Diagnostic Medical Parasitology. Lynne Shore Garcia
much like the 10% formalin described above (Fig. 2.5). The sediment is used to prepare the permanent smear, and it is frequently recommended that the stool material be placed on an albumin-coated slide to improve adherence to the glass (Fig. 2.6).
Figure 2.5 Stool collection vials, one containing SAF fixative and the other containing Z-PVA, one of the non-mercury-based fixatives. This combination of collection vials is an excellent option; concentrations and fecal immunoassays can be performed from the SAF vial, while the permanent stained smear can be performed from the Z-PVA vial. doi:10.1128/9781555819002.ch2.f2.5
Figure 2.6 Albumin used to precoat the slide prior to the application of SAF-fixed stool concentration sediment. Once the smear is dry, it is ready for permanent staining. doi:10.1128/9781555819002.ch2.f2.6
SAF is considered to be a “softer” fixative than mercuric chloride. The organism morphology is not quite as sharp after permanent staining as that of organisms originally fixed in solutions containing mercuric chloride. The pairing of SAF-fixed material with iron hematoxylin staining provides better organism morphology than does staining SAF-fixed material with trichrome (personal observation). However, a change in the trichrome formulation tends to improve the trichrome staining using SAF (see chapter 3 on examination of fecal specimens). Although SAF has a long shelf life and is easy to prepare, the smear preparation technique may be a bit more difficult for less experienced laboratory personnel who are not familiar with fecal specimen techniques. Laboratories that have considered using only a single preservative have selected this option (concentration, permanent stain, fecal immunoassays for Giardia and Cryptosporidium). Helminth eggs and larvae, protozoan trophozoites and cysts, coccidian oocysts, and microsporidian spores are preserved by this method. After centrifugation, special stains for the coccidia (modified acid-fast stains) and the microsporidia (modified trichrome stains) can be used with the concentrate sediment obtained from SAF-preserved stool material.
SAF fixative is prepared as follows:
To make Mayer’s albumin, mix equal parts of egg white and glycerin. Place 1 drop on a microscope slide, and add 1 drop of SAF-preserved fecal sediment (from the concentration procedure). After mixing, allow the smear to dry at room temperature for 30 min prior to staining. Mayer’s albumin is also available commercially.
Schaudinn’s Fluid
Schaudinn’s fluid is designed to be used with fresh stool specimens or samples from the intestinal mucosal surface. Many laboratories that receive specimens from in-house patients (no problem with delivery times) often select this approach. Permanent stained smears are then prepared from fixed material. A concentration technique for Schaudinn’s fluid-preserved material is also available but is not widely used.
Mercuric Chloride, Saturated Aqueous Solution
Use a beaker as a water bath; boil (use a hood if available) until the mercuric chloride is dissolved; let stand for several hours until crystals form.
Schaudinn’s Fixative (Stock Solution)
Immediately before use, add 5 ml of glacial acetic acid per 100 ml of stock solution.
Schaudinn’s fluid containing PVA (mercury base)
PVA is a plastic resin that is normally incorporated into Schaudinn’s fixative (28). The PVA powder is inert and is not a fixative but serves as an adhesive for the stool material; i.e., when the stool-PVA-fixative mixture is spread onto the glass slide, it adheres because of the PVA component. Fixation is still accomplished by the Schaudinn’s fluid itself. Perhaps the greatest advantage of the use of PVA is the fact that a permanent stained smear can be prepared. Although some laboratories may perform a fecal concentration from a PVA-preserved specimen, some parasites do not concentrate well, nor do some exhibit the typical morphology that would be seen in concentration sediment from a formalin-based fixative. Fixatives containing PVA are highly recommended as a means of preserving cysts and trophozoites for later examination. The use of fixatives containing PVA also permits specimens to be shipped (by regular mail service) from any location in the world to a laboratory for subsequent examination. Fixatives containing PVA are particularly useful for liquid specimens and should be used in the ratio of 3 parts PVA to 1 part fecal specimen. The formula is as follows:
Mix the liquid ingredients in a 500-ml beaker. Add the PVA powder (stirring is not recommended). Cover the beaker with a large petri dish, heavy wax paper, or foil, and allow the PVA to soak overnight. Heat the solution slowly to 75°C. When this temperature is reached, remove the beaker and swirl the mixture for 30 s until a homogeneous, slightly milky solution is obtained.
Schaudinn’s fluid containing PVA (copper base, zinc base)
There has been a great deal of interest in developing preservatives without the use of mercury compounds, and substitute compounds now provide the quality of preservation necessary for comparable protozoan morphology on the permanent stained smear. Copper sulfate has been tried (29, 30) but does not provide results equal to those seen with mercuric chloride (29). However, zinc sulfate has proven to be a good mercury substitute and is used with trichrome stain or iron hematoxylin (Fig. 2.5) (31, 32). Although zinc substitutes have become widely available, each manufacturer has a proprietary formula for the fixative.
Copper Sulfate Solution
Add the CuSO4 ∙ 5H2O to 1,000 ml of distilled water heated to 100°C. Mix until dissolved.
Modified PVA Fixative (Stock Solution)
Immediately before use, add 5 ml of glacial acetic acid per 100 ml of stock solution.
Single-Vial Collection Systems (Other than SAF)
Several manufacturers now have available single-vial stool collection systems, similar to SAF or modified PVA methods (11). From