DPIRD approved fluke egg sedimentation test (FEST) procedure

7. Procedure

1. Weigh out 4g (10g for horses and cattle) of faeces into a 500 mL beaker.
2. Add approximately 100mL of tap water and thoroughly mix with a spatula to homogenise the faecal sample. Pre-soaking and vigorous stirring of firm faeces such as sheep, goats and alpaca may be necessary to aid in dispersion of the faecal pellet.
3. Set up the three sieves on top of each other with the 150 micron mesh size on top, followed by the 90 micron and 45 micron mesh size sieves. The sieves should be placed on a surface that will not impede the free flow of water through the sieves, e.g. over a sink drain hole or corrugated sink drainer.
4. Pour the homogenised faeces through the sieves and follow through with a powerful jet of tap water from a suitable hose, until the faeces are thoroughly dispersed and the emergent water is clear. It may be necessary to partially separate the 90 micron and 45 micron sieves to break the air lock and prevent over-flow from the sieves.
5. Remove the 150 micron sieve. Continue to wash through the 90 micron and 45 micron sieves, thoroughly dispersing the faecal material with the jet of water from the hose. Remove the 90 micron sieve and repeat the above process with the 45 micron sieve.
6. Incline the 45 micron sieve at approximately 45° to the horizontal and gently wash the filtrate from the top to the bottom taking care not to spill any material. Tip the filtrate into a numbered sedimentation flask and gently rinse any residues from the sieve into the flask. Make up to the 100mL mark with tap water and start the timer for 6 minutes.
7. Thoroughly reverse-rinse the sieves with a strong jet of water to prevent sample carry-over into subsequent samples (pay particular attention to the lip of each sieve, which may harbour some faecal material).
8. After six minutes has elapsed, suck off the supernatant from the sedimentation flask very carefully with the pump to the 20mL mark. Ensure that the tube or pipette used to siphon off the supernatant remains close to the surface throughout the process, allowing an amount of air to be drawn into the tube, to avoid disturbing the sediment. If the sample is very dirty, then the sample can be split into two sedimentation flasks and combined again before transferring to the petridish.
9. Refill flask with water to 100mL and repeat the above. The process can be repeated as many times as needed in order to clarify the sediment.
10. When the supernatant is clear, suck down to 10mL and add one drop of methylene blue.
11. Leave for five minutes for effective staining of the debris.
12. Transfer all of the material into a viewing chamber or petri dish, ensuring all the sediment is included. View under an inverted microscope using 40x magnification. A dissecting microscope or a compound microscope can also be used providing the sample is cleared of floating debris by repeated sedimentation. A minimum magnification of 20x is suggested for reliable screening for the presence or absence of trematode eggs.
13. For all positive samples add 12.5mL of 2% iodine solution per litre of discarded material and hold for 12 hours before disposal of wastes.