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Haemonchus contortus L3 Ex-Sheathing

Glucose Tyrodes Recipe

Culture Media Component Volume/Mass (for ~500 mL total)
DI Water 500 mL
Glucose Tyrodes 4.8 g
Sodium Bicarbonate 3 g
Penicillin G sodium salt 0.6 g
Streptomycin sulfate salt 1 g
Fluorocytosine 0.005 g

HcL3 Media Recipe

Culture Media Component Volume/Mass (for ~500 mL total)
DI Water 500 mL
Gentamicin 1500 µL
Amphotericin B 5 mL
1M HEPES 1 mL
EZMix Tryptone Pancreatic Digest of Casein 10 g
Yeast Extract 5 g
d-Glucose 28.5 g
Potassium phosphate dibasic 0.4 g
Potassium phosphate monobasic 0.4 g
Penicillin G sodium salt 0.065 g
Streptomycin sulfate salt 0.065 g

Materials

  • 2% bleach solution
  • Glucose Tyrodes buffer
  • HcL3 media
  • 50 mL conical tubes

Protocol

  1. Remove Glucose Tyrodes buffer and HcL3 media from the 4°C fridge and aliquot into 50 mL conical tubes. Place aliquot into 37°C incubator 1hr before starting the ex-sheathing process.

  2. Remove the Haemonchus from the 4°C deli fridge 1hr before starting the ex-sheathing process and draw off the volume needed for your assay and add to a 50 mL conical tube.

  3. Calculate the volume based on the concentration of L3's you will need for your assay.

  4. Add DI water up to the 45 mL line, then add 5 mL of the 2% bleach solution and mix well.

  5. You can use a 15 mL tube or a 1.5 mL tube but use the bleach solution in a 1:9 ratio.

  6. After incubating for 5 minutes, spin down the tube at 1,000 RPM for 3 minutes.

  7. Draw off solution without disturbing the HcL3 pellet and wash with Glucose Tyrodes.

  8. Repeat steps 4 and 5 three times.

  9. After the last spin, draw off as much of the Glucose Tyrodes as possible, then add the HcL3 media up to the appropriate volume needed for your assay.