100 ml distilled water. Allow the cellophane coverslips to soak in the glycerine mixture for at least 24 h. Although the malachite green is recommended, it is not essential for the procedure.
3. Some procedures require a metal or synthetic fiber filter mesh; however, this method is more complex. Just remember that if the fecal specimen is formed, hard, and somewhat dry, you will have to add a few drops of water so the final stool/water emulsion becomes mushy. To obtain 10-, 20-, or 50-mg samples, metal templates containing holes calibrated to deliver these fecal amounts can be used.
Procedure
1. Using an applicator stick, transfer approximately 50 mg of fresh stool to a standard 1 × 3-in. microscope slide. (A 4-mm cube of feces weighs about 65 mg.)
2. Cover the stool with the cellophane coverslip.
3. Turn the preparation upside down on a paper towel (flat, absorbent surface), and press it down until the fecal film covers approximately 20 to 25 mm in diameter. If the amount of feces is too much, the excess usually flows from under the cellophane coverslip and is absorbed by the paper towel.
4. Allow the preparation to stand for 1 h at room temperature to allow clearing of the fecal material but not the eggs. Do not overclear; thin-shelled eggs (hookworm) will tend to collapse and may disappear. Examine the slide at the end of the clearing period (usually not to exceed 1 h). It has been recommended that the clearing time be extended to 24 h for S. mansoni eggs; however, hookworm eggs will probably have collapsed and disappeared after this amount of time.
5. Examine the entire fecal film using the low power objective (10×).
Correlation with Treatment
The following numbers have been compiled to indicate the correlation between egg counts and the need for treatment. The two helminths listed below are generally the only ones for which the egg count will determine whether the patient is treated for the initial infection. As mentioned above, with the current therapeutic agents available, this information has become less clinically relevant, at least in many parts of the world. However, it has been recognized that egg counts do not always correlate with or accurately predict the worm burden of the host.
1. For T. trichiura, about 30,000 eggs/g indicates the presence of several hundred worms, which may cause definite symptoms.
2. For hookworm, about 2,500 to 5,000 eggs/g usually indicates a clinically significant infection.
The effectiveness of therapy for any helminth infection may be evaluated by doing repeated egg counts after treatment. Low egg counts for T. trichiura and hookworm are generally reflected by a lack of clinical signs and symptoms in individuals harboring these parasites. However, the presence of even one Ascaris worm is potentially dangerous because of the active migrating habits of this parasite, which may result in serious clinical manifestations.
When schistosome eggs (Fig. 4.7) are recovered from either urine or stool, they should be carefully examined to determine viability. The presence of living miracidia within the eggs indicates an active infection that may require therapy. The viability of the miracidia can be determined in two ways: (i) the cilia of the flame cells (primitive excretory cells) may be seen on a wet smear by using high dry power and are usually actively moving, and (ii) the miracidia may be released from the eggs by a hatching procedure (Fig. 4.8) (1, 28, 30, 31). The eggs usually hatch within several hours when placed in 10 volumes of dechlorinated or spring water (hatching may begin soon after contact with the water). The eggs that are recovered in the urine (24-h specimen collected with no preservatives) are easily obtained from the sediment and can be examined under the microscope to determine viability. McMullen and Beaver (31) recommended the use of a sidearm flask, but an Erlenmeyer flask is an acceptable substitute.
Figure 4.7 Schistosoma mansoni egg showing flame cells. (Illustration by Nobuko Kitamura; modified from E. C. Faust et al., Craig and Faust's Clinical Parasitology, 8th ed., Lea & Febiger, Philadelphia, PA, 1970.) doi:10.1128/9781555819002.ch4.f7
Figure 4.8 (Upper) Sidearm hatching flask used to recover miracidia from viable schistosome eggs (illustration by Nobuko Kitamura). (Lower) Miracidium larva released from egg during hatching procedure (Armed Forces Institute of Pathology photograph). doi:10.1128/9781555819002.ch4.f8
Quality Control for Hatching of Schistosome Eggs
1. Make sure that the water used is chlorine free; chlorine kills the miracidia. You can use bottled water or leave tap water in an open pan overnight before use (eliminates chlorine in tap water).
2. Check the saline solution (used to prepare the stool concentration) for the presence of any free-living organisms (flagellates or ciliates). This is normally not a problem.
3. Since it is neither realistic nor practical for the majority of laboratories to perform parallel positive-control procedures, review drawings and size measurements of schistosome eggs and/or miracidia.
4. The microscope should be calibrated, and the objectives and oculars used for the calibration procedure should be used for all measurements on the microscope. The calibration factors for all objectives should be posted on the microscope for easy access (multiplication factors can be pasted on the body of the microscope).
5. Record all QC results.
Procedure for Hatching Schistosome Eggs
1. Thoroughly homogenize a fresh stool specimen (40 to 50 g) in 50 to 100 ml of 0.85% NaCl.
2. Strain through two layers of gauze placed on a funnel. Collect material in a small beaker or 50-ml centrifuge tube.
3. Allow the suspension to settle for 1 h. Pour off and discard the supernatant fluid, and repeat this rinse process at least twice.
4. Decant the saline solution, resuspend the sediment in a small quantity of chlorine-free (spring) water (10 to 20 ml), and pour the suspension into a 500-ml sidearm flask or an Erlenmeyer flask.
5. Add chlorine-free water to the flask so that the fluid level rises to 2 to 3 cm in the side arm or to the top 2 cm of the Erlenmeyer flask. Cover the flask with aluminum foil or black paper, leaving the side arm of the flask exposed to light; if an Erlenmeyer flask is used, cover to 1 cm below the level of fluid in the neck of the flask.
6. Allow the flask to stand at room temperature for several hours or overnight in subdued light.
7. Place a bright light at the side of the flask opposite the surface of exposed water. Do not place the light against the glass, to avoid generation of excess heat. As the eggs hatch, the liberated miracidia will swim to the upper layers and collect in the side arm (or neck region of an Erlenmeyer flask). Make sure that the exposed part of the flask or side arm of the hatching flask
