Care, maintenance and manipulation of Drosophila
This section contains the following information:
1. Culturing techniques
a. Introduction
b. Bottles and vials
c. Media
d. Environment
e. Anesthetizing flies
f. Transferring flies from one vial to
another
2. Sexing flies
3. Collecting virgins
4. Crossing flies
5. Diseases
6. The morgue
Culturing techniques
Introduction
In order to incorporate D. melanogaster in the classroom,
it will be necessary to maintain cultures of flies for manipulation in crosses
and as a backup for any mishaps which may occur. Culturing is very easy and
it is recommended to have students maintain their own cultures of flies. In
that way, each student or group would be directly responsible for the care and
long-term maintenance of the flies, including making large culture populations
for their crosses. When directly involved, students gain proficiency and a greater
understanding of the flies requirements and behavior. The teacher should remain
as coach, not lecturer, assisting students in techniques. The instructor needs
to maintain stock cultures of all strains and mutants used by students in case
the afore- mentioned unforeseeable incident occurs and student cultures die
out or become intermixed. Losing cultures is the exception rather than the rule,
and as long as students reculture their flies on a regular basis and no mass
contamination occurs (see pests and diseases section), flies can be maintained
for decades.
Bottles and vials
TH Morgan used glass milk bottles for his experiments
and, indeed, any container will do, including baby jars and assorted containers.
However, for ease of culturing and transferring cultures, uniform bottles and
vials are the best approach. Both can be purchased from a biological supply
store. Bottles are used mainly for the maintenance of large populations of flies
whereas culture vials are useful for maintaining smaller populations and are
the preferred container for constructing student crosses. If there is a desire
to maintain stock cultures for a long period of time, or to reuse bottles and
vials it is important completely clean and sterilize them. This is to prevent
outbreaks of pests and diseases.
To clean bottle and vials freeze them if there are flies in them. Remove any food, wash well, then either autoclave for 20 minutes at 121 degrees C and 15 psi (if plastic be sure it can be autoclaved) or wash vials in a 10% chlorine bleach solution.
Bottles and vials can be purchased in a variety of sizes and materials. Glass is effective, however if dropped a student could lose 2 weeks of data in a single spill. Autoclaved (sterile) plastic vials are available and are preferable for student use. Vial sizes range from 96 mm by 25 mm to larger sizes, however the smaller size is recommended for making crosses and maintaining small cultures. There are a variety of plugs available from soft cotton to foam plugs. This is a matter of preference and costs, however cotton works fine and can be bought at a local drug store in a pinch.
Where to
buy
What
they look like
Media
The first step in preparing culture vials is adding food media.
There are a variety of types of food available for the flies; some require cooking
and others are bought already prepared and dehydrated. The latter can be purchased
from a biological supply company. This is, of course, much quicker and easier
than preparing cooked media, so much so that students can fill their own vials
with media. However, it must be completely rehydrated for best results, since
this is the only water source for adults and larvae. Therefore, follow the suggestions
below to ensure a completely hydrated media:
Add dry media to the bottle or vial to about 1/5 to 2/5 volume. Add water until media appears completely moistened. Allow the vial to sit for a few minutes, adding additional water if necessary until the media is completely hydrated. The surface should be moist with a shiny appearance and there should be no spaces in the media. If the media is not completed hydrated, production of vigorous cultures is compromised.
Flies may be added minutes after media has been hydrated. Remember to add several grains (but not more) of yeast to the media surface before adding flies.
Cooked media can be stored in a refrigerator for several weeks. Be sure to allow media to warm to room temperature before adding flies. Do not allow media to dry out. Media should fill the culture vial, bottle or vial 1/5th to 2/5th full. Keep the media out overnight to cure, being sure to completely cover the vials with cloth to keep flies from laying eggs in them. The next day, add yeeast and plugs. Refrigerate any unused medai vials. Unused media can last up to two weeks.
Environment
The easiest way to grow flies is at room temperature. However,
the optimum rearing condition is a temperature of 25C and 60% humidity. In these
conditions generation time is shorter (9-10 days from egg to adult). Unless
equipment is readily available this is unnecessary for successful rearing and
crossing of flies. It is preferable to keep flies out of drafts and direct sunlight
or heat sources. These will rapidly dry the media, necessitating frequent media
changes and the potential to dehydrate the flies.
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!). Flynap (from Carolina
Biological) is messy and has an odor that some find offensive. Each of these,
however, requires low- cost equipment which can be easily purchased. Carbon
dioxide works very well, keeping flies immobile for long periods of time with
no side effects, however CO2 mats (blocks) are expensive
and a CO2 source (usually a bottle) and delivery system
(vials and clamps) are necessary, increasing the costs. If resourceful, one
can use the CO2 emitted from Alka-Seltzer tablets to anesthetize
flies for short periods of time. Set up a large test tube with a tube and stopper
system. Add water in the tube, then the Alka-Seltzer tablet. Carbon dioxide
gas will be emitted.
