Commission Implementing Regulation (EU) 2015/1375Show full title

CHAPTER I U.K. REFERENCE METHOD OF DETECTION

Magnetic stirrer method for pooled sample digestion U.K.

1. Apparatus and reagents U.K.

(a)

Knife or scissors and tweezers for cutting specimens.

(b)

Trays marked off into 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 blender with a sharp chopping blade. Where the samples are larger than 3 g, a meat mincer with openings of 2 to 4 mm or scissors must be used. In the case of frozen meat or tongue (after removal of the superficial layer, which cannot be digested), a meat mincer is necessary and the sample size will need to be increased considerably.

(d)

Magnetic stirrers with thermostatically controlled heating plate and Teflon-coated stirring rods approximately 5 cm long.

(e)

Conical glass separation funnels, capacity of at least 2 litres, preferably fitted with Teflon safety plugs.

(f)

Stands, rings and clamps.

(g)

Sieves, mesh size 180 microns, external diameter 11 cm, with stainless steel mesh.

(h)

Funnels, internal diameter not less than 12 cm, to support the sieves.

(i)

Glass beakers, capacity 3 litres.

(j)

Glass measuring cylinders, capacity 50 to 100 ml, or centrifuge tubes.

(k)

A trichinoscope with a horizontal table or a stereo-microscope, with a substage transmitted light source of adjustable intensity.

(l)

A number of 9 cm diameter petri dishes (for use with a stereo-microscope), marked on their undersides into 10 × 10 mm square examination areas using a pointed instrument.

(m)

A larval counting basin (for use with a trichinoscope), made of 3 mm thick acrylic plates as follows:

(i)

the bottom of the basin to be 180 × 40 mm, marked off into squares,

(ii)

the sides to be 230 × 20 mm,

(iii)

the end to be 40 × 20 mm. The bottom and the ends must be inserted between the sides, to form two small handles at the ends. The upper side of the bottom must be raised 7 to 9 mm from the base of the frame formed by the sides and the ends. The components must be stuck together with glue suitable for the material.

(n)

Aluminium foil.

(o)

25 % hydrochloric acid.

(p)

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.

(q)

Tap water heated to 46 to 48 °C.

(r)

A balance accurate to at least 0,1 g.

(s)

Metal trays, capacity 10 to 15 litres, to collect the remaining digestive juice.

(t)

Pipettes of different sizes (1, 10 and 25 ml) and pipette holders.

(u)

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

(v)

Siphon for tap water.

2. Collecting of specimens and quantity to be digested U.K.

(a)

In the case of whole carcasses of domestic swine, a specimen weighing at least 1 g is to be taken from a pillar of the diaphragm at the transition to the sinewy part. Special trichinae forceps can be used provided an accuracy of between 1,00 and 1,15 g can be guaranteed.

In the case of breeding sows and boars, a larger sample weighing at least 2 g is to be taken from a pillar of the diaphragm at the transition to the sinewy part.

In the absence of diaphragm pillars, a specimen of twice the size 2 g (or 4 g in the case of breeding sows and boars) is to be taken from the rib part or the breastbone part of the diaphragm, or from the jaw muscle, tongue or abdominal muscles.

(b)

For cuts of meat, a sample weighing at least 5 g of striated muscle, containing little fat is to be taken, where possible from close to bones or tendons. A sample of the same size is to be collected from meat that is not intended to be cooked thoroughly or other types of post-slaughter processing.

(c)

For frozen samples, a sample weighing at least 5 g of striated muscle tissue is to be taken for analysis.

The weight of meat specimens relates to a sample of meat that is free of all fat and fascia. Special attention must be paid when collecting muscle samples from the tongue in order to avoid contamination with the superficial layer of the tongue, which is indigestible and can prevent reading of the sediment.

3. Procedure U.K.

I. Complete pools (100 g of samples at a time) U.K.

(a)

16 ± 0,5 ml of hydrochloric acid is added to a 3 litre beaker containing 2,0 litre of tap water, preheated to 46 to 48 °C; a stirring rod is placed in the beaker, the beaker is placed on the preheated plate and the stirring is started.

(b)

10 ± 0,2 g of pepsin or 30 ± 0,5 ml liquid pepsin is added.

(c)

100 g of samples collected in accordance with point 2 is chopped in the blender.

