Aim: Culturing Drosophila using standard methods

Objective: To set up Drosophila using standard methods.

Principle: Drosophila melanogaster is one of the most widely used model organism in research
on genetics and genome evolution. Mass culture of D. melanogaster is important to produce
enough amounts of flies for research purposes. Drosophila species have extensive literature on
their genetics, development and ecology. Besides, the short generation time of Drosophila aided
the studies on genetics especially studies of the laws of heredity. One of the most extensively used
model organisms is Drosophila melanogaster, which is also known as the Cinderella of genetics.
This species of fruit flies not only possesses of well-defined genetics information, they also have
short generation time which one generation only requires about two weeks. In addition, one pair
of parent flies is able to produce several hundreds offspring which ease the process of genetics. D.
melanogaster are bred in more solid culture media with yeast added manually to ferment those
culture media. One of the most common ingredients added to fruit culture media in order to solidify
the culture media is agar. In addition, sugar content in Drosophila culture media must be sufficient
too. This sugar content is not only vital for the growth of Drosophila, it is also required by yeast
for fermentation. methyl parahydroxy benzoate and propionic acid are also used as antifungal and
antibacterial agents respectively.
Materials: Jaggry, corn flour, Agar, Yeast, methyl parahydroxy benzoate and propionic acid,
ether, culture bottles, nonabsorbent cotton, whatman no 1filter paper, brush, petriplate, BOD
incubator 250C, microscope
Method:
Media preparation:
Ingredients: 27 g agar, 200 g cornmeal (organic, fine ground), 140 g sugar, 50 g yeast, 20 ml
propionic acid (added to prevent bacterial growth), 20 ml of methyl parahydroxy benzoate.
Procedure:
1. Dissovle agar in 2L tap water by boiling
2. Dissolve cornmeal, sugar and yeast in 1L cold tap water so that it is free of lumps.
3. Once agar is dissovled add cornmeal mix and bring to boil. Stir constantly to aviod
caking/burning on bottom.
4. Boil for 15 minutes, stirring constanty.
5. Take off stove and cool. Add Propionic acid and Nipagen. Ensure you are working in
a well ventalted area.
6. Distrubute fly food into vial/bottles as required. Only fill each container up to 1.5 cm.
7. Cover with paper towels and allow to cool and dry (over night) at room temperture.
8. Vials/bottles should be plugged before storing in a fridge.
9. Bring the vials at room temperature before use
10. Transfer required males and females in to the vial and keep vials in horizontal position until
flies revive if etherized.
11. Once flies revive keep the bottles vertical in BOD incubator at 250C
Handling Flies: Once the flies have been knocked you can move the flies around using a clean
paint brush. Using the paint brush carefully means you will not damage the flies when sorting and
manourving. Transferring flies from one container to another involves tapping the bottle gently,
the flies dislodge from the walls and fall to the bottom. Removing the lid from the bottle you want
to transfer the flies to and placing the flies over this bottle, then tap and the flies will fall from one
bottle to another, quickly put the lids on the bottles.
Anesthetizing flies: The problem with fruit flies is that they fly! Therefore a variety of methods
have been developed to anesthetize flies. Include are ether, commercial brands such as Flynap,
carbon dioxide, and cooling. Each has its strengths and weaknesses. Ether is flammable, has a
strong odor and will kill flies if they are over-etherized (and can anesthetize younger students!).
Set up a large test tube with a tube and stopper system. Anesthetizing flies by cooling is also simple
method. In order to incapacitate the flies, place the culture vial in the freezer until the flies are not
moving, generally 8-12 minutes.
Transferring flies from one vial to another: Flies should be transferred every 10 to 14 days.
Students should maintain a backup culture of their flies and the instructor should maintain backup
stock cultures of all fly strains. There are two basic ways to transfer flies when forming new
cultures. One requires no anesthetizing but quick hands.
A) Place a funnel in the mouth of a fresh culture vial that already has media added. In the old vial
(the one with flies in it), gently tap the flies down by softly tamping the vial on a soft surface, such
as a mouse pad. The flies will fall to the bottom and remain there for a few seconds (no more than
that!), enough time to quickly take the plug off the vial, invert it into the funnel, and gently tamp,
together, the two vials to force flies down into the new vial.
B) An alternative way is to put the flies in the freezer for about 8 minutes. This will cause the flies
to fall into a state of stupor. After placing a funnel on the new vial, invert the vial with motionless
flies into the funnel. This is not as much fun but you won’t have any flies flying around the
classroom.
Sexing flies: It is quite easy to tell males from females and with a little practice students will
become confident of their ability to do so. Notice that males are generally smaller and have a
darker and more rounded abdomen. The coloration of the abdomen is the easiest to recognize. In
addition, males have tarsal sex combs on their first pair of legs. These are black and very distinctive
but can only be seen under relatively high magnification. With a little practice, by looking at the
abdomen students will become proficient in accurately sexing flies. Sexing flies is critical when
making crosses, so be sure student are confident in identifying the difference between the sexes.
In order for students to feel comfortable sexing flies, give or have them obtain 25 or more mixed
sex flies and allow them to sort the flies into two piles, male and female. Other students in the
group and the instructor should verify the sorting. Each member of the group should be able to sex
flies.

Male

Female

Collecting virgin females: While it’s a simple matter of placing virgin females with males, it is
important to recognize the time factor involved for obtaining virgins. Females remain virgins for
only 8-10 hours after eclosure and must be collected within this time frame. NOTE: Females have
the ability to store sperm after a single mating.
Removal method: Remove all flies 8-10 hours before collecting (generally this is done first thing
in the morning). Visually inspect surface of food to ensure complete removal of flies. After 8-10
hours (usually before you leave work) collect all females that are present. All will be virgins. Since
they are photoperiod- sensitive, females tend to eclose early in the morning. Therefore early
collections will ensure the greatest number of virgins for experimentation.
Visual method: Note that virgin females are much larger than older females and do not have the
dark coloration of mature females. In addition, in the early hours after eclosure, there will be visible
a dark greenish spot (the meconium, the remains of their last meal before pupating) on the
underside of the abdomen.
Temperature cycling: It is possible to maximize the number of virgins in a morning collection by
using temperature cycling. When cultures are maintained at a temperature of 18°C, development
is slowed so females will not mate until 16 hours after enclosure. By removing flies in the
afternoon/evening and placing the vials in an 18°C incubator, 98% of flies obtained in the morning
will be virgins.

Result:

Media poured in vials Culure established Culture Established