Formalin Solution
Solutions To Induce Relaxation in Adult Helminths ( 13 )
Tap Water or Physiologic Saline
1. Place living worms into a dish containing tap water or 0.85% NaCl solution.
2. Refrigerate the dish for 2 to 4 h.
Note Tap water generally works better than saline; also, the trematodes expel their eggs, thus allowing the internal morphology to be seen more clearly.
Dilute Menthol (14)
1. Dissolve 24 g of menthol crystals in 10 ml of 95% ethyl alcohol, and mix well.
2. Store the solution until needed.
3. Place living worms into a dish containing 100 ml of tap water or 0.85% NaCl.
4. Add 1 drop of menthol solution to the dish containing the worms.
5. Refrigerate the dish for 2 to 3 h.
Note The use of menthol accelerates relaxation of the worms. When this approach is used, the rostellum on the scolex of adult cestodes remains extruded during fixation.
Roundworms can be preserved with several different fixatives (Fig. 9.2). Formalin is usually not recommended, since it tends to harden the tissues. Nematodes can be killed with hot water (60 to 63°C) and then transferred to a preservative, such as alcohol-glycerin or alcohol-formalin-acetic acid (AFA). It is recommended that nematode larval stages be fixed in hot water. Direct fixation in preservatives may cause the cuticle to become “sticky,” and the larvae will be damaged when they adhere to the glass container (15).
Figure 9.2 Adult Ascaris lumbricoides nematode (male worm). doi:10.1128/9781555819002.ch9.f2
Alcohol-Glycerin
Alcohol (70%) containing 5% glycerin is an excellent fixative for most nematodes and should be used hot (60 to 63°C). Specimens can be left in this original fixing solution indefinitely. The glycerin will protect the specimen if the alcohol evaporates. Fixative evaporation is another reason why fixed specimens should be routinely checked every 6 months for possible fixative replacement.
Alcohol-Glycerin
AFA Solution
A solution of alcohol, formalin, and acetic acid can be routinely used for nematodes, trematodes, and cestodes. The fixative should be used hot (60 to 63°C); after fixation for 24 h, parasites can be stored in the alcohol-glycerin mixture.
AFA Solution
Glacial Acetic Acid
Undiluted glacial acetic acid is recommended for fixation of the smaller nematodes (Fig. 9.3). They are killed instantly in an extended position. This fixative is not recommended for the larger worms, such as Ascaris lumbricoides. The acetic acid will clear the worm tissue so that the internal structure becomes visible. Morphologic characteristics can be seen under the microscope if the worm is placed in water on a slide. Worms fixed with acetic acid can be placed in AFA for 24 h and then into alcohol-glycerin for long-term storage.
Figure 9.3 Adult Trichuris trichiura nematode (note the “whiplike” appearance; this is a much smaller adult worm than Ascaris lumbricoides). The small end attaches to the intestinal wall, while the large end is free in the bowel lumen. doi:10.1128/9781555819002.ch9.f3
Dilute Formalin
Although formalin is not recommended as a general fixative for nematodes, a dilute solution (1 to 2%) can be used to kill A. lumbricoides. Higher concentrations should not be used since the differences in osmotic pressure may cause the worms to rupture. After storage in dilute formalin for at least 24 h, the worms can be transferred to 10% formalin for long-term storage.
Note Ascaris, Toxocara, and Baylisascaris eggs continue to develop in formalin (10% or less) to the infective stage and remain viable and infective for a number of months (Fig. 9.4). Worms should 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
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