1977L0096 — EN — 07.06.1984 — 004.001 — 1
This document is meant purely as a documentation tool and the institutions do not assume any liability for its contents
►B COUNCILDIRECTIVE
of21December 1976
on the examination for trichinae (trichinella spiralis) upon importation from third countries of
freshmeatderivedfromdomesticswine
(77/96/EEC)
(OJ L 26, 31.1.1977, p. 67)
Amended by:
Official Journal
No page date
►M1Council Directive 81/476/EEC of 24 June 1981 L 186 20 8.7.1981
►M2Council Directive 83/91/EEC of 7 February 1983 L 59 34 5.3.1983
►M3Commission Directive 84/319/EEC of 7 June 1984 L 167 34 27.6.1984
Amended by:
►A1Act of Accession of Greece L 291 17 19.11.1979
▼B
COUNCILDIRECTIVE
of21December 1976
ontheexaminationfortrichinae(trichinellaspiralis)uponimporta-
tion from third countries of fresh meat derived from domestic
swine
(77/96/EEC)
THE COUNCIL OF THE EUROPEAN COMMUNITIES,
Having regard to the Treaty establishing the European Economic
Community,
Having regard to Council Directive 72/462/EEC of 12 December 1972
on health and veterinary inspection problems upon importation ofbovine animals and swine and freshmeat from th ird countries(
1), as
last amended by Directive 75/379/EEC(2), and in particular Article 21
thereof,
Having regard to the proposal from the Commission,
Whereas in Directive 72/462/EEC the Council provided in Article 21
for the laying down of the method and procedures required fordetecting the presence of trichinae in fresh pigmeat;
Whereas the application of Directive 72/462/EEC will not have the
desired effects as long as disparities exist between the Member Statesas to the guarantees required in respect of the detection of trichinaeupon importation of fresh meat from third countries; whereas it is
therefore necessary to lay down Community arrangements in this field;
Whereas, in order to protect consumer health, it is necessary that fresh
pigmeat be systematically subjected to an examination using methods
recognized as effective, in order to eliminate meat containing trichinae;
Whereas when the examination is carried out in the exporting third
country, it must be carried out in slaughterhouses which comply with
certain conditions and which contain, in particular, a screening labora-tory provided withsuitable equipment;
Whereas in order to be able to distinguish the meat samples examined
from those not examined it is necessary to provide for the affixing of aspecial mark to meat which has been examined with a negative result;
Whereas there should be a procedure establishing close and effective
cooperation between the Commission and the Member States for asses-sing the advisability of permitting establishments in the third countriesto carry out this examination or to work on the meat examined;whereas there should also be a procedure for bringing the technicalprovisions relating in particular to the examination methods, the
requirements concerning the screening laboratories and the procedure
for marking examined meat into line withtech nical progress and withexperience acquired;
Whereas the Member States should be allowed to admit fresh meat
which has not been screened for trichinae in the exporting thirdcountry, provided that this meat, undergoes treatment by freezing;which ensures that any trichinae which may be present are renderedharmless, either in the exporting third country, or in the Member State
for which the meat is intended; whereas this treatment must neverthe-
less be carried out according to certain well defined procedures andestablishments fulfilling certain conditions,1977L0096 — EN — 07.06.1984 — 004.001 — 2
(1) OJ No L 302, 31. 12. 1972, p. 28.
(2) OJ No L 172, 3. 7. 1975, p. 17.
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HAS ADOPTED THIS DIRECTIVE:
Article 1
The definitions used in this Directive are those contained in Directive
72/462/EEC.
Moreover:
(a) ‘freshmeat’ means freshmeat of domestic swine;
(b) ‘examination’ refers to the examination to detect the presence of
trichinae in fresh meat.
Article 2
1. In order to be admitted to intra-Community trade, freshmeat
originating in third countries which contains skeletal muscles (striatedmuscles) shall be examined under the supervision and responsibility ofan official veterinarian.
2. The examination shall be carried out in accordance with one of
the methods provided for in Annex I, on the whole carcase or, failingthis, on each half carcase, quarter carcase or piece to be imported intothe Community.
3. The examination shall take place in a slaughterhouse approved in
the exporting country in accordance with Article 4 of Directive 72/462/
EEC and authorized to carry out this examination in accordance withArticle 4 of this Directive.
4. The examination shall take place before the health marking
provided for in Chapter X of Annex B to Directive 72/462/EEC.
5. If it is not possible to carry out the examination in the exporting
country, the Member State for which the fresh meat is intended mayauthorize its importation provided that the examination is carried outwithin its territory at the time of the public health inspection providedfor in Article 24 (2) of Directive 72/462/EEC, at an inspection post
within the meaning of Article 27 (1) (b) of that Directive.
6. (a) If the outcome of the examination is negative, the fresh meat
shall be marked immediately after the examination, in accor-dance withAnnex III.
(b) In the case of ink stamping, use shall be made of a colorant
within the meaning of Article 17 (3) of Directive 72/462/EEC.
Article 3
1. By way of derogation from Article 2, the Member State for which
it is intended may authorize the exemption from examination of freshmeat from certain third countries or parts of such countries, provided itis frozen in accordance withth e provisions of Annex IV.
2. This treatment shall be carried out in an establishment situated in
the exporting third country, and described in Article 4 (1).
Freezing in the exporting third country must be the subject of certifica-
tion by the official veterinarian on the health certificate accompanyingthe meat, as referred to in Article 22 (3) of Directive 72/462/EEC.
3. If the treatment has not been carried out in the exporting third
country, it must be carried out at an inspection post as described inArticle 2 (5).
Freezing in a Member State must be the subject of certification by the
official veterinarian on the certificates accompanying the meat, as
referred to in Article 25 of Directive 72/462/EEC.
