Taenia saginata after India ink injection of the uterine branches. doi:10.1128/9781555819002.ch9.f10
Euparal Mounts of Tapeworm Proglottids
Most helminth eggs mounted in Euparal (Flatters and Garnett, Ltd., Manchester, UK) exhibit excellent optical and drying properties, and this mounting medium can also be used to mount tapeworm proglottids (25). Proglottids should be placed in 100% ethyl alcohol for 5 to 10 min and then pressed between two slides as described above. They should then be placed in Euparal; transparency will occur in 2 to 3 h. The best results have been obtained by keeping the slides in a 50°C incubator overnight.
Double-Coverglass Method for Microscopic Mounts of Cysts, Eggs, and Larvae
Slides prepared by the double-coverglass technique will last for several weeks to several years, depending on the organisms and the care taken in preparation. The procedure is as follows (16).
1. Place a small drop of 10% formalin or formalin-saline suspension (containing cysts, eggs, and/or larvae) in the center of a 22-mm round or square coverglass.
2. Using forceps, very carefully apply a smaller coverglass (12, 15, or 18 mm round or square) to the suspension so that the fluid flows to the edges of the small coverglass (with no bubbles). If excess fluid flows beyond the small coverglass, it should be blotted dry or allowed to evaporate. Do not allow it to dry too long; otherwise, bubbles will accumulate under the coverglass.
3. Place a large drop of Permount or other permanent mounting medium in the center of a 1- by 3-in. (1 in. = 2.54 cm) microscope slide. Using forceps, place the double-coverglass preparation (small coverglass side down) onto the mounting medium so that the medium flows to the edges of the large coverglass.
4. Allow the preparation to thoroughly dry in a horizontal position (this may take several days).
Mounting of Arthropods for Examination (20, 26–28)
Before being mounted, specimens in which xylene is used as a solvent require dehydration through 50, 70, and 95% ethyl alcohol. Clearing can be done in clove oil, carbol-xylene (3 parts xylene to 1 part phenol crystals), or absolute ethyl alcohol followed by xylene. The specimens should remain in each solution for at least 15 to 20 min. Specimens mounted in balsam or Permount will take several days to harden; however, those mounted in isobutyl methacrylate will dry very rapidly (within a few hours). With this type of permanent mount, ringing or sealing of the coverglass is not necessary. Some specific recommendations are presented below.
Temporary mounts can be made with a drop of 50% ethyl alcohol, which is gently heated. Clearing and extension of the specimen occur, revealing the typical morphology (Fig. 9.11). Permanent mounts of living specimens can be made by placing the specimen in a drop of chloral-gum medium, adding a coverglass, and then gently heating until bubbling begins. Specimens originally preserved in alcohol must first be washed in distilled water to remove the alcohol.
Figure 9.11 Sarcoptes scabiei itch mites in wet mounts. (Upper) Adult mite. (Lower) Skin scraping containing adult mite (black arrow), eggs (red arrow), and mite feces (green arrow). These specimens could be seen using the high dry objective (magnification, ×400). (Lower image courtesy of National Institutes of Health, http://www.nlm.nih.gov/medlineplus/ency/images/ency/fullsize/2471.jpg). doi:10.1128/9781555819002.ch9.f11
Chloral-Gum Medium
Specimens may be preserved in 70% alcohol or mounted on slides for identification (Fig. 9.12).
Figure 9.12 (Left) Body louse, Pediculus humanus. (Right) Flea, Pulex irritans. (Courtesy of the CDC Public Health Image Library.) doi:10.1128/9781555819002.ch9.f12
1. Drop living fleas or preserved specimens into 10% KOH for a few days until sufficiently cleared.
2. Transfer for 30 min to a small volume of water containing a few drops of concentrated HCl.
3. Dehydrate in 50% ethyl alcohol for 30 min.
4. Dehydrate in 95% ethyl alcohol for 30 min.
5. Clear in beechwood creosote for 1 h, or place in several changes of absolute ethyl alcohol and clear in clove oil or xylene.
6. Mount on slides in balsam, Permount, or isobutyl methacrylate.
Specimens should be preserved in 70% alcohol or cleared and mounted on slides. They can be fixed in an extended position by being gently pressed between two slides while immersed in hot water. Clearing in KOH, dehydration in alcohols, and mounting with balsam, Permount, or isobutyl methacrylate can be done as recommended for fleas (Fig. 9.13).
Figure 9.13 Ticks. (Left) Example of a hard tick, Ixodes sp. (Right) Example of a soft tick, Argas sp. (Courtesy of the CDC Public Health Image Library.) doi:10.1128/9781555819002.ch9.f13
Spiders, scorpions, centipedes, lice, bedbugs, maggots and other larvae, nymphs, and soft-bodied insects can be preserved in 70% ethyl alcohol containing a small amount of glycerin to prevent drying and shrinkage. Containers should remain tightly sealed and should be checked periodically. Larger, hard-bodied insects can be pinned, labeled, and stored in boxes containing naphthalene flakes or paradichlorobenzene to prevent damage from mold and living insects (Fig. 9.14) (29).
Figure 9.14 Example of large hard-body insects, which are triatomid bugs. These large insects can be pinned, labeled, and stored in boxes containing naphthalene flakes or paradichlorobenzene. (Courtesy of the CDC Public Health Image Library.) doi:10.1128/9781555819002.ch9.f14
Note Identification of the many species of arthropods can best be handled by an entomologist or specialist working with a particular arthropod group. Additional help with identification may be obtained
