Gastrointestinal nematode egg purification

Adapted from the Kotze and Gilleard labs

• Nemabiome protocol (sheep tab)

• Example in Parsitology

• Example of the assay in action in Veterinary Parasitology

Materials

• 13% NaCl solution
• 300, 180, 106 and 25 μm sieves
• 10% and 25% sucrose solutions
• 5.65-6% sodium hypochlorite
• M9
• 100X Amphotericin B (2.5 μg/mL final)
• 100X Tylosin tartrate (800 μg/ml final)

Equipment

• Benchtop centrifuge

Protocol

1. Weigh out 24 g of fecal matter and homogenize it in 100 mL of 13% NaCl.

2. Filter through 300, 180, 106 and 25 μm stainless steel sieves. Clean eggs will be captured on the 25 μm sieve.

3. Flush the trapped eggs to an edge and use a squirt bottle of water to rinse the eggs into a petri dish.

4. Create a sucrose gradient in a 50 mL conical tube by adding 10 mL of cold 10% sucrose followed by 10 mL of cold 25% (place pipette at the bottom of the tube when adding each layer).

5. Carefully transfer the egg solution to the sucrose gradient.

6. Centrifuge the tubes for 7 min. at 650 x g.

7. The eggs will be at the interface of the gradient. Use a pipette to transfer the eggs to the 25 μm sieve and wash with water.

8. Rinse the eggs into a 50 mL conical tube (final volume of 40 mL water). Add 10 mL of bleach solution and rotate for 10 minutes.

9. Rinse again on the sieve and resuspend eggs in M9 + Amphotericin B + tylosin tartrate (2500 egg/mL).