Article 4
1. The authorization for a slaughterhouse to carry out the examina-
tion and of a cutting plant to cut up or bone meat which has undergonesuch examination, or the authorization for an establishment to carry out1977L0096 — EN — 07.06.1984 — 004.001 — 3
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the freezing treatment referred to in Article 3, shall be decided on in
accordance withth e procedure laid down in Article 9. In addition tothe requirements of Article 4 of Directive 72/462/EEC, account shall
be taken of the guarantees given in respect of compliance with this
Directive and, in the case of slaughterhouses, of:
(a) the presence of the rooms and apparatus necessary for carrying out
the examination;
(b) the qualifications of the personnel responsible for carrying out the
examination.
Authorization shall be granted to a slaughterhouse and cutting plant
only where the competent authorities of the third country concernedhave officially recognized that the slaughterhouse and cutting plant
are in a position to satisfy the conditions laid down in Article 5 and
in Annex III; also, in the case of a slaughterhouse, that it has a labora-tory which complies with the conditions laid down in Annex II,Chapter I, and which is in a position to satisfy the requirements of theother chapters of Annex II and those of Annex I.
Authorization shall be granted to an establishment to carry out the
freezing treatment only if the competent authorities of the third countryconcerned have officially recognized that the establishment is in a posi-tion to satisfy the conditions laid down in Annex IV.
2. On the list(s) referred to in Article 4 (4) of Directive 72/462/EEC,
a special indication shall be inserted against the names of the establish-ments which have been granted an authorization within the meaning ofparagraph1.
Article 5
1. In slaughterhouses which have been granted an authorization in
accordance with Article 4, swine the meat of which is intended for
the Community must be slaughtered in different rooms or in the
absence thereof at different times from swine the meat of which is notintended for the Community, unless the meat of such swine is exam-ined in accordance withth e same procedure.
2. The cutting and boning of meat which has undergone an examina-
tion withnegative results and is intended for th e Community must becarried out in cutting plants, in accordance withArticle 4.
In these cutting plants, the cutting and boning of such meat must be
carried out in different rooms or in the absence thereof, at differenttimes from meat which is not intended for the Community, unless themeat is examined in accordance withth e same procedure.
Article 6
The inspections in third countries provided for in Article 5 of Directive
72/462/EEC must also verify whether the present Directive is beingapplied.
Article 7
The Member States shall draw up and communicate to the Commission
the list of the inspection posts referred to in Article 2 (5) at which:
— the examination,
— the freezing referred to in Article 3,
may be carried out.
They shall ensure that these posts have the equipment necessary for
carrying out the operations in question.
Article 8
Using the procedure laid down in Article 9, decisions may be taken to
adapt or add to the Annexes hereto in the light of technological devel-1977L0096 — EN — 07.06.1984 — 004.001 — 4
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opments; such decisions must ensure that health guarantees are main-
tained at their present standard.
Article 9
1. Where the procedure laid down in this Article is followed, the
matter shall without delay be referred by the chairman, either on his
own initiative or at the request of a Member State, to the StandingVeterinary Committee (hereinafter referred to as ‘the Committee’) setup by the Council Decision of 15 October 1968.
2. Within the Committee the votes of the Member States shall be
weighted as laid down in Article 148 (2) of the Treaty. The chairmanshall not vote.
3. The representative of the Commission shall submit a draft of the
measure to be adopted. The Committee shall deliver its opinion on
these measures within a time limit set by the chairman having regardto the urgency of the questions under examination. Opinions shall bedelivered by a majority of ►
A1forty-five ◄votes.
4. The Commission shall adopt the measures and shall implement
them immediately, where they are in accordance with the opinion of
the Committee. Where the measures envisaged are not in accordance
with the opinion of the Committee, or if no opinion is delivered, theCommission shall without delay submit to the Council a proposal onthe measures to be taken.
The Council shall adopt the measures by a qualified majority.If the Council has not adopted any measures within three months of the
date on which the matter is referred to it, the Commission shall adoptthe proposed measures and shall implement them immediately save
where the Council has decided against such measures by a simple
majority.
Article 11
The Member States shall bring into force on 1 January 1979 at the
latest the laws, regulations and administrative provisions needed for
compliance with this Directive. They shall forthwith inform the
Commission thereof.
Article 12
This Directive is addressed to the Member States.1977L0096 — EN — 07.06.1984 — 004.001 — 5
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ANNEX I
METHODSOFEXAMINATIONFORTRICHINAE
I. TRICHINOSCOPIC EXAMINATION
(a)Apparatus
An incandescent lamp trichinoscope with 50 × and 80 to 100 × magni-
fication.
A pressure glass consisting of two glass plates — one of which is
divided into equal fields — small curved scissors, small forceps, a knife
for cutting specimens, small numbered containers for storing the speci-
mens separately, a dropping pipette, a glass of acetic acid and a glass of
potassium hydroxide solution for brightening any calcifications or soft-
ening dried meat.
(b)Takingofspecimens
In the case of whole carcases, at least one specimen of the size of a
hazelnut is to be taken from both diaphragm pillars at the transition of
the sinewy part.
If there is only one diaphragm pillar, one specimen the size of two
hazelnuts is to be taken. In the absence of both diaphragm pillars, two
specimens approximately the size of a hazelnut are to be taken from the
rib part or the breastbone part of the diaphragm or, as the case may be,
from the lingual muscle or the jaw muscle or from the abdominal
muscles.
For pieces of meat, from eachpiece take th ree samples of skeletal
muscle, containing little fat, if possible the size of a hazelnut, and taken
from different points, where possible near to bones or tendons.
(c)Method
If both diaphragm pillars are present the trichinae inspector must cut,
from each of the above specimens taken from a whole carcase, seven
pieces the size of an oat-kernel making 14 in all; if only one diaphragm
pillar is present, 14 pieces, from different places and if possible from
the transition to the sinewy part; he must then compress them between
the glass plates in such a way that normal print can be clearly read
through the slide preparation. If the flesh of the specimens to be exam-
ined is dry and old, the preparations must be softened in a mixture of
one part potassium hydroxide solution to about two parts water for 10
to 20 minutes before pressing.