The least harmful to the flies is either carbon dioxide or cooling anesthetizing. Of these two choices, cooling is the simplest, requiring only a freezer, ice and petri dishes. In addition, it is the only method which will not affect fly neurology, therefore behavior studies may begin after the flies have warmed up sufficiently.
Anesthetizing flies by cooling
In order to incapacitate the flies, place the culture vial in the freezer until
the flies are not moving, generally 8-12 minutes. Dump the flies onto a chilled
surface. This can be constructed by using the top of a petri dish, adding crushed
ice, then placing the bottom of the petri dish on top. Adding flies to this
system will keep them chilled long enough to do each experiment. Simply place
the flies back into the culture vial when finished. Flies will "wake up" relatively
quickly once off the ice, so keep them cold. There are no long-lasting side
effects to this method, although flies left in the refrigerator too long may
not recover. Another way to keep flies chilled is adding water to zip-lock type
freezer bags, place in the freezer with a petri dish nestled on the bag, and
allow to freeze.
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.
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.
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. Alternatively, it is quite easy to distinguish virgins from mature flies visually. It is strongly suggested that you obtain extra virgins in case a mistake is made in identification or the fly dies before mating and egg lying can occur. In a strong culture, multiple virgin females should be easily obtained. Although females are able to lay eggs as virgins, they will be sterile and no larvae will be produced. Below are three ways to obtain virgins.
Removal method
Remove all flies 8-10 hours before collecting. Visually inspect
surface of food to ensure complete removal of flies. After 8-10 hours collect
all females that are present. All will be virgins. Place in a fresh culture
vial and wait 2-3 days look for larvae. Virgin females can lay eggs, but they
will be sterile. 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. However, collection is possible later in the
day.
Visual method
Being able to recognize virgin females removes the necessity of emptying culture
vials on a timely basis and allows students to collect their own without the
necessity of coming to class at odd times of the day. 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 degrees 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 degrees C incubator, 98% of flies obtained in the morning will
be virgins. Placing virgins in their own vials for 2-3 days will eliminate those
2% that are non-virgins.
Once females are deemed virgins, add males. Generally, males will mate more efficiently if they have matured 3 days or longer. Be sure to select robust, healthy males; the older the flies, the lower the mating efficiency. Mating occurs quickly and the behavior is interesting to watch, but will not be addressed here. Females begin laying fertile eggs soon after mating. Refer to the life cycle chart for evidence of F1 larvae. Remove adults once it has been established that enough larvae are present since you may not be able to distinguish parents from the F1 generation.
Purchased cultures are almost always disease- free. However if you are collecting wild populations, mites and bacteria may be present. In addition, food may develop molds or bacterial growth. These are generally not a major problem for small-scale use, as in a classroom. However, mites are a major concern.
Mites
There are two types of mites to be wary of: food mites and parasitic mites.
For both, immediately quarantine the cultures from the rest of the stocks (put
them in a separate room) to prevent continued contamination. Mites can be persistent
and debilatory on the health of the flies and success of crosses.
Food mites
Food mites, which do no harm to the flies, need to be eradicated none-the less.
These small mites will be seen crawling on the surface of the food and, to a
lesser extent, on the sides of the vial. First, isolate these cultures and destroy
them if you do not need the flies. If you need the flies, follow this procedure.
1. In an empty vial, add moist paper towels to cover the bottom 2. Add the flies
with the mites 3. After an hour, transfer the flies into a new vial. Invert
the new vial over the old one. Flies will climb into the new vial faster then
the mites. You may need to repeat this procedure. Remember to keep the affected
flies in quarantine until you are positive they are mite-free.
Parasitic mites
The second type of mite is parasitic on the flies themselves. These are evident
by looking at the flies and seeing mites on most any body part, including the
legs, abdomen and head. The method of removal is to destroy the afflicted flies.
First, be sure to immediately quarantine any infected flies from other stock
cultures, as with food mites. Then, freeze the vial and use sterilizing techniques
or throw the vial away.
Picture of parasitic mites on a fly
Bacteria
There is little effect of bacteria on the flies themselves, however food media
may become infected and inhibit growth and development of larvae. White food
media take on a reddish-brown color, indicating bacterial contamination. Bacteria
may be transferred via flies, airborne particles, yeast contamination or equipment.
Treatment is to remove flies from contaminated culture vials and, of course,
not use discolored vials to maintain flies. Keeping food in a refrigerator until
ready to use will help keep infection at a minimum. Be sure to allow time for
the food to warm to room temperature before adding flies.
Molds
Much like bacteria, fungal infections are mainly isolated to the media. Most
are inhibitory towards larvae. Treatment is the same as with bacteria - removal
of the flies from the infected culture vial. All media should contain a fungicide
(see media recipes), including dehydrated media.
This is an unfortunate necessity when using flies. A bottle or beaker with mineral oil added is generally used. Dump anesthetized flies directly into the mineral oil where they drown. A bottle (beaker, or screw, capped jar filled) with ethanol or isopropanol can also be used as a morgue.