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

C. Automatic digestion method for pooled samples of up to 35 g

1. Apparatus and reagents

(a)

Knife or scissors for cutting specimens.

(b)

Trays marked off with 50 squares, each of which can hold samples of approximately 2 g of meat, or other tools giving equivalent guarantees as regards the traceability of the samples.

(c)

A Trichomatic 35® blender with filtration insert.

(d)

Hydrochloric acid 8,5 ± 0,5 % weight.

(e)

Transparent polycarbonate membrane filters with a diameter of 50 mm and a pore size of 14 microns.

(f)

Pepsin, strength 1:10 000 NF (US National Formulary) corresponding to 1:12 500 BP (British Pharmacopoeia) and to 2 000 FIP (Fédération internationale de pharmacie), or stabilised liquid pepsin with minimum 660 European Pharmacopoeia units/ml.

(g)

A balance accurate to 0,1 g.

(h)

Tweezers with a flat tip.

(i)

A number of microscope slides with a side-length of at least 5 cm or a number of petri dishes at least 6 cm in diameter, marked on their undersides into 10 × 10 mm square areas using a pointed instrument.

(j)

A (stereo-)microscope with transmitted light (magnification 15 to 60 times) or a trichinoscope with a horizontal table.

(k)

A bin for collection of waste liquids.

(l)

A number of 10 litre bins to be used for decontamination of apparatus, e.g. with formol, and for digestive juice remaining where specimens test positive.

(m)

A thermometer accurate to 0,5 °C within the range 1 to 100 °C.

2. Collecting of specimens

As stipulated in Chapter I(2).

3. Procedure

I. Digestion procedure

(a)

Place the blender with the filtration insert, connect the waste tube and place the tube so it drains into the waste bin.

(b)

When the blender is switched on, heating will start.

(c)

Before this is done, the bottom valve located below the reaction chamber must be opened and closed.

(d)

Up to 35 samples weighing approximately 1 g each (at 25 to 30 °C) taken from each individual sample in accordance with point 2 are then added. Ensure that larger pieces of tendons are removed as they may clot the membrane filter.

(e)

Pour water up to the edge of a liquid chamber connected to the blender (approximately 400 ml).

(f)

Pour about 30 ml hydrochloric acid (8,5 %) to the edge of the smaller, connected liquid chamber.

(g)

Place a membrane filter under the coarse filter in the filter holder in the filter insert.

(h)

Lastly, add 7 g of pepsin or 21 ml liquid pepsin. This order must be followed strictly to avoid decomposition of the pepsin.

(i)

Close the lids of the reaction and liquid chambers.

(j)

Select the period of digestion. A short digestion period (5 minutes) must be set for pigs at the normal slaughter age and a longer time (8 minutes) for other samples.

(k)

When the start button on the blender is turned on, the process of dispensing and digestion starts automatically, followed by filtration. After 10 to 13 minutes the process is completed and stops automatically.

(l)

Open the lid of the reaction chamber after checking that the chamber is empty. If there is foam or any digestion liquid remaining in the chamber, repeat the procedure in accordance with Section V.

II. Recovery of larvae

(a)

Remove the filter holder and transfer the membrane filter to a slide or petri dish.

(b)

Examine the membrane filter using a (stereo-)microscope or a trichinoscope.

III. Cleaning equipment

(a)

Where the result is positive, fill the blender reaction chamber with boiling water until it is two-thirds full. Ordinary tap water is poured into the connecting liquid chamber until it covers the lower sensor. Automatic cleaning then takes place. Decontaminate the filter-holder and any other equipment, e.g. using formol.

(b)

After work is completed for the day, fill the blender liquid chamber with water and put it through a standard cycle.

IV. Use of membrane filters

Each polycarbonate membrane filter may be used no more than five times. The filter is to be turned between each use. In addition, the filter must be checked after each use for any damage which would make it unsuitable for further use.

V. Method to be applied when digestion is incomplete and filtration cannot be carried out

Once the blender has been put through an automatic cycle in accordance with Section I, open the lid of the reaction chamber and check whether there is foam or any liquid remaining in the chamber. If this is the case, proceed as follows:

(a)

close the bottom valve below the reaction chamber;

(b)

remove the filter holder and transfer the membrane filter to a slide or petri dish;

(c)

put a new membrane filter in the filter holder and attach the filter holder;

(d)

fill the blender liquid chamber with water until the lower sensor is covered;

(e)

carry out the automatic cleaning cycle;

(f)

after the cleaning cycle has ended, open the lid of the reaction chamber and check whether any liquid remains;

(g)

if the chamber is empty, remove the filter holder and transfer the membrane filter to a slide or petri dish with tweezers;

(h)

examine the two membrane filters in accordance with Section II. If the filters cannot be examined, repeat the entire digestion process with a longer digestion time in accordance with Section I.

VI. Positive or doubtful results

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