If, in the case of whole carcases, specimens have to be taken from the
rib part or the breastbone part of the diaphragm, the lingual muscle or
jaw muscle or the abdominal muscles, then 14 pieces the size of an oat-
kernal are to be cut from eachspecimen, i.e. a total of 28.
From each of the samples taken from pieces of meat, the trichinae
inspector must cut four pieces the size of an oat-kernal, making 12
pieces in all.
The trichinoscopic examination must be carried out in such a manner
that each preparation is scanned slowly and carefully. If the trichino-
scopic examination reveals suspect areas, the nature of which cannot
be definitely ascertained even withth e most powerful magnification of
the trichinoscope, these must be checked by microscope.
The microscopic examination should be carried out in such a manner
that each preparation is scanned slowly and carefully at a magnification
of 30 to 40 ×.
In the case of an uncertain result, the examination must be continued on
a further number of specimens and slide preparations, if necessary with
the aid of higher magnifications, until the information required is
obtained. The trichinoscopic examination must be carried out for at
least three minutes.
The trichinoscopic examination must be carried out for at least six
minutes in the case of substitute specimens taken from the rib part or
breastbone part of the diaphragm pillars, the lingual muscle or the jaw
muscle or the abdominal muscles.1977L0096 — EN — 07.06.1984 — 004.001 — 6
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The minimum time fixed for the examination does not include the time
necessary for sample-taking and for making the preparations.
As a general rule, the trichinoscopic examiner should not inspect more
than 840 pieces a day, though by way of exception he may inspect up
to 1050.
II. THE ARTIFICIAL DIGESTION METHOD
(a)Apparatusandmaterial
— knife for taking samples,
— small numbered containers, withclosures, for storing samples, if
necessary until repetition of the examinations,
— incubator,— 2 to 3 litre glass funnel withstand, a connecting h ose in rubber,
clamps for fastening the connecting hose,
— a plastic sieve (approximately 18 cm in diameter, and approximately
1 mm mesh),
— gauze,— a small tapering tube witha sealed end,— a glass block dish,— a meat mincer,
— a stereo-microscope (magnification 15 to 40 ×) witha suitable ligh t
source,
— digestive liquid made up as follows:
10 g of pepsin (80 u/g FIP: Fédération internationale de pharmacie),
5 ml HCl (at least 37 %) made up to a litre withtap-water.
(b)Takingofspecimens
1. In the case of whole carcases a sample of at least 20 g to be taken
from a diaphragm pillar at the transition to the sinewy part. In the
absence of diaphragm pillars a specimen of at least 20 g to be taken
from the rib part or the breastbone part of the diaphragm or from the
lingual muscle or the jaw muscle or the abdominal muscles.
2. For pieces of meat, a sample of at least 20 g of skeletal muscle to be
taken, containing little fat and where possible near to bones or
tendons.
(c)Method
For the examination of a collective specimen from 10 pigs, a 10 g
specimen shall be prepared from each individual 20 g sample. The
remaining 10 g shall be kept for additional single-specimen examination
should this be necessary.
10 specimens, each weighing 10 g, shall be combined into a collective
specimen; it shall be minced in a meat mincer (with 2 mm perforations)
and placed loosely in a sieve lined witha layer of gauze. Th e sieve
shall then be placed in a funnel connected by a length of rubber hose
to a small tapered tube with a sealed end; the funnel shall be filled
from the edge with digestive liquid until the matter for analysis is
completely covered. The proportion of matter for analysis to digestive
liquid must be approximately 1:20 to 1:30.
After 18 to 20 hours of incubation at 37 to 39 ºC, the small tapered tube
shall be disconnected and removed. After carefully drawing off the
supernatant liquid, the sediment present in the tip of the tube is to be
carefully rinsed in a dish. Then to be examined for the presence of
trichinae with a stereo-microscope at 20 to 40 × magnification.
In the case of a positive or doubtful result from the examination of a
collective specimen, the remaining single specimens to be examined
individually after addition of a further 20 g from each pig, or in the
case of pieces of meat, the addition of 20 g taken from each piece,
according to (b) above.1977L0096 — EN — 07.06.1984 — 004.001 — 7
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III. METHOD USING THE ARTIFICIAL DIGESTION OF COLLECTIVE
SAMPLES
(a)Apparatusandreagents
— knife and tweezers for collecting specimens,
— meat mincer with2 to 3 mm diameter perforations,
— a 3 litre Erlenmeyer flask witha rubber or cotton-wool plug,
— a conical separation funnel of 2000 ml capacity,
— an ordinary A-base stand of approximately 28 cm lengthwithan
80 cm stem,
— a ring, diameter approximately 10 to 11 cm which can be fixed to
the stand,
— a clamp with a flat vice (23 × 40 mm) which can be attached to the
stand by means of a double coupling,
— a sieve (meshsize 177 microns) withan external diameter of 11 cm
fitted withbrass or stainless steel mesh ,
— a funnel withan internal diameter of not less th an 12 cm,
— 100 ml glass measuring cylinders,
— a stereo-microscope (magnification 15 to 40 ×) witha suitable ligh t
source, or a trichinoscope with horizontal table for the compressor
withsuitable ligh t source,
— when using the trichinoscope: a larval counting basin is used which
may be described as follows:
the larval counting basin is made from 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 should be
inserted between the sides, thus forming a basin with two small
handles in both ends. The upper side of the bottom should be
raised 7 to 9 mm from the base of the frame formed by the
sides and the ends. The parts should be fixed by using glue
appropriate for the material,
— a number of 9 cm diameter Petri dishes (when using stereo-micro-
scope) marked on their undersides into 10 × 10 mm square
examination areas using a pointed instrument,
— a number of 10 litre bins to be used when applying decontamina-
tion, such as formol treatment, to the apparatus and for the
remaining digestive juice in the case of positive results,
— concentrated (37 %) hydrochloric acid,
— pepsin strength: 1: 10000 NF (US National Formulary)
corresponding to 1: 12500 BP (BritishPh armacopoea)
corresponding to 2 000 FIP (Fédération internationale de phar-
macie),
— a number of trays which can hold 50 samples of approximately 2 g
each,
— a balance accurate to 0,1 g.
