🦠 Trichinella larvae in meat — the artificial digestion method with a magnetic stirrer

Microbiology PN-EN ISO 18743

Overview

ISO 18743:2015 specifies a method for the detection of Trichinella larvae in meat by artificial digestion using a magnetic stirrer. The samples must be free of fat, tendons and fasciae (the hard layer of connective tissue is removed from the tongue); the size of the individual sample is laid down by the competent authority and the samples from individual animals may be pooled — the maximum mass of muscle in a pool for digestion is 100 g, with up to 15 g more where necessary; with a smaller pool (e.g. 50 g) the volume and the composition of the digestion fluid may be reduced proportionally to a minimum of 1 l. The samples are to be taken from the predilection muscles (in the pig — the pillars of the diaphragm or the masseter; in the horse — the masseter, the diaphragm, the tongue; in the wild boar — the diaphragm, the foreleg, the tongue) in twice the mass needed for the test, and are to be examined as soon as possible or stored at 2–8 °C, not frozen. Frozen samples (e.g. from the monitoring of wild animals) are examined with the sedimentation time extended to 60 min, because dead larvae uncoil from their spiral shape and settle more slowly; the size of the test portion should then be increased. The result is given as the presence or absence of Trichinella larvae in x grams of sample; all suspect findings are examined at a magnification of 60–100×, and positive larvae should be sent for microscopic and molecular (PCR) identification to the species/genotype.

In the European Union Commission Implementing Regulation (EU) 2015/1375 (as amended by Regulation 2022/1418) names, in annex I chapter I, the standard ISO 18743:2015 as the reference method for the detection of Trichinella, and in Chapter II describes equivalent methods (e.g. digestion in a Stomacher 3500 homogeniser with one and a half litres of water at 40–41 °C, 25 ml of 17.5 % HCl and 6 g of pepsin for 25 min; a 180 µm sieve 11 cm in diameter; a thermometer accurate to 0.5 °C in the range 20–70 °C; a balance accurate to ± 0.1 g). The pepsin is to be of a strength of 1:10 000 NF (equivalent to 1:12 500 BP and 2 000 FIP) or, in liquid form, of at least 660 units/ml; a different activity is allowed if the final activity of the fluid corresponds to 10 g of pepsin 1:10 000 NF (5.3 of ISO 18743). The Commission guidelines for the audits of Trichinella laboratories check, among other things, the use of the magnetic-stirrer method, the 180 µm mesh of the sieves, the quality of the pepsin and of the acid and the sedimentation times. In Poland the standard is in force as PN-EN ISO 18743:2015-11 with amendment A1:2024-02.

Method principle

The meat is comminuted in a blender or a mincer with the addition of 50–100 ml of digestion fluid or of tap water (45 ± 2 °C) per 100 g — usually three pulses of 5–10 s, no longer, so as not to damage the larvae. The digestion fluid is prepared freshly: to 2 l of tap water heated to 45 ± 2 °C 16 ml of 25 % hydrochloric acid is added (for 37 % HCl — 11 ml), the stirring is switched on and then 10 g of pepsin in powder or granules (1:10 000 NF) or 30 ml of liquid pepsin (660 U/ml) is added; the order water → acid → pepsin is obligatory, because it protects the pepsin from being decomposed by the concentrated acid. The comminuted meat is transferred into a 3 l beaker with the fluid (the equipment is rinsed with the fluid), the beaker is covered with aluminium foil and the digestion is carried out for 30 min at 45 ± 2 °C, stirring so that a deep vortex forms without splashing; the time may be extended to a maximum of 60 min. The fluid is poured through a 180–200 µm sieve into a separating funnel (the beaker and the sieve are rinsed with ≥ 100 ml of water); the digestion is regarded as satisfactory when what remains on the sieve is mainly indigestible tissue (fasciae, connective tissue) in an amount not greater than 5 % of the mass of the sample — otherwise the procedure is repeated. After 30 min of sedimentation the tap of the separating funnel is opened quickly and completely and about 40 ml (the primary sediment) is run off into a tube, left to stand for 10 min, 30 ml of the supernatant is drawn off, leaving ≤ 10 ml (the secondary sediment), which is poured into a gridded Petri dish together with 10 ml of the rinsings; after at least 1 min the transparency is checked (the sharpness of the lines of the grid) and it is examined systematically, square by square, under a stereomicroscope or a trichinoscope at a magnification of 10–20× for at least 10 min; a turbid sediment is clarified by diluting it again to 40 ml, allowing it to settle for 10 min and drawing it off to 10 ml. The examination is carried out directly after the digestion (at the latest on the same day, the dish being kept in a refrigerator); the larvae that are found are transferred into a 1–2 ml vial with 70–90 % ethanol.

