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STUDENT OBJECTIVE

Students observe the life cycle and anatomy of Drosophila. Over a period of four weeks, they verify the Mendelian concepts of dominance-recessiveness, segregation, independent assortment, and sex linkage by setting experimental crosses. Students may also dissect the third instar larvae and make slides of the giant chromosomes found in the salivary glands.

EQUIPMENT	AMOUNT
	(Class of 24 with 8 groups)
Incubator or growth chamber, 22—24°C; timed fluorescent lights can help synchronize emergence Compound microscope Dissecting microscope	1/lab 1/student 1/student
MATERIALS
Drosophila cultures Red-eyed, normal wing homozygous (Wild) (CBS#17-2100) White-eyed, vestigial wing homozygous (Mutant) (CBS#17-2720)* Stock culture chambers Student culture chambers, plastic vials, 5" x 1" diameter (CBS#17-3085)* Student culture stoppers, nonabsorbent cotton wrapped in cheesecloth, or foam (CBS#17-3080)* Nylon netting, 1" x 4" (CBS#17-3090)* White cards, 3" x 5" Camel hair brush, #00 Anesthetizer (See lab manual figure 9.3 for construction.) or Fly Nap® (CBS 17-3010) 8 oz glass bottle rubber stopper centrifuge tube cotton funnel, plastic, 2 1/2" diameter	2 cultures/lab 2 cultures/lab 2/lab 6/group 6/group 6/group 1/student 1/student 1/student 1/group
Pads, tapping (12 cm x 12 ¥ 30 mm paper pad) Lens paper Tissue, blotting Morgue, 4 oz glass container (screw-lid) with 50% alcohol Slides, glass Coverslips, #1 medium square glass Dissecting needles Forceps Alcohol lamp Prepared slide—Drosophila giant chromosomes (CBS#30-9066)*	1/group 4 pkg/lab 2 boxes/lab 1/lab 1/student 1/2 oz/lab 1/student 1/student 1/lab Demonstration

*Please refer to the Appendix for name and address of supplier.

SOLUTIONS

Ether, anesthesia, C₂H₅OC₂H₅. !!CAUTION!! Explosive! No flames or sparks should be in the laboratory during use. Provide adequate ventilation. An alternative to ether is Fly Nap® available from CBS.

Instant Drosophila food—Blue medium (CBS)

Distilled water

Baker’s yeast

50% ethanol (C₂H₅O)
Acetocarmine stain
0.8% saline

PREPARATION

Six Weeks before Lab

1. Stock cultures should be ordered for arrival three weeks before the scheduled lab. Request that the adults emerge on arrival date.
2. For culturing Drosophila, order supplies to arrive at the same time that the stock arrives. To cover emergencies, order 20% more than needed. Extra culture vials and stoppers are needed for sub-culturing parental stocks. CBS provides an excellent guide to subculturing fruit flies, and it is shipped with each order of flies.
3. To insure minimum contamination of cultures by fungi and bacteria, autoclave glass culture containers and stoppers for 15 minutes at 15 psi. Stoppers should be lightly packed into a large grocery sack with the top folded and stapled. All plastic containers should be scrubbed with strong detergent, rinsed, and put through a cycle in a dishwasher. Use of antimite paper (CBS#17-3115)* on shelves of culture storage can prevent mite contamination.

Three Weeks before Lab

1. To raise the needed number of flies from the commercial cultures, prepare stock culture bottles with an adequate amount of medium, according to the supplier’s instructions. From each commercial vial, remove ten females and five males and place them in a prepared culture bottle. To prevent crossbreeding, handle mutant- and wild-type flies separately. Make certain that all equipment and work areas are free of flies before handling a different strain.
2. Place the jars of flies in a 22—25 ^oC growth chamber with a pan of water on the bottom. If available, the timer should be set for the lamps to deliver a 12-hour day with dawn at 9:00 a.m. (see One Week before Lab).