(b)Collectionofspecimens
1. In the case of whole carcases, a specimen to be taken of approxi-
mately 2 g from a pillar of the diaphragm at the transition to the
sinewy part. In the absence of diaphragm pillars, a specimen of the
same size to be taken from the rib part of the breastbone part of the
diaphragm, from the lingual muscle or the jaw muscle or the abdom-
inal muscles.
2. For cuts of meat, a sample of approximately 2 g of skeletal muscle
to be taken, containing little fat and, where possible, near to bones or
tendons.
(c)Method
1. (i)Complete pools (100 samples at a time)
Approximately 1 g of sample shall be taken from each of the
100 individual samples from the pigs. The pooled sample is put
once through the mincer.
The minced meat shall be placed in the 3 litre Erlenmeyer flask
together with 7 g of pepsin, approximately 2 litres of tap-water1977L0096 — EN — 07.06.1984 — 004.001 — 8
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heated to about 40 to 41 ºC and 25 ml of concentrated hydro-
chloric acid. The mixture shall be shaken to dissolve the pepsin.
The pH of the solution will be about 1,5 to 2.
— For digestion, the Erlenmeyer flask shall be incubated at 40
to 41 ºC for approximately four hours. The flask shall be
regularly shaken during the time of incubation, at least twice
every hour.
— The digested solution shall be filtered through the sieve into
the conical 2 litre separation funnel and left undisturbed on
the stand for at least one hour.
— A total volume of approximately 45 ml should be run off
into a measuring cylinder and divided between three Petri
dishes, the bottoms of which should be marked off into
squares, with15 ml in eachdish .
— EachPetri dishsh all be minutely examined for larval
trichinae under the stereo-microscope.
— Where larval counting basins are employed, the 45 ml shall
be distributed between two larval counting basins and exam-
ined under the trichinoscope.
The larvae appear as identifiable organisms in the deposit
and often, when the water is lukewarm, rolling and unrolling
movements of the ‘spiral’ may be observed.
— Digests should be examined as soon as they are ready. Under
no circumstances should examination be postponed until the
following day.
If the digests are unclear or not examined within 30 minutes
of their preparation, they should be clarified as follows. The
final sample of 45 ml is poured into a measuring cylinder
and allowed to stand for 10 minutes. At the end of this
time, 30 ml of supernatant fluid is removed by suction and
the remaining 15 ml is made up to 45 ml with tap-water.
After a further settling period of 10 minutes, 30 ml of super-
natant fluid is removed by suction and the remaining 15 ml
is poured into a Petri dishor larval counting basin for exam-
ination. The measuring cylinder should be washed with 10 ml
of tap-water and these washings should be added to the
sample in the Petri dish or larval counting basin for exami-
nation.
(ii)Pools of less than 100 samples
Up to 15 single samples could be added to a total pool of 100
samples and be examined together with these samples. If more
than 15 samples and less than 100 samples are examined, thedigestion fluid should be reduced proportionately.
2. In the case of a positive or doubtful result following the examination
of a collective sample, a further 20 g sample should be taken from
eachpig in accordance with(b) above. Th e 20 g samples from five
pigs should be pooled and examined by the method described above.
In this way samples from 20 groups of five pigs will be examined.
When trichinellae are detected in a pooled sample from five pigs,
further 20 g samples should be collected from the individual pigs in
the group and each should separately be examined using the method
described above.
IV. THE MECHANICALLY ASSISTED POOLED SAMPLE DIGESTION
METHOD/SEDIMENTATION TECHNIQUE
(a)Apparatusandreagents
— knife or scissors for cutting specimens,
— trays marked off with 50 squares each of which can hold samples of
approximately 2 g of meat,
— a Stomacher Lab-blender 3 500 Thermo model,
— plastic bags suitable for the Stomacher Lab-blender,
— conical separation funnels of 2 litre capacity, preferably fitted with
teflon safety plugs,
— stands, rings and clamps,
— sieves, meshsize 177 microns, external diameter 11 cm withstain-
less steel mesh,
— funnels with an internal diameter not less than 12 cm, to support the
sieves,1977L0096 — EN — 07.06.1984 — 004.001 — 9
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— 100 ml glass measuring cylinders,
— a 25 ml dispenser,
— beakers of 3 litre capacity,— spoon or glass rod for stirring the digestion fluid in the beaker,
— a plastic syringe and tube for suction,
— a measuring spoon for 6 g,— a thermometer accurate to ± 0,5 ºC within the range 1 to 100 ºC,— a vibrator, e.g. an electric shaver with the head removed,
— a relay which will switch on and off at one minute interval,
— a trichinoscope with a horizontal table or a stereo-microscope, with
a suitable light source,
— larval counting basin (when using trichinoscope): the larval counting
basin is made from 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 should be
inserted between the sides, thus forming two small handles in
both ends. The upper side of the bottom should be raised 7 to
9 mm from the base of the frame formed by the sides and the
ends. The parts should be fixed by using glue appropriate for
the material,
— a number of 9 cm diameter Petri dishes (when using stereo-micro-
scope) marked on their undersides into 10 × 10 mm square
examination areas using a pointed instrument,
— 17,5 % hydrochloric acid solution,
— pepsin strength1: 10000 NF (US National Formulary)
corresponding to 1: 12500 BP (BritishPh armacopoea)corresponding to 2 000 FIP (Fédération internationale de phar-
macie),
— a number of 10 litre bins to be used when applying decontamina-
tion, such as formol treatment, to the apparatus and for the
remaining digestive juice in the case. of positive results,
— a balance accurate to 0,1 g.
