Culturing and Treatment of Tester Strains

Set up the following two crosses by mating 80 virgin females with 40 males per vial:

1. Standard cross (ST): Cross flr3/TM3, BdSfemales to mwh/mwh males.

2. High-bioactivation cross (HB): Cross ORR/ORR; flr3/TM3, BdS females to mwh/ mwh males.

3.1.1. Chronic Exposure: 3-D-Old Larvae Treated for 48 H

1. After 3 d put the parental flies in culture bottles containing a solid agar base (3% [w/v] agar-agar in water) covered completely with an approx 5-mm layer of live baker's yeast supplemented with sucrose.

2. Eight hours later remove the flies from the bottles.

3. Wash out the 3-d-old larvae (72 ± 4 h after the beginning of oviposition) with tap water through a fine-meshed stainless-steel strainer.

4. Put the larvae, in equal batches of approx 100, into plastic vials containing 1.5 g of Drosophila Instant Medium.

5. Use 5 mL of the test compound solutions to rehydrate the 1.5 g of dry instant medium (see Note 1). Include a negative control, using water or solvent.

The treated individuals remain in the vials until the emergence of the surviving adult flies.

3.1.2. Acute Exposure: 3-D-Old Larvae Treated for 2-6 H

2. Put the 3-d-old larvae into plastic tubes that have one end covered with fine nylon gauze.

3. Place the tube into a 50-mL beaker containing 0.3 g of powdered cellulose and 1.5 mL of mutagen solution. The larvae will immediately start to feed through the gauze on the wet powdered cellulose.

4. Two to six hours later, remove the larvae by rinsing them with tap water.

5. Flush the larvae into a culture vial containing 1.5 g of dry Drosophila Instant Medium wetted with 5 mL of distilled water.

The standard procedure for the wing spot assay employs the chronic exposure. However, when the compound under investigation is chemically unstable, the acute exposure must be used.

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