05 - Heavy Metal: Effects of Environmental Toxins on C. elegans
Module III: Collection of C. elegans and Toxicity Bioassay Excerpts from Edvo-Kit #856
TRANSFER
WT
MS
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3.5 mL S-Buffer
RINSE
REPEAT steps 2 - 4 for Mutant worms (MS).
WT
MS
WT
WT
MS
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10 min.
Wild-Type Worms
Wild-Type Worms
100 µL HM solution
TAP
RECORD your heavy metal in TABLE 1.
WT
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MS REMOVE ~3 mL DO NOT
WT
100 µL S-buffer
Disturb worms
HM-WT
HM-MS
C-WT
C-MS
1. LABEL one 15 mL tube of S-buffer, large transfer pipet, and snap top tube with “WT” (Wild- Type) and the other 15ml tube of S-buffer, large transfer pipet, and snap top tube with “MS” (Mutant Strain). 2. Using the WT large transfer pipet, TRANSFER 3.5 mL of S-buffer from the WT conical tube to the Petri dish containing the wild-type C. elegans . 3. DISLODGE worms by rinsing the dish several times. RINSE the dish by either (a) swirling the plate or (b) holding the plate at a slight angle and allowing the buffer to collect near the bottom. Next suck up the buffer using the WT transfer pipet and then expel the buffer near the top so that the buffer runs down the plate. 4. Once most worms are suspended in the buffer, TRANSFER the worms and buffer back to the WT 15 mL conical tube using the WT transfer pipets. 5. REPEAT steps 2-4 for mutant worms using the MS labeled items. Record in Table 1. 6. Keep the conical tubes still and upright to ALLOW the worms to settle to the bottom of the tubes (~10 minutes). While you wait, REVIEW Appendix B and complete Steps 7 through 9. 7. LABEL four 1.5 mL snap-top test tubes, counting chambers, and small transfer pipets with “HM-WT”, “HM-MS”, “C-WT”, and “C-MS”. 8. RECORD which heavy metal you plan to test in your lab book or in Table 1 (page 9). 9. ADD 100 µL of your heavy metal solution to both “HM” tubes and 100 µL of the S-buffer to both “C” tubes. 10. Using the large transfer pipets, slowly REMOVE ~3 mL of the cleared S-buffer supernatant without disturbing the worms that have settled to the bottom of the tubes. Between 300 and 500 µL of buffer with worms should remain at the bottom of both tube. 11. MIX the WT tube by tapping the bottom 3-4 times or until worms are uniformly resuspend- ed throughout. TAP HM-WT COUNT WAIT REPEAT steps 16-17 twice for counts at 2 min. and 4 min. - - - - - - - - - - - - - - - - WT 20 µL
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