(b)Collectionofspecimens
1. In the case of whole carcases, a specimen to be taken of approxi-
mately 2 g from a pillar of the diaphragm at the transition to the
sinewy part. In the absence of diaphragm pillars, a specimen of the
same size to be taken from the rib part or the breastbone part of the
diaphragm, from the jaw muscle or the abdominal muscle.
2. For cuts of meat, a sample of approximately 2 g of skeletal muscle
to be taken, containing little fat and, where possible, near to bones or
tendons.
(c)Method
1.Digestion procedure
(i)Complete pools (100 samples at a time)
— The Stomacher Lab-blender 3 500 should be fitted with a
double plastic bag and the temperature control set at 40 to
41 ºC
— One and a half litres of water preheated to about 32 to 35 ºC
is poured into the inner plastic bag and the water heated to
40 to 41 ºC.
— 25 ml of 17,5 % hydrochloric acid is then added to the water
in the Stomacher.
— 100 samples of approximately 1 g each(at 25 to 30 ºC)
taken from eachof th e individual samples, in accordance
with(b), are th en added.
— 6 g pepsin is finally added. This order of addition should be
strictly adhered to in order to avoid decomposition of the
pepsin.
— The Stomacher is then allowed to pound the content of the
bag for 25 minutes.
— The pastic bag is then removed from the Stomacher and the
digestion fluid is filtered through the sieve into a 3 litre
beaker.1977L0096 — EN — 07.06.1984 — 004.001 — 10
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— The plastic bag is washed with approximately 100 ml of
water, which is then used to rinse the sieve and finally added
to the filtrate in the beaker.
Up to 15 single samples could be added to a total pool of 100
samples and be examined together with these samples.
(ii)Smaller pools less than 100 samples
— The Stomacher Lab-blender 3 500 should be fitted with a
double plastic bag and the temperature control set at 40 to
41 ºC.
— A digestion fluid is prepared by mixing about one and a half
litres of water and 25 ml of 17,5 % hydrochloric acid. 6 g of
pepsin is added and the whole mixed at a temperature of 40
to 41 ºC. This order of addition should be strictly adhered to
in order to avoid decomposition of the pepsin.
— Of the digestion fluid, a volume corresponding to 15 ml per
gram of sample is measured (e.g. for 30 samples the volume
required is 30 × 15 ml or 450 ml) and transferred to the
inner of the two plastic bags together with the meat samples
of approximately 1 g (at 25 to 30 ºC) taken from eachof th e
individual samples in accordance with(b).
— Water at a temperature of approximately 41 ºC is poured into
the outer bag to a total volume in the two bags of one and a
half litres.
— The Stomacher is then allowed to pound the content of the
bag for 25 minutes.
— The plastic bag is then removed from the Stomacher and the
digestion fluid is filtered through the sieve into a 3 litre
beaker.
— The plastic bag is washed with approximately 100 ml of
water, which is then used to rinse the sieve and finally added
to the filtrate in the beaker.
2.Recovery of larvae by sedimentation
— Ice (300 to 400 g of ice flakes, scaly ice or crushed ice) is added
to the digestion fluid, bringing its volume up to about 2 litres.
The digestion fluid is then stirred until the ice has melted.
In the case of smaller pools (see 1 (ii)), the amount of ice should
be reduced correspondingly.
— The chilled digestion fluid is transferred to a 2 litre separation
funnel, equipped witha vibrator in an extra clamp.
— Sedimentation for 30 minutes, during which time the sedimenta-
tion funnel is vibrated intermittently, i.e. one minute vibration
followed by one minute pause.
— After 30 minutes, a 60 ml sample of the sediment is quickly run
off into a 100 ml measuring cylinder. (The funnel is rinsed with
detergent solution after use).
— The 60 ml sample is allowed to stand for at least 10 minutes,
after which time the supernatant should be withdrawn by suction,
leaving a volume of 15 ml to be examined for the presence of
larvae.
— For suction, a disposable syringe can be used, equipped witha
plastic tube.
The length of the tube should be such that 15 ml will remain in
the measuring cylinder when the flanges of the syringe rest on
the cylinder's rim.
— The remaining 15 ml is poured into a larval counting basin or
two Petri dishes and examined using a trichinoscope or stereo-
microscope respectively.
— Digests should be examined as soon as they are ready. Under no
circumstances should examination be postponed until the
following day.
If the digests are unclear, or are not examined within 30 minutes
of their preparation, they should be clarified as follows. The final
sample of 60 ml is poured into a measuring cylinder and allowed
to stand for 10 minutes. At the end of this time, 45 ml of super-
natant fluid is removed by suction and the remaining 15 ml is
made up to 45 ml withtap-water. After a furth er settling period
of 10 minutes, 30 ml of supernatant fluid is removed by suction
and the remaining 15 ml is poured into a Petri dish or larval
counting basin for examination. The measuring cylinder should
be washed with 10 ml of tap-water and these washings should1977L0096 — EN — 07.06.1984 — 004.001 — 11
▼M3
be added to the sample in the Petri dish or the larval counting
basin for examination.
3. In the case of a positive or doubtful result, following the examina-
tion of a collective sample, a further 20 g sample should be taken
from eachpig in accordance with(b) above. Th e 20 g samples
from five pigs should be pooled and examined by the method
described above. In this way, samples from 20 groups of five pigs
will be examined. When trichinellae are detected in a pooled sample
from five pigs, further 20 g samples should be collected from the
individual pigs in the group and each should separately be examined
using the method described above.
V. THE MECHANICALLY ASSISTED POOLED SAMPLE DIGESTION
METHOD/ ‘ON FILTER ISOLATION’ TECHNIQUE
(a)Apparatusandreagents
Those indicated in method IV (a).