Two Weeks before Lab

1. The adult flies (parent generation) should be removed from the cultures and discarded. Emerging flies will appear in five or six days and should be harvested for the lab.
2. If the food medium appears to be too moist, finely pulverized commercial food can be sprinkled lightly on top. The commercial food should be pulverized with a mortar and pestle. Store in a shaker for use in prep laboratory and lab. A shaker can be fashioned from a plastic 35 mm film canister by piercing the top several times with a hot needle.

One Week before Lab

1. A schedule should be established to collect virgin females. Timing is of utmost importance. Females are not receptive to males until four to six hours past emergence. Emergence is apparently controlled by a biological clock that is set by the beginning of light at dawn. If the stock flies are kept on a light-dark cycle of twelve hours each, 60% of the flies will emerge between "DAWN" and four hours into the day. If artificial dawn is set for early in the working day and the bottles are dumped one hour before dawn, plenty of flies should be available and can be collected at four-hour intervals for several days. This artificial lighting is not necessary (only convenient) and flies can be collected at four-hour intervals during the workday.

   One hour before DAWN. In a dimly lit room, dump and discard the flies. Check container twice to make certain no adult flies remain in the container.

   DAWN plus four hours. Dump flies of one phenotype and sort according to sex. Store in separate vials with food medium. They can be held for 7—14 days. Repeat the procedure for the other phenotype. Adequately mark each container with sex, phenotype, and date.

   DAWN plus eight hours. Follow the same procedure as at "DAWN plus four hours" and thereafter at four-hour intervals during the workday. Procedure can be repeated on subsequent days until enough flies are collected.

   You should collect six virgin females of wild and mutant types and four males of each type for each student group in the lab. Package these in separate culture chambers in sufficient multiples to supply all groups in one lab. That way flies of one type can be anesthetized and dispensed before another type is dispensed. Dispense mutants first because they will not fly away if they recover first in vials II and III.

2. 0.8% saline preparation:

   0.8 g NaCl

   Mix salt with 100 ml water. Package in four dropper bottles for students to use.

3. Acetocarmine stain preparation:

   STOCK SOLUTION

   0.5 g carmine
   100 ml 45% acetic acid
   Boil 2—4 minutes. Cool and filter.

   WORKING SOLUTION

   Dilute stock with 45% acetic acid, 1:2

   Wear goggles and prepare in fume hood!

4. During the collection of virgin flies, the discards should be put into a morgue filled with 1:1 ethanol-water. Students can examine these flies to practice sexing and identifying the characteristics of Drosophila.
5. Students can prepare commercial culture medium correctly when adequate directions are provided. Type the following procedure and attach paper to the supply table.

Day of the Lab

Just before the start of the lab class, stock growth cultures should be selected that have all the stages of the life cycle for study and dissection. The dissecting of the salivary gland will depend on the number of larvae available and the skill of the student. A commercially prepared Drosophila giant chromosome slide can help students see the expected results.

One Week following Lab

1. Students anesthetize and remove adults of the parent generation and discard in morgue. Warn students about the explosive nature of ether.
2. Moisture content of students’ vials can be adjusted as prescribed above.
3. Cultures should be returned to the growth chambers and checked periodically for adequate temperature and moisture.

Two Weeks following Lab

1. Students will prepare a new set of culture vials according to the laboratory manual.
2. The adults from the F₁ generation should be lightly anesthetized and the next cross be made with the proper number and sex of flies. Warn students about ether.
3. The remaining flies should be returned to the anesthetizer and killed with an overdose of ether. They then can be sexed and their phenotypes scored. The dead flies should be put into the morgue and saved for the next year’s students to use in their morphology studies. If there were less than 15 flies per vial, these vials should be held another week and another count made. All student vials from the first cross should be emptied, washed, and rinsed thoroughly.

Three Weeks following Lab

1. Students will remove the parental flies from F₂ generation vials.
2. If necessary, students can again count for additional emerging adults in the F₁ generation vials.

Four Weeks following Lab

1. Students remove the F₂ generation and count the numbers of each phenotype. If numbers are low, repeat counting in fifth week.
2. Thoroughly wash, rinse, and dry culture vials and equipment before storing. Glassware and foam articles should be autoclaved for 15 minutes at 15 psi.

