Commission Implementing Regulation (EU) 2015/1375Dangos y teitl llawn

B. Mechanically assisted pooled sample digestion method/'on filter isolation' technique

1. Apparatus and reagents

As stipulated in Section A(1).

Additional equipment:

(a)

1 litre Gelman funnel, complete with filter holder (diameter 45 mm);

(b)

filter discs, consisting of a circular stainless steel mesh with an aperture of 35 microns (disc diameter: 45 mm), two rubber rings 1 mm thick (external diameter: 45 mm; internal diameter: 38 mm), the circular mesh being placed between the two rubber rings and bonded to them using a two-component glue suitable for the two materials;

(c)

an Erlenmeyer flask, capacity 3 litres, fitted with a side tube for suction;

(d)

a filter pump;

(e)

plastic bags, capacity at least 80 ml;

(f)

equipment for sealing the plastic bags;

(g)

rennilase, strength 1:150 000 Soxhlet units per gram.

2. Collecting of specimens

As stipulated in Chapter I(2).

3. Procedure

I. Grinding

Grinding the meat samples in a meat mincer beforehand will improve the digestion quality. If an electrical blender is used, the blender must be operated three to four times for approximately one second each time.

II. Digestion procedure

This procedure may involve complete pools (100 g of samples at a time) or pools of less than 100 g.

(a)

Complete pools (100 samples at a time)

See Section A(3)(II)(a).

(b)

Smaller pools (less than 100 samples)

See Section A(3)(II)(b).

III. Recovery of larvae by filtration

(a)

Ice (300 to 400 g of ice flakes, scaly ice or crushed ice) is added to the digestion fluid to bring its volume up to about 2 litres. In the case of smaller pools, the amount of ice must be reduced correspondingly.

(b)

The digestion fluid is stirred until the ice has melted. The chilled digestion fluid is then left for at least three minutes to let the larvae coil.

(c)

The Gelman funnel, fitted with a filter holder and filter disc, is mounted on an Erlenmeyer flask connected to a filter pump.

(d)

The digestion fluid is poured into the Gelman funnel and filtered. Towards the end of filtration, the digestion fluid can be helped to pass through the filter by applying suction with the filter pump. Suction must cease before the filter becomes dry, i.e. when 2 to 5 ml of fluid is left in the funnel.

(e)

Once all the digestion fluid has been filtered, the filter disc is removed and placed in an 80 ml capacity plastic bag, together with 15 to 20 ml of rennilase solution. The rennilase solution is made by adding 2 g of rennilase to 100 ml of tap water.

(f)

The plastic bag is sealed twice and placed between the inner and outer bags in the Stomacher.

(g)

The Stomacher is allowed to pound for three minutes, e.g. while it is working on a complete or incomplete pool.

(h)

After three minutes, the plastic bag, complete with filter disc and rennilase solution, is removed from the Stomacher and opened with scissors. The liquid contents are poured into a larval counting basin or petri dish. The bag is washed out with 5 to 10 ml of water, which is then added to the larval counting basin for examination by trichinoscope or to the petri dish for examination by stereo-microscope.

(i)

Digests must be examined as soon as they are ready. Under no circumstances is examination to be postponed until the following day.

Note: Filter discs must never be used when not completely clean. Unclean discs must never be allowed to dry out. Filter discs can be cleaned by leaving them in rennilase solution overnight. Before use, they must be washed in fresh rennilase solution using the Stomacher.

IV. Positive or doubtful results

Where the result is positive or uncertain, the provisions laid down in Chapter I(3)(III) shall apply.