METHODS
MATERIALS
1 M sodium azide – to paralyze the worms for epifluorescence microscopy
2% agarose – to mount worms on a microscope slide
Alcohol lamp and lighter – to sterilize the pick
Beaker – for a water bath
4 plates of C. elegans transgenic strain CL6412, which has pan-neuronal expression of GFP.
2 control- no heat shock but does get vortex
2 experimental- heat shock and vortex
1 plate of C. elegans mutant worms on NGM plates (daf-16::GFP mutants)
Heat/stir plate – for heating a beaker of water to use for heat-shocking plates of worms
Hot pot – for heating agarose
ImageJ – software you will use to quantify the pattern of gene expression in the worms
Microscopes (dissecting/epifluorescence), slides, slide spacers, coverslips – to observe your worms
Parafilm – to seal NGM worm plates prior to heat shocking in water
Pasteur pipette – for applying agarose to a slide
Pick – for transferring worms
Sharpie marker – to label your plates and slides
Thermometer – to measure the actual water temperature used to heat shock the worms
Timer – to time the heat shock stimulus
The vortex and beads setup used in the worm abuse experiment
S-basal buffer for washing worms off the plate
15 ml conical tubes
PROCEDURE
Prepare 6 agar slides
Begin heating water bath to 34C
Isolate 3 DAF-16 worms and 3 CL6412 worms on 2 of the prepared slides
Take pictures and add to spreadsheet
Wrap DAF-16 and CL6412 plates in parafilm and begin heating for 30 min
Set up vortex for non-heated CL6412 worms
Wash plate with DI water Into microcentrifuge tube
Vortex for 10 seconds
Replate the worms onto fresh agar plate
Allow plate to dry
Isolate 3 abused non-heated CL6412 worms onto one agar slide
Take pictures and add to spreadsheet
After heating wait 30 min then isolate 3 DAF-16 and 3 heated CL6412 worms onto agar slides
Take pictures and add to spreadsheet
Set up vortex for heated CL6412 worms
Wash plate with DI water Into microcentrifuge tube
Vortex for 10 seconds
Replate the worms onto fresh agar plate
Allow plate to dry
Isolate 3 abused heated CL6412 worms onto an agar slide
Take pictures and add to spreadsheet
Email unlabeled pictures from 1st and 2nd experiments to group
Count number of lesions on neurons
Send all group members email with attached images
Attach positive and negative control images
Ask group members to count number of suspected lesions
Group members will have access to a spreadsheet to write their numbers in​​
Aggregate data
Math magic
CONTROLS
Positive Controls:
Non-heated vortexed worms; heated DAF-16 worms.
Negative Controls:
Both groups of worms that were heated and not heated but never vortexed; non-heated DAF-16 worms.
STATISTICAL TESTS USED
A single-factor ANOVA test was run on all four experimental groups of worms as well as the on the individual counts of worms.