jelly to begin to solidify, and apply the coverglass. If the specimen is large, place the coverglass onto small pieces of broken glass to provide more depth under the coverglass.
6. Allow the preparation to gel overnight (horizontal position). The following day, the coverglass can be sealed with Vaspar or enamel paint. These preparations usually last for several years, particularly if no unnecessary pressure is applied to the coverglass.
Note If personnel are not experienced in preparation of these permanent mounts, remember that the worms can be stored indefinitely in vials of pure glycerin. They can be studied in this medium as temporary mounts and then carefully returned to the vial of glycerin.
Glycerin
Nematode specimens can be examined as temporary mounts and subsequently stored in pure glycerin. This approach is particularly helpful for larger specimens (Fig. 9.8).
Figure 9.8 Semipermanent mounts of small nematodes. (Left) Small nematode from soil sample. (Right) Male Enterobius vermicularis (pinworm) nematode. doi:10.1128/9781555819002.ch9.f8
Lactophenol
Lactophenol is recommended when larger specimens are to be examined as temporary mounts. The worms can be placed into lactophenol from alcohol or formalin; clearing will occur, with the length of time depending on the size of the worm. The worms can then be washed in alcohol.
Lactophenol (Refractive Index, 1.44)
Trematodes are usually studied as stained whole mounts (Fig. 9.9). Two stains that are recommended are carmine and hematoxylin. Many modifications are found in the literature, and most give excellent results. The stains are usually available commercially, and they can also be easily prepared in the laboratory.
Figure 9.9 Stained trematodes. (Upper) Eurytrema sp. (Lower) Heterophyes sp. Note that the morphologic details are clearly visible after staining. doi:10.1128/9781555819002.ch9.f9
Semichon’s Acid Carmine
Add glacial acetic acid slowly to the water. Add the carmine powder, and heat to 95 to 100°C for 15 min. Cool and filter (stock solution). Add few drops of stock solution to 70% alcohol, making the working stain pink.
Van Cleave’s Combination Hematoxylin Stain
Van Cleave’s combination hematoxylin stain is a combination of Delafield’s hematoxylin and Ehrlich’s hematoxylin that rarely overstains trematode specimens.
Delafield’s Hematoxylin Stain
Dissolve the hematoxylin in ethyl alcohol, add aluminum alum solution, and let stand for 1 week exposed to air and light (in a paper-capped container). Filter; add glycerin and methyl alcohol. Age for 6 to 8 weeks in a tightly capped bottle in the refrigerator. This is the working stain solution (no dilution is needed).
Ehrlich’s Hematoxylin Stain
Dissolve hematoxylin powder in alcohol; add the other ingredients. Expose to air and light for at least 2 weeks (in a paper-capped container). The solution can be ripened immediately by the addition of 0.4 g of sodium iodate (NaIO3). Store refrigerated in a tightly capped bottle. This is the working solution (no dilution is needed).
Van Cleave’s Hematoxylin Staining Solution
Dissolve the potassium alum in water and add the two hematoxylin stains. This is the working solution.
1. Staining should be done in dilute solutions of carmine or hematoxylin (at least overnight). Specimens stained in carmine are placed into the dilute stain from 70% ethyl alcohol. Those stained in hematoxylin are placed in the stain from water. It is best to overstain and then destain (regressive stain approach).
2. Rinse in 70% ethyl alcohol.
3. Destain in weak acid-alcohol (2 to 4 drops of concentrated HCl in 100 ml of 70% ethyl alcohol). Leach the color from tissues until they are clear but the internal organs remain well stained. Destaining may take several minutes to hours; however, the specimens must be periodically checked to avoid overdestaining.
4. Rinse in 70% alcohol.
5. Place the specimens for 30 min to 1 h in a solution of 70% ethyl alcohol containing 1 or 2 drops of saturated aqueous Na2CO3, NaHCO3, or LiCO3. This step neutralizes the acid step and prevents continued destaining.
6. Rinse in 70% ethyl alcohol.
7. Dehydrate through 80, 95, and 100% ethyl alcohol, with 11 to 15 min for each alcohol change.
8. Clear the specimens in xylene for at least 15 min.
9. Mount in Permount or other permanent mounting medium.
Cestodes can also be examined as stained whole mounts, although the Taenia tapeworms can be examined more rapidly with India ink in a temporary mount (see chapter 4 for India ink injection of tapeworm proglottids). The same carmine and hematoxylin stains as used for trematodes can be used for cestodes.
India Ink Procedure for Tapeworm Proglottids
Identification of adult worms usually involves examination of a tapeworm proglottid. A Taenia proglottid must be gravid, containing the fully developed uterine branches. Using a syringe (1 ml or less) and a 25-gauge needle, India ink is injected into the central uterine stem of the proglottid, filling the uterine branches with ink, or into the uterine pore. The proglottid can then be rinsed in water or saline, blotted dry on paper towels, pressed between two slides, and examined. After the identification has been made, the proglottid can be left between the two slides (place a rubber band around the slides), dehydrated through several changes of ethyl alcohol (50, 70, 90, and 100%), cleared in two changes of xylene, and mounted with Permount for a permanent record. After the xylene step, the proglottid will be stuck to one of the two slides; do not try to remove it (it is very brittle and will crack), but merely add the Permount to the proglottid and then add the coverglass (Fig. 9.10).
Figure 9.10 Taenia gravid proglottids. (Upper) Taenia solium stained with carmine stain.
