collection of large numbers of virgin females.
For many mating schemes virginity is desirable for efficiency's sake, but not essential
because the progeny of non-virgin females can be distinguished phenotypically from the
progeny of interest. If your scheme requires virginity (e.g., male fertility testing, or
the progeny of non-virgin females are indistinguishable from those of the intended
mating), hold females for 3-4 days and check for larvae in the holding vial before using
the females in matings. Don't overcrowd females in holding vials - 50 or so in an 8 dram
vial, fewer if you aren't sure of their virginity (you'll have to discard all of the females
in the holding vial if any have mated). The peak of female fertility is genotype-
dependent, but on average females are best used between 4 and 10 days old.

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