1. C. Elegans Unit: Lab Activities

2. Overview of Techniques
Basic Worm Handling Techniques
Pouring NGM Agar plates
Nematode Growth medium
Innoculating broth with OP50 E. coli for feeding
“Seeding” NGM Agar plates with OP50
Chunking worms to propagate more worms
Picking worms to move exact numbers of worms
Making a worm pick
Chemotaxis Assay – Biorad Kit

3. Basic Technique: Pouring NGM Plates
Melt down 1-2 bottles of NGM agar
Hot plate, water bath, microwave
About 14 plates per group of 2
Label with
Initials
Date
NGM agar
INSTRUCTOR NOTE: HAVE a student pour 16 extra plates for instructor use with Chemotaxis kit (worm thawing)
Instructor Prep: Petri Dishes, NGM, sharpies

4. Basic Technique: Innoculating with OP50
Using sterile technique, transfer 3mls of LB broth (premade) to a clean bacterial culture tube (the clear ones with loose caps)
Dip a sterile innoculating loop into the OP50 stock broth or touch to surface of stock plate culture
Put loop into your LB broth and swirl
Label with your initials, date, and OP50
Put in tube racks in 37°incubator
NOTE: All materials that touch bacteria go in bacterial waste pans
Instructor prep: Thaw OP50 stock, get LB broth (premade – if not left make some, autoclave it), Set up bleach pans

5. Basics: Seeding NGM plates with OP50
Add OP50 in red sharpie to the labels on your plates
Once OP50 has grown for at least 24 hours, Add 200 μl of OP50 broth culture to the surface of 6 of your NGM plates (2 for chunking, 2 for picking, 2 for making worm stocks for chemotaxis assay later)
Leave any remaining plates empty for later –store in fridge
Using an L-spreader, spread the broth around, avoiding the very edges of the dish.
Let soak in about 15 min, then parafilm
Invert and incubate at ROOM TEMP.
NOTE: All materials that touch bacteria go in bacterial waste pans
Instructor prep – Bacterial waste pans

6. Basic Technique: Chunking worms
Obtain a stock plate of N2 (wild-type) worms from instructor
Get 2 of your NGM + OP50 plates, Add to the labels “N2”
Flame-sterilize a scalpel, and touch to surface of agar to cool
Cut a 1cm2 square of agar from the stock plate
Transfer it to the surface of the NGM-OP50 plate
Parafilm and invert
Incubate at room temperature
Instructor prep: scalpels, lighters

7. Basic Technique: Picking Worms
Instructor will demonstrate how to make a worm pick
Make your worm pick and label it with your initials
Get a plate of worms that was chunked a few days ago and has many worms on it
Transfer 5-10 worms to an NGM + OP50 plate
Practice!
Video: hOw&feature=related
Instructor prep: Platinum wire, clay, pliers

8. Biorad Chemotaxis Assay: Quick Guide
Pour NGM agar plates, Seed with OP50
To make stocks of N2 and Mutant worms for assay
Use Wash buffer (S-buffer) method to transfer worms to make a new stock plate
Pour assay plates - 20 total per kit
Prepare Assay plates
Perform assay
Collect & record data
Perform Statistical analysis

9. Biorad Kit Prepare NGM seeded with OP50
You have extra from our basic worm techniques – you’ll need just 2 plates for this assay
You should have 2 plates left with NGM & OP50, no worms – if not seed more.
NOTE: the kit calls for use of OP50-pBAD which has an amp resistance gene, and requires culture on NGM+amp. We are skipping this as it is not necessary and gives us more flexibility.
Instructor Prep: None

10. Biorad Kit: Pour assay plates
These have a small amount of Wash buffer in the agar instead of NaCl (a component of NGM)
We have just 20 per class (per kit)
Instructor has prepped solution & autoclaved it; Each team come pour 2 plates
Label with your initials, date and ASSAY plates
Instructor prep: Make the assay buffer from kit – comes powdered – see instruction in kit, Autoclave it.

11. Biorad Kit: Making stocks of WT and Mut (daf-18) worms
Use wash buffer transfer method (S-buffer technique) to make new stocks of your worms onto NGM + OP50 (NOT ASSAY PLATES)
Follow instructions on pp of your kit manual
Make sure you label plates with WT worms (N2) or Mutant (daf-18)
Don’t cross contaminate them
Be sure to incubate at room temperature
Need 5 days to make enough worms for next steps
Instructor prep: Call in Biorad kit coupon in advance to to get 2 worm strains, then when they arrive, thaw and plate onto fresh NGM (see kit) – no OP50 is necessary on plates, frozen worm stocks have it. The worms need to grow for 4-7 days before they can be used by students. Dilute wash buffer.

13. Sec.

16. BioRad Kit: Finishing assay plates – adding salt gradient
Instructor has prepared wash buffer and 2.5M NaCl solution
Follow instructions on next slide to set up plates
Let plates incubate overnight before use.
Instructor Prep: Dilute wash buffer

18. BioRad Kit: Chemotaxis Assay
Follow the instructions on pp of your handout to set up chemotaxis assay
Allow 30 minutes for worms to move
Collect data & record in notebook
Next class: Perform Statistical analysis

29. Chi Square Analysis Follow along with BioRad kit Chi-Square supplement
Read it
Try practice problems
Analyze your own data
Record in notebook