‘(f)

Pepsin, strength 1: 10 000 NF (US National Formulary) corresponding to 1:12 500 BP (British Pharmacopoeia) and to 2 000 FIP (International Pharmaceutical Federation), or stabilised liquid pepsin with minimum 660 European Pharmacopoeia units/ml’;

(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

(i)

(j)

(k)

(l)

(m)

(n)

(o)

(p)

(q)

(r)

(s)

(t)

(u)

(v)

(w)

(x)

(y)

(z)

(aa)

(a)

The 20 microns nylon mesh filter is placed on the filtration support. The conical filtration steel funnel is fixed to the support with the block system and the steel sieve of 180 microns mesh size is placed on the funnel. The vacuum pump is connected with the filtration support and with the metal or plastic tank, to collect the digestive fluid.

(b)

Stop stirring and pour the digestion fluid into the filtration funnel through the sieve. Rinse the beaker with 250 ml of warm water. The rinsing liquid must be poured into the filtration ramp after the digested fluid has been successfully filtrated.

(c)

With the forceps, take the filtration membrane holding it by an edge. Fold the filtration membrane in four minimum and put it in the 15 ml conical tube.

(d)

The filtration membrane is pushed at the bottom of the 15 ml conical tube with the help of the pestle and strongly pressed by doing successive back and forth movements with the pestle which should be positioned inside the filtration membrane folding according to the manufacturer’s instructions.

(e)

The sample diluent is added into the 15 ml conical tube by pipette and the filtration membrane is homogenised with the pestle by doing successive low amplitude back and forth movements, avoiding abrupt movements to limit liquid splashes according to the manufacturer’s instructions.

(f)

Each sample, the negative control, and the positive control, are dispensed into different fields of the agglutination card by pipettes, according to the manufacturer’s instructions.

(g)

The latex beads are added into each field of the agglutination card by a pipette, according to the manufacturer’s instructions, without making them come into contact with the sample/s and controls. In each field, the latex beads are then gently mixed with a disposable stick until the homogeneous liquid covers the entire field.

(h)

The agglutination card is put on the 3D rocker and is rocked according to the manufacturer’s instructions.

(i)

After the time established by the manufacturer’s instructions, the rocking is stopped and the agglutination card is put on a plane surface and the reaction results are read. In the case of a positive sample, the beads aggregates must appear. In the case of a negative sample, the suspension remains homogeneous without beads aggregates.

(j)

All equipment in contact with meat must be carefully decontaminated between runs by soaking for a few seconds in warm water (60-90 °C). Surfaces upon which meat residues or inactivated larva/e remain may be cleaned with a clean sponge and tap water. Once the procedure is finalised, a few drops of detergent may be added to degrease the equipment. Then each piece must be thoroughly rinsed several times in order to remove all traces of detergent.

(k)

The pestle must be carefully decontaminated between runs by soaking for a few seconds in at least 250 ml of warm water (60-90 °C). Meat residues or inactivated larvae that could remain on its surface must be eliminated with a clean sponge and tap water. Once the procedure is finalised, a few drops of detergent may be added to degrease the pestle. Then the pestle must be thoroughly rinsed several times in order to remove all traces of detergent.