Supplementary equipment to the abovementioned:
— 1 litre Gelman funnel, complete withfilter h older (diameter 45 mm),
— filter discs; the filter discs consist of:
a circular stainless steel meshwithan aperture of 35 microns (th e
diameter of the disc should be 45 mm),
two rubber rings made of 1 mm thick rubber (the external diameter
should be 45 mm and the internal diameter 38 mm),
the circular mesh is placed between the two rubber rings and
bonded to them using a two-component glue suitable for the two
materials,
— an Erlenmeyer flask witha capacity of 3 litres and fitted witha side
tube for suction,
— a filter pump,— plastic bags witha capacity of at least 80 ml,
— equipment for sealing the plastic bags,
— rennilase, strength1: 150000 soxh let units per gram.
(b)Collectionofspecimens
See method IV (b).
(c)Method
1.Digestion procedure
(i)Complete pools (100 samples at a time)
See method IV (c) (1) (i).
(ii)Smaller pools less than 100 samples
See method IV (c) (1) (ii).
2.Recovery of larvae by filtration
— Ice (300 to 400 g of ice flakes, scaly ice or crushed ice) is added
to the digestion fluid, bringing its volume up to about 2 litres.
In the case of smaller pools, the amount of ice should be reduced
correspondingly.
— The digestion fluid is then stirred until the ice has melted. The
chilled digestion fluid is then left for at least three minutes to
let the larvae coil.
— The Gelman funnel, fitted with a filter holder and filter disc, is
mounted on the Erlenymeyer flask connected to a filter pump.
— The digestion fluid is poured into the Gelman funnel and filtered.
Towards the end of filtration, the passage of the digestion fluid
through the filter can be assisted by applying suction with the
filter pump. Suction should cease before the filter becomes dry,
i.e. when 2 to 5 ml of fluid are left in the funnel.
— When all the digestion fluid has been filtered, the filter disc is
removed and placed in an 80 ml capacity plastic bag, together
with15 to 20 ml of rennilase solution. Th e solution of rennilase
is made by adding 2 g of rennilase to 100 ml of tap-water.
— The plastic bag is sealed twice and placed in the Stomacher
between the inner and outer bag.
— The Stomacher is allowed to pound for three minutes, e.g. while
it is working on a complete or incomplete pool.1977L0096 — EN — 07.06.1984 — 004.001 — 12
▼M3
— After three minutes, the plastic bag, complete with filter disc and
rennilase solution, is removed from the Stomacher and opened
withscissors. Th e 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 exami-
nation by stereo-microscope.
— Digests should be examined as soon as they are ready. Under no
circumstances should examination be postponed until the
following day.
Note
Filter discs should never be used when not completely clean.
Unclean discs should never be allowed to dry out.
Filter discs can be cleaned by leaving them in rennilase solution
overnight. Before use, they should be washed in fresh rennilase solu-
tion using the Stomacher.
3. In the case of a positive or doubtful result, following the examina-
tion of a collective sample, a further 20 g sample should be taken
from eachpig in accordance with(b) above. Th e 20 g samples
from five pigs should be pooled and examined by the method
described above. In this way, samples from 20 groups of five pigs
will be examined. When trichinellae are detected in a pooled sample
from five pigs, further 20 g samples should be collected from the
individual pigs in the group and each should separately be examined
using the method described above.
VI. THE MAGNETIC STIRRER METHOD FOR POOLED SAMPLE DIGES-
TION
(a)Apparatusandreagents
— knife and tweezers for cutting specimens,
— trays marked off into 50 squares each of which can hold samples of
approximately 2 g of meat,
— a Moulinette blender,
— magnetic stirrers, with thermostatically controlled heating plate and
teflon coated stirring rods, approximately 5 cm long,
— conical separation funnels of 2 litre capacity,
— stands, rings and clamps,
— sieves, meshsize 177 microns, external diameter 11 cm withstain-
less steel mesh,
— funnels with an internal diameter not less than 12 cm, to support the
sieves,
— beaker of 3 litre capacity,
— measuring cylinders of approximately 50 ml capacity, or centrifuge
tubes,
— a trichinoscope with horizontal table or a stereo-microscope, with a
suitable light source,
— larval counting basin (when using trichinoscope): the larval counting
basin is made from 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 should be
inserted between the sides, thus forming two small handles in
both ends. The upper side of the bottom should be raised 7 to
9 mm from the base of the frame formed by the sides and the
ends. The parts should be fixed by using glue appropriate for
the material,
— a number of 9 cm diameter Petri dishes (when using stereo-micro-
scope) marked on their undersides into 10 × 10 mm square
examination areas using a pointed instrument,
— aluminium foil,
— 25 % hydrochloric acid,
— pepsin strength: 1: 10000 NF (US National Formulary)
corresponding to 1: 12500 BP (BritishPh armacopoea)
corresponding to 2 000 FIP (Fédération internationale de phar-
macie),
— tap-water heated to 46 to 48 ºC,1977L0096 — EN — 07.06.1984 — 004.001 — 13
▼M3
— a number of 10 litre bins to be used when applying decontamina-
tion, such as formol treatment, to the apparatus and for the
remaining digestive juice in the case of positive results,
— a balance accurate to 0,1 g.
(b)Collectionofspecimens
1. In the case of whole carcases, a specimen to be taken of approxi-
mately 2 g from a pillar of the diaphragm at the transition to the
sinewy part. In the absence of diaphragm pillars, a specimen of the
same size to be taken from the rib part or the breastbone part of the
diaphragm, from the jaw muscle or the abdominal muscles.
2. For cuts of meat, a sample of approximately 2 g of skeletal muscle
to be taken, containing little fat and, where possible, near to bones or
tendons.