(d)

The chopped meat is transferred to the 3 litre beaker containing the water, pepsin and hydrochloric acid.

(e)

The mincing insert of the blender is immersed repeatedly in the digestion fluid in the beaker and the blender bowl is rinsed with a small quantity of digestion fluid to remove any meat still adhering.

(f)

The beaker is covered with aluminium foil.

(g)

The magnetic stirrer must be adjusted so that it maintains a constant temperature of 44 to 46 °C throughout the operation. During stirring, the digestion fluid must rotate at a sufficiently high speed to create a deep whirl without splashing.

(h)

The digestion fluid is stirred until the meat particles disappear (approximately 30 minutes). The stirrer is then switched off and the digestion fluid is poured through the sieve into the sedimentation funnel. Longer digestion times may be necessary (not exceeding 60 minutes) in the processing of certain types of meat (tongue, game meat, etc.).

(i)

The digestion process is considered satisfactory if not more than 5 % of the starting sample weight remains on the sieve.

(j)

The digestion fluid is allowed to stand in the funnel for 30 minutes.

(k)

After 30 minutes, a 40 ml sample of digestion fluid is quickly run off into the measuring cylinder or centrifuge tube.

(l)

The digestion fluids and other liquid waste are kept in a tray until reading of the results is completed.

(m)

The 40 ml sample is allowed to stand for 10 minutes. 30 ml of supernatant is then carefully withdrawn by suction to remove the upper layers and leave a volume of not more than 10 ml.

(n)

The remaining 10 ml sample of sediment is poured into a larval counting basin or petri dish.

(o)

The cylinder or centrifuge tube is rinsed with not more than 10 ml of tap water, which has to be added to the sample in the larval counting basin or petri dish. Subsequently, the sample is examined by trichinoscope or stereo-microscope at a 15 to 20 times magnification. Visualisation using other techniques is allowed, provided examination of positive control samples has been shown to give an equal or better result than traditional visualisation methods. In all cases of suspect areas or parasite-like shapes, higher magnifications of 60 to 100 times must be used.

(p)

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

Where the digests are not examined within 30 minutes of preparation, they must be clarified as follows. The final sample of about 40 ml is poured into a measuring cylinder and allowed to stand for 10 minutes. 30 ml of the supernatant fluid is then removed, leaving a volume of 10 ml. This volume is made up to 40 ml with tap water. After a further settling period of 10 minutes, 30 ml of the supernatant fluid is withdrawn by suction, leaving a volume of no more than 10 ml for examination in a petri dish or larval counting basin. The measuring cylinder is washed with no more than 10 ml of tap water and these washings are added to the sample in the petri dish or the larval counting basin for examination.

If the sediment is found to be unclear on examination, the sample is poured into a measuring cylinder and made up to 40 ml with tap water and then the procedure described in this Section is followed. The procedure can be repeated 2 to 4 times until the fluid is clear enough for a reliable reading.

II. Pools of less than 100 g U.K.

Where needed, up to 15 g can be added to a total pool of 100 g and examined together with these samples in accordance with Section I. More than 15 g must be examined as a complete pool. For pools of up to 50 g, the digestion fluid and the ingredients may be reduced to 1 litre of water, 8 ml of hydrochloric acid and 5 g of pepsin.

III. Positive or doubtful results U.K.

Where examination of a collective sample produces a positive or uncertain result, a further 20 g sample is taken from each pig in accordance with point 2(a). The 20 g samples from five pigs are pooled and examined using the method described in this Chapter. In this way samples from 20 groups of five pigs will be examined.

When Trichinella is detected in a pooled sample from five pigs, further 20 g samples are collected from the individual pigs in the group and each is examined separately using the method described in this Chapter.

Parasite samples are to be kept in 90 % ethyl alcohol for conservation and identification at species level at the EU or national reference laboratory.

After parasite collection, positive fluids (digestive juice, supernatant fluid, washings, etc.) are to be decontaminated by heating to at least 60 °C.

IV. Cleaning and decontamination procedure after a positive or doubtful result U.K.

When the examination of a collective or individual sample produces a positive or doubtful result, all material in contact with meat (blender bowl and blade, beaker, stirring rod, temperature sensor, conical filtration funnel, sieve and forceps) must be carefully decontaminated by washing in warm water (65 to 90 °C). It is recommended to rinse each piece thoroughly to remove the detergent if a detergent is used during washing.