Applications

• Official examination of the meat of pigs, horses and wild boar for Trichinella in slaughterhouses and cutting plants (the reference method under Regulation (EU) 2015/1375)
• Examination of pooled samples of up to 100 g (e.g. 100 samples of 1 g) with the possibility of tracing back to the source carcase by digesting ever smaller pools
• Monitoring of wild animals (foxes, raccoon dogs, bears) — also of frozen samples with extended sedimentation
• Veterinary and official laboratories subject to audits under the guidelines of the European Commission
• Quality documentation: the laboratory record (batch number of the pepsin, digestion time, residues on the sieve, result)

Key parameters

Standard

Standard number

PN-EN ISO 18743:2015-11 + A1:2024-02 (ISO 18743:2015 + Amd 1:2023)

Title (PL)

Mikrobiologia łańcucha żywnościowego — Wykrywanie larw włośni Trichinella w mięsie metodą wytrawiania

Title (EN)

Microbiology of the food chain — Detection of Trichinella larvae in meat by artificial digestion method

Step-by-step procedure

1. Sampling and preparation of the samples

   Take the predilection muscles (pig: the pillars of the diaphragm or the masseter) in twice the mass needed for the test; remove the fat, the tendons and the fasciae. Check the mass, the condition and the marking; examine at once or store at 2–8 °C without freezing. Pool for digestion: at most 100 g (+ up to 15 g).

2. Comminution

   Comminute the meat in a blender/mincer with 50–100 ml of digestion fluid or of water at 45 ± 2 °C per 100 g, usually with three pulses of 5–10 s — no longer, so as not to damage the larvae.

3. Digestion fluid (fresh)

   To 2 l of tap water at 45 ± 2 °C in a beaker add 16 ml of 25 % HCl (11 ml in the case of 37 %), switch on the stirring on the magnetic stirrer and add 10 g of pepsin 1:10 000 NF or 30 ml of liquid pepsin 660 U/ml. The order: water, acid, pepsin.

4. Digestion

   Transfer the meat into the 3 l beaker with the fluid, rinse the blender with the fluid, cover it with foil, maintain 45 ± 2 °C (check with the thermometer) and stir so that a deep vortex forms without splashing. Digest for 30 min; extend it if necessary, to a maximum of 60 min in total.

5. Filtration

   With the tap closed, pour the fluid through the 180–200 µm sieve into the separating funnel; rinse the beaker and the sieve with ≥ 100 ml of water. If more than 5 % of the mass of the sample or undigested muscle remains on the sieve — repeat the digestion (digest the remainder further and examine it in addition, or take new samples).

6. Sedimentation and collection of the sediment

   Allow it to settle for 30 min (the funnel may be tapped gently every 10 min; frozen samples — up to 60 min). Open the tap quickly and completely, run off about 40 ml of the primary sediment into a tube, leave it to stand for 10 min, draw off 30 ml of the supernatant, leaving ≤ 10 ml of the secondary sediment (in the case of turbidity: add 30 ml of water, 10 min, repeat).

7. Microscopic examination

   Pour the secondary sediment into a gridded Petri dish, rinse the tube with 10 ml of water and wait ≥ 1 min. Check the transparency (the sharpness of the grid); clarify it if necessary (make up to 40 ml, 10 min, draw off to 10 ml). Examine it square by square at 10–20× for ≥ 10 min without disturbing the fluid; suspect objects at 60–100×. Examine at once, at the latest on the same day (the dish in a refrigerator).

8. Result and handling of positives

   Give the presence/absence of Trichinella larvae in x g. Transfer the larvae into a 1–2 ml vial with 70–90 % ethanol and send them for identification (microscopy, PCR). Where a pool is positive, digest ever smaller pools of larger samples until the carcase is identified. Fill in the laboratory record (pepsin — batch number, digestion time, residues on the sieve, result).

9. Decontamination

   Decontaminate the fluids, the rinsings, the glassware and the equipment that may contain larvae by heating them to ≥ 70 °C or chemically (e.g. sodium hypochlorite with 0.01 % of active chlorine for ≥ 3 h) before pouring them away or washing them.

Required equipment and apparatus

Reagents, media and consumables

Health and safety (OHS)

• Trichinella larvae are infective for humans — decontamination of all the fluids and of the equipment (≥ 70 °C or hypochlorite), gloves, no eating in the laboratory
• Concentrated hydrochloric acid (25–37 %) — goggles, gloves, a fume hood when measuring it out; always add the acid to the water
• Pepsin in powder — avoid inhaling it (sensitisation); add the pepsin after the acid, never to the concentrated acid
• Hot fluid at 45 °C and the stirrer with a hotplate — take care when pouring into the separating funnel
• Glassware of large capacity (3 l beakers, 2.5 l separating funnels) — risk of breakage and of spilling infectious material

Frequently asked questions