a collection system that includes both a preserved specimen and the remainder of the fresh stool. Certainly cost is a factor, because several vials in the collection system cost more than a single vial containing preservative. Each laboratory will have to decide for itself, often basing the decision on the types of procedures ordered by the physicians who use the laboratory service, the test method selected (traditional methods, immunoassay detection kits, or molecular methods), and the lag time between specimen collection and submission to the laboratory.
With increased emphasis on continuous quality improvement, managed-care contracts, cost containment, and the clinical relevance of diagnostic test results generated, compliance with specimen acceptance or rejection criteria has become more important and a necessary part of overall quality performance. The generation of patient data begins with the quality of the specimen; anything that is done to compromise that quality should not be acceptable within the laboratory setting.
If the specimen has not been preserved immediately after passage, it is important to know the age of the specimen when it reaches the laboratory. Freshly passed specimens are necessary for the detection of trophic amebae, flagellates, and ciliates. Liquid specimens must be examined within 30 min of passage (not 30 min from the time the specimen reaches the laboratory or is clocked in by the computer). Soft specimens should be examined within 1 h of passage. Immediate examination of a formed specimen is not as critical; however, if the stool cannot be examined on the day of collection, portions of the specimen should be preserved. In a routine laboratory setting, these time frames are often neither practical nor possible. Thus, the routine use of stool preservatives for diagnostic parasitology is highly recommended and has been widely accepted.
Microscopic Examination (Ova and Parasite Examination)
The microscopic examination of the stool specimen, normally called the ova and parasite examination, consists of three separate techniques: the direct wet smear, the concentration, and the permanent stained smear. Each of these methods is designed for a particular purpose and forms an integral part of the total examination (1–22, 49). With increased emphasis on proper specimen collection and cost containment, the approach to the ova and parasite examination has changed somewhat during the last few years. Many laboratories are requesting that all fecal specimens be collected in preservatives prior to delivery to the laboratory to decrease the lag time between specimen passage and fixation, thus providing better organism morphology and subsequent identification.
Because preserved organisms do not exhibit motility, the direct wet smear is no longer considered a mandatory part of the routine ova and parasite examination. However, if fresh fecal specimens are delivered to the laboratory, the direct wet smear should be performed, particularly on liquid or very soft stools.
In addition to normal specimen debris, the microscopic examination of fecal material may reveal the following:
1. Trophozoites and cysts of intestinal protozoa
2. Oocysts of coccidia and spores of microsporidia
3. Helminth eggs and larvae
4. Red blood cells (RBCs), which may indicate ulceration or other hemorrhagic problems
5. White blood cells (WBCs), specifically polymorphonuclear leukocytes (PMNs), which may indicate inflammation
6. Eosinophils, which usually indicate the presence of an immune response (which may or may not be related to a parasitic infection)
7. Macrophages, which may be present in bacterial or parasitic infections
8. Charcot-Leyden crystals, which may be found when disintegrating eosinophils are present (and may or may not be related to a parasitic infection)
9. Fungi (Candida spp.) and other yeasts and yeastlike fungi
10. Plant cells, pollen grains, or fungal spores, which may mimic some helminth eggs, protozoan cysts, coccidian oocysts, or microsporidial spores
11. Plant fibers or root or animal hairs, which may mimic helminth larvae
Normal mixing in the intestinal tract usually ensures an even distribution of organisms. However, depending on the level of infection, examination of the fecal material as a direct smear may or may not reveal organisms. The direct wet smear is prepared by mixing a small amount of stool (about 2 mg) with a drop of 0.85% NaCl; this mixture provides a uniform suspension under a 22- by 22-mm coverslip. Some workers prefer a 1.5- by 3-in. (1 in. = 2.54 cm) slide for the wet preparations rather than the standard 1- by 3-in. slide, which is routinely used for the permanent stained smear. A 2-mg sample of stool forms a low cone on the end of a wooden applicator stick. If more material is used for the direct mount, the suspension is usually too thick for an accurate examination; any sample of less than 2 mg results in the examination of too thin a suspension, thus decreasing the chances of finding organisms. If present, blood or mucus should always be examined as a direct mount. The entire 22- by 22-mm coverslip should be systematically examined with the low-power objective (10×) and low light intensity (Fig. 3.1); any suspicious objects may then be examined with the high dry objective (40×). At least one-third to one-half of the coverslip should be examined under high dry power (total magnification, ×400), even if nothing suspicious has been seen. Use of an oil immersion objective (100×) on mounts of this kind is not routinely recommended unless the coverslip is sealed to the slide (a no. 1 thickness coverslip is recommended for oil immersion). For a temporary seal, a cotton-tipped applicator stick dipped in equal parts of heated paraffin and petroleum jelly should be used. Nail polish can also be used to seal the coverslip. Many workers think that the use of the oil immersion objective on this type of preparation is impractical, especially since morphological detail is more readily seen by oil immersion examination of the permanent stained smear. This is particularly true in a busy clinical laboratory situation.
Figure 3.1 Method of scanning direct wet film preparation with a 10× objective. Note that the entire coverslip preparation should be examined before indicating the examination is negative. (Illustration by Nobuko Kitamura.) Note: All methods contained in the figures can be found in reference 48. doi:10.1128/9781555819002.ch3.f1
The direct wet mount is used primarily to detect motile protozoan trophozoites. These organisms are very pale and transparent, two characteristics that require the use of low light intensity. Protozoan organisms in a saline preparation usually appear as refractile objects. If suspicious objects are seen on high dry power, at least 15 s should be allowed to detect motility of slowly moving protozoa. Application of heat by placing a hot penny on the edge of a slide may enhance the motility of trophic protozoa. Tapping on the coverslip can also stimulate the fluid to move; objects will roll over, thus providing a better view of the parasite or artifact. Helminth eggs and/or larvae, protozoan cysts, and coccidian oocysts may also be seen on the wet film, although these forms are more likely to be detected after fecal concentration procedures (Fig. 3.2).
Figure 3.2 Direct wet smear with saline. (Top row) Giardia lamblia (G. duodenalis,
