Diagnostic Medical Parasitology. Lynne Shore Garcia
highly suggestive.
Streck tissue fixative has also been tested as a substitute for formalin and polyvinyl alcohol in fecal preservation. Stool samples were examined microscopically as follows: (i) in wet mounts (by bright-field and epifluorescence microscopy), (ii) in modified acid-fast-, trichrome-, and safranin-stained smears, and (iii) with two commercial test kits. Specific results showed that Cyclospora oocysts retained full fluorescence, modified acid-fast- and safranin-stained smears of Cryptosporidium and Cyclospora oocysts were equal in staining quality, and results were comparable in the immunofluorescence assay and enzyme immunoassay commercial kits. However, stool fixed in Streck tissue fixative and stained with trichrome showed unacceptable staining quality compared with stool fixed in preservatives, many of which contained PVA (fecal adhesive). Thus, Streck fixative is an excellent substitute for formalin; however, modifications to the trichrome procedure will be required to improve the staining characteristics of protozoan parasites (12). With the introduction of the Universal Fixative, TOTAL-FIX, formalin is no longer a consideration.
Formalin-Saline Solution
Although formalin is generally used at room temperature for routine diagnostic work, hot (60 to 63°C) 5% formalin in a ratio of 3 parts fixative to 1 part stool is recommended for bulk specimens containing intestinal protozoa. Long-term storage of protozoa is enhanced with buffered formalin; the solution should be replaced every 6 months. Acceptable morphology for teaching purposes can be maintained for 6 months to several years, depending on the organism and the lag time between specimen collection and fixation. The organism cytoplasm will tend to become glassy or granular with very poor nuclear definition. Cysts of Entamoeba coli and Giardia lamblia (G. duodenalis, G. intestinalis) tend to maintain their morphologic characteristics for years, whereas cysts of E. histolytica and E. hartmanni do not preserve well (Fig. 9.1). All of these cysts retain their ability to take up iodine in a wet, direct smear.
Figure 9.1 (Left) Entamoeba coli cyst (unstained). (Right) Giardia lamblia cyst (unstained). doi:10.1128/9781555819002.ch9.f1
The standard 10% formalin will fix protozoan cysts; however, it does not preserve morphology as well as does the buffered 5% formalin.
Formalin-Saline Solution
Formaldehyde is normally purchased as a 37% HCHO solution; however, for dilution, it should be considered to be 100%.
Buffered Formalin-Saline Solution
10% 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