Diagnostic Medical Parasitology. Lynne Shore Garcia
examination would include the concentration and the permanent stained smear (1, 2). doi:10.1128/9781555819002.ch2.f2.3
Note When selecting an appropriate fixative, keep in mind that a permanent stained smear is mandatory for a complete examination for parasites (1, 2, 26). It is also important to remember that disposal regulations for compounds containing mercury are becoming more restrictive; each laboratory will have to check applicable state and federal regulations to help determine fixative options.
Note It is important to remember that when using fecal immunoassays for the Entamoeba histolytica/E. dispar group or Entamoeba histolytica, fresh or frozen stool is required (in some cases Cary-Blair may be acceptable, but check the package insert prior to use). To date, stool submitted in routine fecal fixatives is not acceptable for these particular fecal immunoassays.
Formalin
Formalin has been used for many years as an all-purpose fixative that is appropriate for helminth eggs and larvae and for protozoan cysts, oocysts, and spores. Two concentrations are commonly used: 5%, which is recommended for preservation of protozoan cysts, and 10%, which is recommended for helminth eggs and larvae. Although 5% is often recommended for all-purpose use, most commercial manufacturers provide 10%, which is more likely to kill all helminth eggs (Fig. 2.4). To help maintain organism morphology, the formalin can be buffered with sodium phosphate buffers, i.e., neutral formalin. Selection of specific formalin formulations is at the user’s discretion. Aqueous formalin will permit the examination of the specimen as a wet mount only, a technique much less accurate than a permanent stained smear for the identification of intestinal protozoa. However, the fecal immunoassays for Giardia lamblia (G. duodenalis, G. intestinalis) and Cryptosporidium spp. can be performed from the aqueous formalin vial. Fecal mmunoassays for the Entamoeba histolytica/E. dispar group and Entamoeba histolytica are limited to fresh or frozen fecal specimens (some kits may work with stool submitted in Cary-Blair; check the package insert). After centrifugation, special stains for the coccidia (modified acid-fast stains) and the microsporidia (modified trichrome stains) can be performed from the concentrate sediment obtained from formalin-preserved stool material. The most common formalin preparation is 10% formalin, prepared as follows:
Figure 2.4 When the stool specimen is added to the vial, the final ratio of stool to preservative is about 1:3. doi:10.1128/9781555819002.ch2.f2.4
Dilute 100 ml of formaldehyde with 900 ml of 0.85% NaCl solution. (Distilled water may be used instead of saline solution.)
Note Formaldehyde is normally purchased as a 37 to 40% HCHO solution; however, for dilution, it should be considered to be 100%.
If you want to use buffered formalin, the recommended approach (1, 3) is to mix thoroughly 6.10 g of Na2HPO4 and 0.15 g of NaH2PO4 and store the dry mixture in a tightly closed bottle. Prepare 1 liter of either 10 or 5% formalin, and add 0.8 g of the buffer salt mixture.
Protozoan cysts (not trophozoites), coccidian oocysts, microsporidian spores, helminth eggs, and larvae are well preserved for long periods in 10% aqueous formalin. Hot (60°C) formalin can be used for specimens containing helminth eggs, since in room-temperature formalin, some thick-shelled eggs (e.g., Ascaris lumbricoides) continue to develop, become infective, and remain viable for long periods. Several grams of fecal material should be thoroughly mixed in 5 or 10% formalin.
To collect large numbers of cysts, eggs, or larvae relatively free from other debris, the whole stool specimen is mixed in water and then strained through several layers of gauze. The suspension is allowed to sediment in a cone-shaped glass or flask for 1 h or more, and the supernatant fluid is discarded. The specimen may be washed several times in this manner before the sediment is finally fixed in hot 10% formalin, as mentioned above. When working with watery diarrhea specimens from patients with suspected cases of coccidiosis or microsporidiosis, the specimen should not be strained through gauze (oocysts and small bits of mucus may cling to the gauze); centrifugation (500 × g for 10 min) is necessary to sediment the oocysts and/or spores.
MIF
MIF (5) is a good stain preservative for most kinds and stages of parasites found in feces; it is especially useful for field surveys. It is used with all common types of stools and aspirates; protozoa, eggs, and larvae can be diagnosed without further staining in temporary wet mounts, either made immediately after fixation or prepared several weeks later. Although some laboratories maintain that a permanent stained smear can be prepared from specimens preserved in MIF, most laboratories using such a fixative examine the material only as a wet preparation (direct smear and/or concentration sediment). For a good discussion of this technique, see reference 27.
The MIF preservative is prepared in two stock solutions, stored separately, and mixed immediately before use.
Solution I (stored in a brown bottle)
Solution II (Lugol’s Solution) (good for several weeks in a tightly stoppered brown bottle)
Combine 9.4 ml of solution I with 0.6 ml of solution II just before use.
1. Add about one-quarter teaspoon (1 g) of fresh feces to the solution, and mix with an applicator stick. Fecal material should be formed or soft if egg counts are to be made later; liquid stool does not work very well for worm burden estimates.
2. Within 24 h, if undisturbed, the specimen forms three well-defined layers. The top layer, a clear orange fluid, consists mainly of formalin, Merthiolate, and water; it does not trap eggs or protozoa. The interface is a thick, pale orange or creamy yellow layer, usually 1 to 2 mm thick; this layer may trap some protozoa and helminth eggs. The bottom layer consists of deeper-staining particulate matter; eggs and protozoa are found throughout this layer.
3. With a glass pipette, MIF direct smears can be made from both the interface and bottom layers. Best results are obtained by making smears from both layers.
4. It has been suggested by some workers that a concentration technique applied to the MIF method (referred to as the MIFC or TFC method) gives satisfactory results. This contention is debatable, and Dunn (27) suggests that it is not nearly as reliable as the MIF direct smear method.
SAF
SAF lends itself to the concentration technique, the permanent stained smear, and fecal immunoassays for Giardia and Cryptosporidium and has the advantage of not containing mercuric chloride, as is found in Schaudinn’s fluid and some of the other fixatives containing PVA (20, 21).