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
- 13% NaCl solution
- 300, 180, 106 and 25 μm sieves
- 10% and 25% sucrose solutions
- 5.65-6% sodium hypochlorite
- 100X Amphotericin B (2.5 μg/mL final)
- 100X Tylosin tartrate (800 μg/ml final)
- Benchtop centrifuge
Weigh out 24 g of fecal matter and homogenize it in 100 mL of 13% NaCl.
Filter through 300, 180, 106 and 25 μm stainless steel sieves. Clean eggs will be captured on the 25 μm sieve.
Flush the trapped eggs to an edge and use a squirt bottle of water to rinse the eggs into a petri dish.
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).
Carefully transfer the egg solution to the sucrose gradient.
Centrifuge the tubes for 7 min. at 650 x g.
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.
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.
Rinse again on the sieve and resuspend eggs in M9 + Amphotericin B + tylosin tartrate (2500 egg/mL).