Magnetic Stirrer Method for the Detection of Trichinella Larvae in Muscle Samples

The overall goal of this diagnostic method is to identify meat, or meat products, that are infested with the food-borne zoonotic parasite Trichinella. This method is the gold standard for the direct detection of Trichinella larvae in meat. The main advantage of the technique is that it is easy to perform and does not require specialized equipment.

Generally, individuals new to this method may struggle, as internal controls cannot be used and stringent quality management must be applied throughout the test. Demonstrating the procedure will be Nora Thaben, a technician from my laboratory.Hello! To prepare a muscle sample for digestion, place 100 grams of the sample in a blender or grinder.

And add two milliliters of preheated water to facilitate blending. Blend at maximum speed for two to three seconds. Before the second blending, open the blender and transfer any muscle sample at the top of the blending bowl edge margin to the bottom.

Repeat the blending, and continue blending with bursts of two to three seconds until no visible pieces of meat remain. Prior to starting this procedure, prepare the digestion fluid in a three-liter beaker as described in the text protocol. Transfer one liter of the preheated digestion fluid into a flask.

Transfer the ground meat into the beaker. And use a spoon or a fork to disseminate the meat in the digestion fluid. Thoroughly rinse the blender bowl, lid, blades, and fork with the remaining digestion fluid.

Place the beaker on a hot plate to keep a constant temperature of 44 to 46 degrees Celsius while monitoring with a thermometer. And then cover the beaker with aluminum foil. Stir the digestion fluid for 30 minutes to create a deep vortex without splashing until the meat particles disappear.

Following digestion of the muscle sample, the digestion fluid must be filtered and the amount of undigested tissue determined. Adjust the scale to zero, weigh a clean 180-micrometer sieve, and note its weight. Check that the separation funnel is leveled vertically.

To allow good larvae sedimentation, the width of the separation glass funnel should not be larger than 55%of its length. Carefully pour the digestion fluid through the sieve, into the separation glass funnel, avoiding any overflow. To avoid loss of sensitivity, thoroughly rinse the glass beaker and the sieve with approximately 200 milliliters of tap water.

And transfer the rinse water into the separation glass funnel. Lift the sieve, and rinse the area under the sieve thoroughly using a squeeze bottle. The rinse water should also be collected in a separation glass funnel.

Leave the sieve on the separation funnel for drying throughout the sedimentation procedure. To begin the sedimentation procedure, keep the filtered digestion fluid in a separation funnel for 30 minutes for the Trichinella larvae to settle at the bottom of the funnel. Once sedimentation is complete, fully open the stopcock of the separation funnel to let at least 20 milliliters of the digestion fluid run off quickly into a measuring cylinder.

Then, fully open the stopcock in the opposite direction to allow 10 milliliters of digestion fluid to pass. Finally, open the stopcock in the opposite direction for another 10 milliliters to pass into the measuring cylinder. Leave the 40 milliliters of digestion fluid in the cylinder for 10 minutes to allow larvae to sediment.

Proceed to determine the amount of undigested tissues. Lift the sieve from the sedimentation funnel and dry the sieve with a paper towel. Subtract the sieve weight before filtration from the sieve weight with undigested tissues.

The digestion process is considered satisfactory if not more than 5%of the starting sample weight remains on the sieve. If the amount of undigested tissue exceeds 5%repeat the digestion using fresh muscle samples. If fresh samples are not available, digest the remaining material again.

After the digestion fluid has been in the cylinder for 10 minutes, remove 30 milliliters of supernatant by suctioning with a pipette without disturbing the 10-milliliter sediment. To reduce the amount of tissue fibers in the sample, add 30 milliliters of tap water to the sediment and leave for another 10 minutes. Repeat this wash until the liquid is clear.

Once the liquid is clear, remove the supernatant and transfer the 10 milliliters of washed sediment to a gridded Petri dish. Rinse the cylinder with 10 milliliters of tap water and add the water to the washed sediment. Immediately after the washing steps, examine the samples using a trichinoscope or a stereo microscope, with a substage-transmitted light source of adjustable intensity.

Using a magnification of 15 to 20x, examine the Petri dish systematically, grid-by-grid. If no suspect structures are found, gently move the fluid in the dish in a circular fashion to allow any Trichinella larvae, which were potentially overlooked, to accumulate in the center of the dish. Repeat the examination.

If a suspect structure is found, increase the magnification to 40 to 100x. Remove Trichinella larvae from the dish with a pipette and collect in a vessel. After the entire dish has been examined, repeat the microscopic examination procedure, starting from the gentle shaking of the dish.

Repeat the procedure at least three times. Or until no more larvae are found. Determine the number of larvae per gram of muscle tissue.

If too many larvae are present in the digestion fluid to determine the larval number reliably, return the fluid containing the larvae to the measuring cylinder. Rinse the dish with tap water from a squeeze wash bottle, and leave the larvae to settle to the bottom. After a 10 minute sedimentation time, reduce the digestion fluid to a defined volume by transferring the larvae from the bottom of the measuring cylinder in one milliliter increments into a new cylinder.

Suspend the larvae homogeneously in the 10 milliliters by vigorous shaking. During the shaking motion, pipette 12 drops of 20 microliters of larval suspension onto a Petri dish. Examine each 20-microliter drop by microscopy, and calculate the total number of larvae as described in the text protocol.

Prior to microscopic examination of Trichinella larvae, it is important to wash each sample sufficiently to remove debris that could prevent the identification of the larvae. Trichinella larvae are 0.7 to 1.5 millimeters long and approximately 0.3 millimeters in width. The esophagus is narrow with a slightly rounded end.

The stichosome is a structure in the anterior half of the body cavity, compromised of a long slender tube surrounded by a row of 45 to 55 large cells. The rectum is also rounded without any projections or appendages. After digestion, the shape of the infective muscle larvae can vary, making identification more difficult.

Typical forms include tightly coiled and lightly coiled moving larvae, or C-shaped, or completely uncoiled larvae. Other structures besides Trichinella larvae can be found after muscle digestion. Examples of incidental findings from wild boar include a bristle-like hair of an earthworm, the trematode Alaria alata, a muscle fiber, a nematode from the genus Metastrongylus, plant fiber, and larvae of the roundworm Toxocara.

Once mastered, the technique can be done in approximately two hours, if it is done properly. After watching this video, you should have a good understanding of how to detect Trichinella larvae in meat by the magnetic stirrer method. Taking into account the most important critical control points.