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Trichinae Export Program - Laboratory Methods  

The following methods have been reviewed and found acceptable for use in detecting larvae of Trichinella spiralis in pork and horse meat. The Acid Digestion methods (the Magnetic Stirrer and Stomacher methods) have been adopted into this program because they are the current standard procedures for use in detecting larvae of Trichinella spiralis in pork exported to the EU, Russia, and Singapore.


Sample Collection Digestion Methods


Those persons assigned to take samples from pork carcasses on the kill floor shall obtain a 5-gram sample from the pillars of the diaphragm (Crus Muscle). A 1-gram sample from each carcass is used when analyzing pork samples. A total of 100 1-gram pork samples may be pooled and analyzed at the same time. Persons assigned to take samples of horse meat on the kill floor shall obtain a 10-gram sample from the masseter muscle (Jaw) of each carcass. A 5-gram sample from each carcass is used when analyzing horse meat. A total of 10 5-gram horse meat samples may be pooled and analyzed at the same time. Alternative sites of collection acceptable for testing exported products in this program are the rib or breastbone part of the diaphragm, the jaw or masseter muscle, or the abdominal muscles.


Method I


The Mechanically Assisted Pooled Sample Digestion Method, Sedimentation Technique (Commonly Referred to as the Stomacher Method).


Materials


Knife or scissors for cutting samples
Trays for collecting samples
Balance for weighing samples
Stomacher Lab-blender 3500
Plastic stomacher bags for the Stomacher 3500
Beakers (3 Liter)
Beakers (1,500 mL)
Pepsin (1:10,000 NF)
Hydrochloric acid (17.5%)
Separatory Funnels (2 Liter)
Ring Stands
Sieves (180 m mesh, #80)
Funnels to support the sieves
Graduated cylinder (100 ml)
Stereomicroscope with light source and 1540 X Magnification
Gridded 9 cm Petri dishes for larval counting (10X10 mm squares)
A set of test weights (especially a 0.1 gram test weight)


Procedures


1. At the beginning of each day, the laboratory analyst should immediately turn on the Stomacher Lab-blender 3500 so that the unit heats up to a set temperature of 40 41 C.


2. The Stomacher Lab-blender 3500 is to be fitted with double plastic bags. This is done by placing one of the Stomacher 3500 plastic bags inside of another. This will help contain the sample digestion fluid should the inner bag break while in the Stomacher.


3. Fill the double plastic bags from step 2 with 1,500 mL (one and one-half liters) of tap water that has been heated to 40 41 C.


4. Add to the heated tap water, from step 3, 25 mL of 17.5% hydrochloric acid (HCl) and mix. Then add 6 grams of pepsin and mix to dissolve. Then add the meat sample to be tested. Make sure that the meat sample has been prewarmed to 25 30 C.


5. Place the double plastic bags containing the digestion fluid into the Stomacher Lab-blender 3500 and allow the Stomacher to pound the contents of the bags for 25 minutes.


6. Following digestion in the Stomacher 3500 for 25 minutes, the bags are removed and the contents poured through a 180 Micron (m) mesh sieve into a 3-liter beaker. Use an additional 100 mL of tap water to rinse the bags and the sieve into the 3-liter beaker. Note: As an alternative, the contents of the bags may be poured through the sieve, which has been placed into a funnel, and the sieve/funnel assembly is placed directly into a 2-liter separatory funnel.


7. Add ice or cold tap water to the digestion mixture to bring the volume to 2 liters. Note: If ice is used, the digestion fluid must be stirred until the ice melts. The chilled digestion fluid is then transferred to a 2-liter separatory funnel.


8. The digestion fluid is then allowed to sit for a 30-minute sedimentation period. During this 30 minutes, the separatory funnel should be lightly tapped with an object (such as scissors, plastic rod, etc.) that will setup vibrations within the separatory funnel, helping larvae settle to bottom of the funnel.


9. After the 30-minute sedimentation period, 60 mL of the digestion fluid is drawn from the bottom of the separatory funnel into a 100 mL graduated cylinder.


10. The 60 mL digestion fluid in the graduated cylinder is allowed to stand for 10 minutes, after which time the upper 75 percent of the contents of the cylinder are aspirated by suction to a final volume of 15 mL. Note: If after 10 minutes the sample is too cloudy, refill the graduated cylinder to 60 mL with warm tap water and let stand another 10 minutes. Then aspirate to 15 mL and proceed with the following steps.


11. The final 15 mL volume of sediment is poured into a gridded Petri dish and examined under a stereomicroscope (15-40X) for the presence of Trichinella larvae.


12. The presence of larvae in the Petri dish denotes a positive sample. The presence of a positive sample requires further analysis of smaller pools or individual samples as described in the EU directive. Note: The presence of a positive sample requires that appropriate Plant Management and the USDA, FSIS Inspector-In-Charge be notified. Notification of these individuals must be documented.