NOTES

1. Ether should be dated when opened. It is better to buy 1/4 lb cans rather than 1 lb ones because less ether will be left after the experiment is finished. Dispose of excess ether, in accordance with local and community safety standards, after the completion of the exercise. Fly Nap® is often a better choice since flies will remain "out" for longer periods of time.
2. Lab cleanup is easy if adequate detergent and the appropriate-sized bottle brushes are provided. When the nylon netting is removed, it should be thoroughly rinsed and soaked in a dilute solution of bleach to kill pupae. Otherwise, flies emerge into the room from pupae caught in sink traps and waste baskets.
3. When flies escape into the room, they can be caught by placing small beakers of red semisweet wine around the room. (Drosophila seem to be selective for dryness of wine.) Theory suggests that the flies are attracted to the scent, crawl down the sides, drink, become senseless, and incapable of returning to the top. Periodically, a palm quickly placed over the top and turned upside down, will "drown" those on the side.
4. To reduce any possibility of error in collection of virgins, the schedule as outlined earlier has been moved up one week. Collecting and holding (20^oC) the virgins in a culture vial with food, water, and yeast will give sufficient time to see any development of larvae due to harvesting non-virgins.
5. The health of the cultures depends on cleanness and the viability of the yeast. The yeast should be fresh to help prevent any increase of foreign mold. This also becomes the major food source for the adults.
6. Commercial anesthetizers are available, however, larger bottles are easier to handle. A simple anesthetizer can be constructed using an 8 oz, widemouthed bottle fitted with a rubber stopper. A hole is bored in the stopper to accommodate a 4" plastic centrifuge tube with a lip. After inserting the tube into the stopper, the centrifuge tube should be pierced in several places with a hot teasing needle; avoid piercing the bottom 1 cm. Add a 2.5 cm layer of cotton to the bottom of the bottle to absorb ether. A wide-bore funnel should rest securely in the tube to allow transfer of flies from culture vial to anesthetizer. When the anesthetizer is not in use, plug the tube with a #0 rubber stopper to prevent the escape of ether.
7. To anesthetize the flies, add eight to ten drops of ether to the cotton in the anesthetizer and cap with the stopper until the ether diffuses into the tube. Flies can be transferred to the tube by tapping the bottom of a culture vial on a table top until all flies fall to the bottom of the vial. Quickly remove the stopper and invert the vial into the funnel. The entire assemblage should then be tapped until all the flies are transferred to the bottom of the anesthetizer tube. Sight down the tube and observe leg movements. When all the legs stop moving, count slowly to five. Then dump the flies onto a 3" x 5" white card for the sexing and scoring. Too much ether can kill or render the flies sterile. Those flies that are dead will have their wings at right angles to their bodies.
8. Dislodging flies from the culture requires some tapping against the lab bench. To avoid breaking the bottle, tap against a paper pad. A little more expensive but permanent pad can be cut from a sponge composition floor mat available at hardware stores.
9. CBS has an excellent booklet on the care and handling of fruit flies.
10. Vestigial wings are often difficult for students to detect. You may consider the apterous trait for ease of identification.

Classroom suggestions

1. Check out the links for this lab topic at http://auth.mhhe.com/biosci/genbio/dolphin/ You will find useful materials for developing your lab introduction or summary, and in some cases, you may want to tell students to connect to a particular site for further information.

ANSWERS TO CRITICAL THINKING QUESTIONS

1. The fruit fly emerges from the pupa 10 to 14 days after fertilization. Therefore, in one fly culture it’s possible to have flies emerging at widely spaced intervals. If you counted the flies before all had emerged, your results may be skewed by factors affecting emergence. Also, fruit flies mate within 6—8 hours of emerging and if the F₁ flies were not removed within 4 hours of emerging, chances are random mating will give unexpected F₂ results.
2. "True breeding" flies are homozygous for a particular trait, and when mated with one another will always produce homozygous offspring, phenotypically the same as the parents in regard to that trait.

SUPPLEMENTAL MATERIALS