Diagnostic Medical Parasitology. Lynne Shore Garcia
be handled with caution, particularly if they are to be dissected.
Figure 9.4 Ascaris lumbricoides fertilized eggs. Note the fully developed larva within each egg; these eggs were viable when photographed. In some cases, the moving larva can be seen, even in formalin-preserved specimens. doi:10.1128/9781555819002.ch9.f4
Most trematodes are very muscular and may contract when placed in fixatives. Living specimens should be placed in cold 0.85% saline for 30 min to several hours (depending on the size of the worm) before fixation. Specimens can be placed on a slide in a petri dish and then covered with another slide or coverglass to flatten the worm. Do not apply pressure, since doing so may distort internal organs (Fig. 9.5).
Figure 9.5 Adult Fasciola hepatica trematodes. (Upper) Fresh trematode (unfixed, unstained). (Middle) Unstained flukes. (Bottom) Stained fluke (note the morphologic details that can be seen after staining). doi:10.1128/9781555819002.ch9.f5
Note Formalin is never recommended for the fixation of trematodes (16).
AFA fixative (see above) should be used hot (60 to 63°C); the worms can be stored in AFA or transferred to 70% alcohol for long-term storage.
Acid fixatives dissolve the characteristic calcareous corpuscles found in tapeworm tissue and should not be used. Since these corpuscles may be used to diagnose tapeworm tissue in histologic sections, buffered formalin or non-acid-containing fixatives are recommended (16).
Formalin
Hot (60 to 63°C) buffered formalin is recommended for the fixation of cestodes. If the whole worms are immediately swirled around in the container of fixative, they will be rapidly fixed with minimal contraction of the proglottids. If placed in cold formalin, the proglottids tend to contract; subsequent India ink injection will be more difficult to accomplish.
AFA Solution
AFA fixes tapeworm tissue well; however, the acid dissolves the calcareous corpuscles. The worms can be transferred to 70% alcohol for long-term storage (Fig. 9.6).
Figure 9.6 Taenia saginata proglottids. (Upper) Note that morphologic details cannot be seen in this preserved but unstained preparation. (Lower) After India ink injection, the uterine branches are now visible and can be counted. doi:10.1128/9781555819002.ch9.f6
Note All tapeworms and proglottids should be handled with care, especially if the species has not been determined. The eggs of Taenia solium (cysticercosis) and Hymenolepis nana are infectious for humans (Fig. 9.7).
Figure 9.7 (Left) Taenia spp. egg (note the striated shell and hooklets seen within the embryo (oncosphere). (Right) Hymenolepis nana egg (note the six-hooked embryo and the polar filaments that are found between the oncosphere and the shell). doi:10.1128/9781555819002.ch9.f7
Whole fecal specimens can be mixed directly with an appropriate fixative, although it is recommended that the organisms be concentrated before fixation. The use of formalin-saline as described for the fixation of protozoa is recommended for helminth eggs and larvae. If well preserved, most helminth eggs maintain their morphologic characteristics indefinitely.
Clinical laboratory personnel may receive arthropods for identification from various patient sources (surface of the body, stool, sputum, etc.) (16, 17). Specimens may be submitted when the actual source is unknown (implicated in a bite, found in the house or yard, etc.). Small, wingless insects and other arthropods should be placed in alcohol (70 to 95%), where they can remain indefinitely. Formalin fixative is not recommended for such specimens. Most flying insects should be killed in a chloroform tube or cyanide bottle and then preserved as a dry mount. Large maggots and other larvae can be killed in hot water first to prevent body shrinkage and contraction when placed in alcohol (18). A number of books also contain detailed information on the collection, preservation, and mounting of arthropods (19–21).
DNA sequencing has become much more common in surveys of archival research collections, particularly in reviewing rare taxa of arthropods. As an example, marine invertebrates have historically been maintained in ethanol following initial fixation in formalin. These collections often represent rare or extinct species or populations, provide detailed time series samples, or come from presently inaccessible or difficult-to-sample localities. Results obtained from preserved crustaceans in archival research collections indicate that in the absence of fresh or frozen tissues, archived formalin-fixed, ethanol-preserved specimens will prove a useful source of material for gene sequence data analysis by PCR and direct sequencing (22). It has also been determined that the now widespread use of critical-point drying of wasps and other insects from alcohol is advocated as a potential source of DNA from rare taxa (23).
Another problem that has been documented involves morphology changes seen with the use of various fixatives. Thus, fixation and mounting can significantly influence the morphometric analysis of mites and other arthropods. It is recommended that morphometric studies be conducted using consistent methods to reduce experimental bias and that the methods used be reported in publications dealing with morphometric analyses (24).
Modified Berelese’s medium is an all-purpose medium that kills, fixes, and preserves many arthropod specimens. No dehydration in alcohol is necessary.
Modified Berelese’s Medium
Mounting and Staining of Parasite Specimens for Examination
Following preservation, many staining and mounting techniques for the preparation of permanent or semipermanent mounts of helminth eggs and larvae and of arthropods may be used.
Because the cuticle of nematodes prevents the uptake of stain , these worms are usually rendered transparent with glycerin or lactophenol. In this way, the internal morphology can be observed for identification purposes. Specific morphologic features that can be seen include the cuticle, alimentary tract, and reproductive structures. Small nematodes can be mounted in glycerin jelly. Standard mounting media containing