Method II


The Magnetic Stirrer Method for Pooled Sample Digestion (Commonly referred to as the Magnetic Stirrer Method)


Materials


Knife or scissors for cutting samples
Trays for collecting samples
Balance for weighing samples
Laboratory blender (Waring or equivalent)
Beakers (3 Liter)
Magnetic stir bars
Stirring hot plate
Pepsin (1:10,000 NF)
Hydrochloric acid (25%)
Separatory funnels (2 Liter)
Ring stands
Aluminum foil
Sieves (180 m mesh, #80)
Funnels to support the sieves
Graduated cylinder (100 mL)
Plastic centrifuge tubes (50 mL)
Stereomicroscope with light source and 15-40 X Magnification
Gridded 9 cm Petri dishes for larval counting (10X10 mm squares)
Thermometer, Mercury or Alcohol, 1 to 100 C in 0.5 increments (immersion type)
A set of test weights (especially a 0.1 gram test weight)


Procedures


1. At the beginning of each day, the laboratory analyst should immediately turn on the magnetic stirring hot plates at a setting that has been pre-determined to give optimum temperature of 46 -48 C. This will allow the hot plates to heat up to the desired temperature.


2. It may be desirable to measure 2 liters of tap water into 3-liter beakers and heat tap water to be within the range for testing.


3. To the heated tap water in step 2 add 16 mL of 25% hydrochloric acid (HCl) and mix, then add 10 grams of pepsin to create the digestion fluid.


4. Using samples collected as described, the laboratory should proceed to weigh samples of approximately 1 gram for pork, or 5 grams for horsemeat. Once a 100-gram sample pool is obtained, the sample pool is placed in a blender (meat samples should be warmed to 25 -30 C). To the blender is also added a small amount of digestion fluid from step 3 above and the samples are chopped by three or four short burst of the blender.


5. The chopped meat sample is then combined with the remaining prepared heated digestion fluid from step 3. The blender is rinsed to ensure the entire meat sample is recovered. A magnetic stirring bar is added to the 3 liter beaker and the beaker is covered with aluminum foil and placed on a heated stirring hot plate (make sure that hot plate is adjusted to maintain a constant temperature of 44 - 46 C. Under no circumstances allow the digestion fluid to heat to a temperature of 50 C.). The digestion fluid should be stirred vigorously for 30 minutes.


6. Following digestion on the stirring hot plate, the 3-liter beaker is removed and checked for completion of the digestion (no large chunks of meat are visible at the bottom of the flash after setting aside for one or two minutes to allow the vortex to settle). The contents of the beaker are poured through a 180 m mesh sieve into a 2-liter separatory funnel. An additional 100 mL of tap water is used to rinse the beaker and the sieve into the 2-liter separatory funnel. Remember the contents of the beaker are poured through the sieve, which has been placed into a funnel, and the sieve/funnel assembly is placed directly into a 2-liter separatory funnel.


7. The digestion fluid is then allowed to sit for a 30-minute sedimentation period. During this 30 minutes, the separatory funnel should be lightly tapped with an object (such as scissors, plastic rod, etc.) that will setup vibrations within the separatory funnel, helping larvae settle to the bottom of the funnel.


8. After the 30-minute sedimentation period, 40 mL of the digestion fluid is drawn from the bottom of the separatory funnel into a 50 mL centrifuge tube.


9. The 40 mL digestion fluid in the centrifuge tube is allowed to stand for 10 minutes, after which time the contents of the centrifuge tube are aspirated by suction to a final volume of 10 mL. Note: If after 10 minutes the sample is too cloudy, refill the centrifuge tube to 40 mL with warm tap water and let stand another 10 minutes. Then aspirate to 10 mL and proceed with the following steps.


10. The final 10 mL volume of sediment is poured into a gridded Petri dish and examined under a stereomicroscope (15-40X) for the presence of Trichinella larvae.


11. The presence of larvae in the Petri dish denotes a positive sample. The presence of a positive sample requires further analysis of smaller pools or individual samples as described in the EU directive. Note: The presence of a positive sample requires that appropriate Plant Management and the USDA, FSIS Inspector-In-Charge be notified. Notification of these individuals must be documented.


Results


The confirmation of the presence of Trichinella spiralis in either acid digestion method (Methods I and II) is determined visually by examination of samples under a stereomicroscope at a magnification of 15-40X. Live larvae will have a characteristic spiral shape when cold (hence T. spiralis) and will be very active and motile when warm. Dead larvae have a characteristic C-shape.


The prevalence of Trichinella infection is quite low in pigs (1/1,000 to 1/100,000 regionally) in the U.S. and even lower in most of Europe. It is likely that the prevalence is even lower in horses. Similarly, swine veterinary inspectors in Europe and some in the U.S. may never find a Trichinella positive carcass in a slaughterhouse. Irrespective of the statistics, it is very important that each sample and pool be handled and examined with technical accuracy, as the consequences of missed larvae can result in human disease. Analysts certified/accredited in this program are advised that the presence of a single larva in a one-gram (pork) or five-gram (horse) sample represents an infection level in a normal serving of meat sufficient to cause clinical human disease.


The EU, Russia, Singapore, and Chile have established specific guidelines for measures to take should a positive pool or sample be obtained. These procedures involve the repeated sampling of all animals represented in the positive pool in smaller pools and individual samples until the infected carcass has been identified. See EU Directive 77/96/EEC, Page No. L 26 73.

 
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  Last Modified Date: 02/08/2011