A. Mechanically assisted pooled sample digestion method/sedimentation technique
1. Apparatus and reagents
Knife or scissors for cutting specimens.
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.
Meat mincer or electrical blender.
A Stomacher lab-blender 3 500 thermo model.
Plastic bags suitable for the Stomacher lab-blender.
Conical separation funnels, capacity 2 litres, preferably fitted with Teflon safety plugs.
Stands, rings and clamps.
Sieves, mesh size 180 microns, external diameter 11 cm, with stainless steel or brash mesh.
Funnels, internal diameter not less than 12 cm, to support the sieves.
100 ml glass measuring cylinders.
A thermometer accurate to 0,5 °C within the range 1 to 100 °C.
A vibrator, e.g. an electric shaver with the head removed.
A relay which will switch on and off at one-minute intervals.
A trichinoscope with a horizontal table or a stereo-microscope, with a sub-stage transmitted light source of adjustable intensity.
A larval counting basin and a number of 9 cm diameter petri dishes as in Chapter I(1), points (l) and (m).
17,5 % hydrochloric acid.
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.
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.
A balance accurate to 0,1 g.
2. Collecting of specimens and quantity to be digested
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.
Complete pools (100 samples at a time):
The Stomacher lab-blender 3 500 is fitted with a double plastic bag and the temperature control set at 40 to 41 °C.
One and a half litres of water preheated to 40 to 41 °C is poured into the inner plastic bag.
25 ml of 17,5 % hydrochloric acid is added to the water in the Stomacher.
100 samples weighing approximately 1 g each (at 25 to 30 °C) taken from each individual sample in accordance with point 2 are added.
Lastly, 6 g pepsin or 18 ml liquid pepsin is added. This order must be followed strictly to avoid decomposition of the pepsin.
The Stomacher is then allowed to pound the content of the bag for 25 minutes.
The plastic bag is 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 lastly added to the filtrate in the beaker.
Up to 15 individual samples can be added to a total pool of 100 samples and examined together with these samples.
Smaller pools (less than 100 samples):
The Stomacher lab-blender 3 500 is fitted with a double plastic bag and the temperature control set at 40 to 41 °C.
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 °C. This order must be followed strictly 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 = 450 ml) and transferred to the inner of the two plastic bags, together with the meat samples weighing approximately 1 g (at 25 to 30 °C) taken from each individual sample in accordance with point 2.
Water at a temperature of approximately 41 °C is poured into the outer bag to make up 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 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 (at 25 to 30 °C), which is then used to rinse the sieve and lastly added to the filtrate in the beaker.
III.Recovery of larvae by sedimentation
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. The digestion fluid is then stirred until the ice has melted. In the case of smaller pools (see Section II(b)), the amount of ice must be reduced correspondingly.
The chilled digestion fluid is transferred to a 2 litre separation funnel, equipped with a vibrator in an extra clamp.
Sedimentation is allowed to proceed for 30 minutes, during which time the sedimentation funnel is vibrated intermittently, i.e. one minute vibration followed by a 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 is withdrawn by suction to leave a volume of 15 ml, to be examined for presence of larvae.
For suction, a disposable syringe, equipped with a plastic tube, can be used. The length of the tube must be such that 15 ml remains 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.
The measuring cylinder is washed with 5 to 10 ml of tap water and the washings are added to the sample.
Digests are to be examined as soon as they are ready. Under no circumstances is examination to be postponed until the following day.
Where the digests are unclear or they are not examined within 30 minutes of their preparation, they must be clarified as follows:
the final sample of 60 ml is poured into a measuring cylinder and allowed to stand for 10 minutes; 45 ml of supernatant fluid is then 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 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 is washed with 10 ml of tap water and these washings are added to the sample in the petri dish or the larval counting basin for examination.
IV.Positive or doubtful results
Where the result is positive or uncertain, the provisions laid down in Chapter I(3)(III) shall apply.