Practical Guide to Diagnostic Parasitology. Lynne Shore Garcia
spores, and 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 cold formalin some thick-shelled eggs may continue to develop, become infective, and remain viable for long periods; however, this approach is not practical for routine clinical laboratories. Several grams of fecal material should be thoroughly mixed in 5 or 10% formalin.
Summary: Formalin
Sodium Acetate-Acetic Acid-Formalin (SAF)
Both the concentration and the permanent stained smear can be performed from specimens preserved in SAF, and the formula has the advantage of not containing mercuric chloride, as is found in Schaudinn’s fluid and mercuric chloride-based PVA fixatives. It is a liquid fixative, much like the 10% formalin described above. 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.
SAF is considered to be a “softer” fixative than Schaudinn’s or mercuric chloride-based PVA fixatives. The organism morphology is not quite as sharp after staining as are 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 of SAF-fixed material with trichrome (personal observation). 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 personnel who are not familiar with fecal specimen techniques. Laboratories that have considered using only a single preservative have selected this option. Helminth eggs and larvae, protozoan trophozoites and cysts, and coccidian oocysts and microsporidian spores are preserved using this method.
SAF fixative is prepared as follows:
Sodium acetate. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 g
Acetic acid, glacial. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.0 ml
Formaldehyde, 37–40% solution. . . . . . . . . . . . . . . . . . . . . . . 4.0 ml
Distilled water. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92.0 ml
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.
Summary: SAF
Schaudinn’s Fluid
Schaudinn’s fluid (which contains mercuric chloride) was one of the original stool fixatives and is used with fresh stool specimens or samples from the intestinal mucosal surface. Many laboratories that receive specimens from in-house patients (which have fewer problems with delivery times) often select this approach. Permanent stained smears are then prepared from fixed material. A concentration technique using Schaudinn’s fluid-preserved material is also available but is not widely used. Due to the difficulties (sources and cost) related to mercury disposal, this fixative is being phased out by most laboratories. Although mercury substitutes are available, the overall protozoan morphology does not tend to be as precise as that seen when mercury-based fixatives are used.
Mercuric chloride, saturated aqueous solution
Mercuric chloride (HgCl2). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110 g Distilled water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1,000 ml
Use a beaker as a water bath; boil (use a hood if available) until the mercuric chloride is dissolved; let stand several hours until crystals form.
Schaudinn’s fixative (stock solution)
Mercuric chloride, saturated aqueous solution . . . . . . . . . 600 ml
Ethyl alcohol, 95% . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 300 ml
Immediately before use, add glacial acetic acid, 5 ml/100 ml of stock solution.
Summary: Schaudinn’s Fluid
Polyvinyl Alcohol (PVA)
PVA is a plastic resin that can be incorporated into Schaudinn’s fixative (or other liquid fixatives). The PVA powder is not a fixative but serves as an adhesive for the stool material; i.e., when the stool-PVA 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 in the use of PVA is the fact that a permanent stained smear can be prepared. PVA fixative solution is highly recommended as a means of preserving cysts and trophozoites for later examination. The use of PVA also permits specimens to be shipped (by regular mail service) from any location in the world to a laboratory for subsequent examination. PVA is 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:
PVA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.0 g
Ethyl alcohol, 95% . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62.5 ml
Mercuric chloride, saturated aqueous . . . . . . . . . . . . . . . . . 125.0 ml
Acetic acid, glacial . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.0 ml
Glycerin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.0 ml
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.
Summary: PVA
Modified PVA (Mercury Substitutes)
Although preservatives have been developed that do not contain mercury compounds, substitute compounds have not provided the same quality of preservation necessary for good protozoan morphology on the permanent stained smear. Copper sulfate has been tried but does not provide results equal to those seen with mercuric chloride. Zinc sulfate has proven to be an acceptable mercury substitute and is used with trichrome stain. Although zinc substitutes have become widely available, each manufacturer has a proprietary formula for the fixative. Compared with mercuric chloride-based fixatives, there is much less margin for error when using modified PVA fixatives. Rapid fixation, proper stool-to-fixative ratios, and adequate mixing are mandatory for good protozoan morphology, particularly on the permanent stained smear.
Note: The important question is not “How beautiful are the organisms?” but “Can you tell which organisms are present?” With some training, microscopists can identify the organisms, although the morphology is not as clear as that seen using mercury compounds. Unfortunately, parasitology microscopy is not a perfect science; we probably miss rare organisms even when