(c)Method
1. (i)Complete pools (100 samples at a time)
— 100 samples of approximately 1 g each, taken from each of
the individual samples in accordance with (b), are chopped
in the Moulinette blender. The blender should be operated
three to four times for approximately one second each time.
— The chopped meat is transferred to a 3 litre beaker and
sprinkled with10 g of pepsin. 2 litres of tap-water, preh eated
to 46 to 48 ºC, is poured into the beaker, together with 16 ml
of hydrochloric acid.
— The mincing insert of the Moulinette blender is immersed
repeatedly in the digestion fluid in the beaker to remove
any meat still adhering.
— The stirring rod is placed in the beaker and the beaker is
covered withaluminium foil.
— The beaker is placed on the preheated heating plate of the
magnetic stirrer and the stirring process started. Before
starting the stirring process, the magnetic stirrer should be
adjusted so that it maintains a constant temperature of 44 to
46 ºC throughout the period of operation. During the stirring
process, the digestion fluid should rotate at a sufficiently
high speed to create a deep whirl without splashing.
— The digestion fluid is stirred for 30 minutes, at the end of
which the stirrer is switched off and the digestion fluid is
poured through the sieve into the sedimentation funnel.
— The digestion fluid is allowed to stand in the funnel for 30
minutes.
— After 30 minutes, a 40 ml sample of digestion fluid is
quickly run off into the measuring cylinder or centrifuge
tube.
— The 40 ml sample is allowed to stand for 10 minutes, at the
end of which time 30 ml of supernatant is withdrawn by
suction, leaving a volume of 10 ml.
— The remaining 10 ml sample of sediment is poured into a
larval counting basin or Petri dish.
— Then the cylinder or centrifuge tube is rinsed with about
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 stereomicroscope
respectively.
— Digests should be examined as soon as they are ready. Under
no circumstances should examination be postponed until the
following day.
If the digests are not examined within 30 minutes of their
preparation, they should be clarified as follows. The final
sample of about 40 ml is poured into a measuring cylinder
and allowed to stand for 10 minutes, after which time 30 ml
of the supernatant fluid is 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 10 ml for examination in a Petri dishor larval counting
basin. The measuring cylinder should be washed with 10 ml
of tap-water and these washings should be added to the
sample in the Petri dish or the larval counting basin for
examination.1977L0096 — EN — 07.06.1984 — 004.001 — 14
▼M3
If the sediment is found to be unclear on examination, the
sample should be poured into a measuring cylinder and
made up to 40 ml with tap-water and then the above proce-
dure should be followed.
(ii)Pools of less than 100 samples
When needed, up to 15 samples of 1 g each may be added to a
total pool of 100 samples and examined together with these
samples according to (c) (1) (i). More than 15 samples should
be examined as a complete pool. For pools up to 50 samples,
the digestion fluid may be reduced to 1 litre.
2. In the case of a positive or doubtful result following the examination
of a collective sample, a further 20 g sample should be taken from
eachpig in accordance with(b) above. Th e 20 g samples from five
pigs should be pooled and examined by the method described above.
In this way samples from 20 groups of five pigs will be examined.
When trichinellae are detected in a pooled sample from five pigs,
further 20 g samples should be collected from the individual pigs in
the group and each should separately be examined using the method
described above.1977L0096 — EN — 07.06.1984 — 004.001 — 15
▼B
ANNEX II
CHAPTER I
CONDITIONSFOR THEAUTHORIZATIONOF TRICHINAE(TRICHI-
NELLASPIRALIS)LABORATORIES
1. Trichinae laboratories must be situated next to the swine slaughterhouses
and, if the establishment does not already possess such facilities to fulfil the
other requirements of the Directive on third countries, have at least the
following facilities available:
(a) a lockable room adequately equipped for the preparation of specimens;
its walls must be smooth and must be lined with a washable light-
coloured covering or paint up to a height of 2 m. A preparation room
must be provided for eachmeth od of examination used;
(b) an adequately equipped lockable examination room which can be
darkened when examination is performed by trichinoscope;
(c) equipment providing adequate ventilation and, if necessary, air condi-
tioning equipment which ensures that the room temperature does not
exceed + 25 ºC;
(d) adequate natural or artificial lighting which does not alter colours; direct
sunlight must be avoided;
(e) in the preparation room, adequate equipment for cleaning and disin-
fecting hands;
►M3(f)◄possibly, refrigerators in which to store meat samples;
(g) a washroom for cleansing and disinfecting examination equipment (e.g.
containers for samples, compressors, knives and scissors), with:
— a waterproof floor-covering which is rot-proof and easy to clean and
disinfect,
— smooth walls which, up to a height of at least 2 m, are lined by a
washable and light-coloured covering or paint.
This provision need not be applied when using the methods indicated
under points II, III, IV, V, VI of Annex I provided the laboratories are
equipped witha large suitably plumbed sink;
►M3(h)◄changing rooms, wash basins and rest rooms and flush lava-
tories;
►M3(i)◄washbasins withh ot and cold running water, provided with
cleansing and disinfecting materials and disposable towels;
►M3(j)◄watertight corrosion-resistant containers, with hermetically
sealed lids, for collecting the samples after examination, and so designed
as to prevent unauthorized removal of the contents;
►M3(k)◄adequate supplies of hot and cold drinking water;
►M3(l)◄equipment for removing waste water which fulfils the condi-
tions for the authorization of slaughterhouses;
►M3(m)◄proper equipment for protection against pests (insects,
rodents, etc.).
CHAPTER II
REQUIREMENTS APPLICABLE TO STAFF, PREMISES, EQUIPMENT
ANDINSTRUMENTSINTRICHINAELABORATORIES
2. Absolute cleanliness is required at all times of laboratory staff, premises,
equipment and instruments:
(a) staff must wear clean working clothes and wash their hands several times
during working hours and after each break;
(b) no animal may enter trichinae laboratories;
(c) equipment and instruments used must be kept clean and in good repair.
