are visual proof of a positive specimen).
* Dientamoeba fragilis trophozoites can be found in formed stool specimens.
Preservation of Stool Specimens
Overview of Preservatives
If there are likely to be delays from the time of specimen passage until examination in the laboratory, the use of preservatives is recommended. To preserve protozoan morphology and to prevent the continued development of some helminth eggs and larvae, the stool specimens can be placed in preservative either immediately after passage (by the patient using a collection kit) or once the specimen is received by the laboratory. Several fixatives are available: formalin, sodium acetate-acetic acid-formalin (SAF), Schaudinn’s fluid, polyvinyl alcohol (PVA), and single-vial systems (Table 3.3). Regardless of the fixative selected, use of the appropriate ratio of fixative to stool (3 parts fixative to 1 part stool) and adequate mixing of the specimen and preservative are mandatory. Although many products are commercially available, the most commonly used preservatives are discussed below. They are all available from various scientific supply houses.
When selecting an appropriate fixative, keep in mind that a permanent stained smear is required for a complete examination for parasites. If the physician orders a fecal immunoassay such as FA, EIA, or the rapid-flow method, you will need to confirm that the fixative you are using is compatible with the immunoassay you have selected. It is also important to remember that disposal regulations for compounds containing mercury are becoming more strict; each laboratory will have to check applicable state and federal regulations to help determine fixative options.
Formalin
Formalin is an all-purpose fixative that is appropriate for helminth eggs and larvae and for protozoan cysts. 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. 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 permits examination of the specimen as a wet mount only, a technique much less accurate than a stained smear for the identification of intestinal protozoa. The most common preparation is 10% formalin, prepared as follows:
Formaldehyde (USP) . . . . . . . . . . . . . 100 ml (or 50 ml for 5%)
Saline solution, 0.85% NaCl . . . . . 900 ml (or 950 ml for 5%)
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 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. For 1 liter of either 10 or 5% formalin, 0.8 g of the buffer salt mixture should be added.
Protozoan cysts (not trophozoites), coccidian oocysts, microsporidian 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
