(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 resins and dehydrating agents are generally not satisfactory because the worms tend to collapse and become distorted during dehydration.
Glycerin Jelly Preparation
Specimens are transferred from pure glycerin directly into the glycerin jelly, a medium that will gel at room temperature, thus providing a semipermanent mount.
Glycerin Jelly (Refractive Index, 1.47)
Dissolve the gelatin in water in a beaker in a water bath. Add the glycerin and melted phenol. Store in small, wide-mouth bottles, and refrigerate. Remelt in a hot water bath before use. Dispense with a dropper onto the slide.
1. Nematodes should first be killed in glacial acetic acid, AFA, or alcohol-glycerin.
2. Place nematodes in a small dish containing 70% alcohol−5% glycerin solution (several millimeters deep).
3. Partially cover the dish to allow gradual evaporation of the alcohol and water for approximately 24 to 36 h. The larger the nematodes, the longer the time needed to complete the evaporation.
Note The evaporation procedure should be done slowly to prevent collapse of the worm; the larger the specimen, the longer the evaporation time.
4. Transfer the worms to another dish containing a few milliliters of glycerin. The specimen should be placed just below the surface; it will eventually (within a few hours) sink to the bottom of the dish. It is then ready to be mounted.
5. Place a drop of liquefied glycerin jelly on the slide, and transfer the specimen into the drop. When the coverglass is added, the glycerin jelly should flow out to the edges of the coverglass.