They must be carefully cleansed and disinfected several times during and
at the end of the day's work.
3. Drinking water must be used for all purposes.1977L0096 — EN — 07.06.1984 — 004.001 — 16
▼M3
▼B
▼M3
▼B
▼M3
▼B
▼B
4. As regards health, staff taking meat samples for examination must comply
with the rules in Annex B, Chapter IV (11) and (12) of Directive 72/462/
EEC.
5. The meat samples required for examination must be taken immediately after
slaughter and examined without delay in the trichinae laboratory of the
slaughterhouse.
These examinations are forbidden away from the slaughterhouse in which the
animals were slaughtered.
6. To avoid fatigue and its consequences, inspection staff should be given short
breaks during the working day.
CHAPTER III
REQUIREMENTSINRESPECTOFTRICHINOSCOPES
The construction and design of trichinoscopes must meet the following
minimum criteria:
1. Simple operation.2. Highligh t intensity:
— accurate results must be obtainable even in a room which is not comple-
tely dark,
— a projector bulb of 100 W (12 V) must be used as the light source.
3. Adequate magnification:
— normal working magnification: 50 x,
— 80 to 100 x magnification for more precise assessment of objects not
clearly identifiable under normal working magnification.
4. Resolving power:
— a clear sharp picture of well-defined colour must be obtainable at each
magnification.
5. Switchmech anism:
— eachch ange of magnification must be accompanied by automatic adjust-
ment of the brightness of the image.
6. Increase of contrast:
— the condensor must be provided with an iris diaphragm enabling the
contrast to be increased for the closer inspection of difficult cases,
— the iris diaphragm must be easy to operate (e.g. control lever on the
platform of the trichinoscope).
7. Easy focusing:
— rapid focusing by means of an adjusting ring,
— precise focusing by means of a control lever.
8. Voltage regulation:
— so that brightness may be adjusted as required.
9. One-way movement of the compressor:
— an automatic blocking mechanism must ensure that the compressor
moves in only one direction, to prevent unintentional displacement.
10. Free view of the projector screen.
11. Projector screen:
— at least 54 cm in diameter,
— high reflecting capability,— durable,— can be dismantled,
— easy to clean.1977L0096 — EN — 07.06.1984 — 004.001 — 17
▼B
ANNEX III
MARKING OF MEAT WHICH HAS BEEN EXAMINEDFOR
TRICHINAE
1. Marking of the meat must be carried out under the responsibility of the offi-
cial veterinarian. For this purpose, he shall keep and maintain:
— the instruments intended for marking which he may hand over to the
assistant staff only at the time of marking and for the length of time
required for this purpose,
— the tags mentioned in paragraph 5. These tags shall be given to the assis-
tant staff at the time when they must be used and in the required number.
2. The mark must be round with a diameter of 2·5 cm. The following informa-
tion must appear on the mark in perfectly legible characters:
— towards the centre the capital letter ‘T’ with arms 1 cm long and 0·2 cm
wide,
— under the letter ‘T’ one of the following sets of initials: CEE, EEG,
EWG, EØF, EEC ►A1or EOK◄. The letters must be 0·4 cm high.
3. Carcases must be marked in ink or hot-branded on the inside of the thighs, in
accordance withparagraph2.
4. Heads must be marked in ink or hot-branded, with a mark meeting the
requirements of paragraph2.
With the exception of cuts exempt from health marking by virtue of Annex
B, Chapter X (43) of Council Directive 72/462/EEC, those taken in cutting
plants from carcases marked in accordance with the rules must, where they
bear no stamp, be marked in accordance with paragraph 2 before the health
mark is affixed.
The label provided for in the second subparagraph of the abovementioned
paragraph43 must comply withth e conditions of paragraph6 below.
5. Marking may also be effected by means of a round tag. This tag, to be
affixed to eachpiece or to eachcarcase must not be reusable, must be made
of strong materials and must meet all hygiene requirements. The following
information must appear on the stamp seals in perfectly legible characters:
— towards the centre the capital letter ‘T’,
— under the letter ‘T’ one of the following sets of initials: CEE, EEG,
EWG, EØF, EEC ►A1or EOK◄. The letters must be 0·2 cm high.
6. The label provided for in Annex B, Chapter X (44) of the Directive
mentioned in paragraph 4 above must, in addition to the health mark, bear a
clearly legible mark identical to that provided for in paragraph 2.1977L0096 — EN — 07.06.1984 — 004.001 — 18
▼B
ANNEX IV
THEFREEZINGOFMEAT
1. Meat brought in already frozen must be kept in this condition.
2. The technical equipment and energy supply of the refrigerating room, must
be such as to ensure that the temperature referred to under paragraph 6 is
reached very rapidly and maintained in all parts of the room and of the meat.
3. Insulated packaging should be removed before freezing, except for meat
which has already reached throughout the temperature referred to under para-
graph 6 when it is brought into the refrigeration room.
4. Consignments in the refrigeration room must be kept separately and under
lock.
5. The date and time when each consignment is brought into the refrigeration
room must be recorded.
6. The temperature in the refrigeration room must be at least − 25 ºC. It should
be measured withcalibrated th ermo-electric instruments and continuously
recorded. It may not be measured directly in the cold air flow. The instru-
ments must be kept under lock. The charts must include the relevant
numbers from the meat inspection register on importation and the date and
time of the commencement and completion of freezing, and must be retained
for one year after compilation.
7. Meat witha diameter or th ickness of up to 25 cm must be frozen for at least
240 consecutive hours, and meat with a diameter or thickness of between 25
and 50 cm must be frozen for at least 480 consecutive hours. This freezing
process may not be applied to meat which has a larger diameter or is thicker.
The freezing time shall be calculated from the point when the temperature
referred to in paragraph 6 is reached in the freezing room.1977L0096 — EN — 07.06.1984 — 004.001 — 19