DPIRD approved fluke egg sedimentation test (FEST) procedure

3. Quality control

4. Principle

Trematode eggs and oocysts of Eimeria leuckarti are very dense and flotation in high-density (density > 1.3) media usually causes distortion of the eggs due to osmotic pressure. The higher density of the fluke eggs and oocysts of Eimeria leuckarti compared to faecal debris allows the separation of faecal debris from eggs. Faeces are filtered through two sieves to remove coarse debris, collected on a fine sieve and then processed through several rapid sedimentation steps. The more rapid sedimentation of the eggs separates them from the less dense faecal material. The supernatant is siphoned off and the whole sediment examined for the presence of trematode eggs or oocysts.

5. Reagent

1. 0.5% methylene blue: Weigh 0.5 g of methylene blue stain into an appropriate beaker. Add 50 millilitres of deionised (DI) or reverse osmosis (RO) water and dissolve on a magnetic stirrer. Make up to 100mL with DI or RO water and filter. Store in a dropper bottle at room temperature.
2. 2% iodine solution: Dissolve 100g of potassium iodide and 20g of iodine crystals in 1000mL of DI or RO water. Dissolve the potassium iodide first, the iodine will then easily go into solution. (Use 12.5mL per litre for positive egg solutions discards). Hold for 12 hours before discarding. This solution keeps indefinitely.

Caution:

Iodine - highly irritating to skin, eyes and mucous membranes; harmful vapour; causes burns. Avoid contact with eyes and skin. Wear gloves and eye protection when making up iodine solution.

Potassium iodide - highly toxic intravenously; moderately toxic orally.

6. Equipment

1. Dropper bottle.
2. Magnetic stirrer.
3. Sedimentation flasks (250mL or 500mL) - the vertical distance between the base of the flask and the 100mL mark should not exceed 80mm.
4. Electronic balance.
5. Spatula.
6. 70mL specimen jars plastic.
7. Wash bottle.
8. Laboratory timer.
9. Vacuum pump.
10. 150 micron, 90 micron and 45 micron mesh 20 cm diameter sieves.
11. Pasteur pipettes.
12. Pasteur pipette bulbs.
13. Glass petri dish (45mm diameter) or other suitable viewing chamber e.g. Universal egg counting slides.
14. Inverted microscope with measuring graticule or stereo microscope with measuring graticule.
15. 500mL beakers.