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 using mercury-based fixatives.
Summary: Modified PVA (Mercury Substitutes)
Single-Vial Collection Systems (Other Than SAF)
Several manufacturers now have available single-vial stool collection systems, similar to the SAF or modified PVA methods. Some of these formulations are advertised as “ecologically friendly” and do not contain either mercury or formalin. From the single vial, both the concentration and permanent stained smear can be prepared. It is also possible to perform fecal immunoassays (EIA, FA, or the rapid-flow assay) with samples from some of these vials. Make sure to ask the manufacturer about all three assays (concentrate, permanent stained smear, and immunoassay procedures) and for specific information indicating that there are no formula components that would interfere with any of the three methods. Like the zinc substitutes, these formulas are proprietary.
Summary: Single-Vial Collection Systems
Quality Control for Preservatives
Preservatives for fecal specimens are checked for quality control by the manufacturer prior to sale, generally with living protozoa. If you prepare your own fixatives, you can use the following approach for quality control. The specimen used for quality control presented below is designed to be used with fixatives from which permanent stained smears will be prepared (Schaudinn’s fluid, PVA, modified PVA, SAF, or the single-vial systems). However, this quality control specimen can also be used in a concentration; the white blood cells (WBCs) can be seen in the concentrate sediment (sedimentation concentration) or in the surface film (flotation concentration).
1. Obtain a fresh anticoagulated blood specimen, centrifuge, and obtain a buffy coat sample (try to find a specimen with a high WBC count).
2. Mix approximately 2 g of soft, fresh fecal specimen (normal stool, containing no parasites) with several drops of the buffy coat cells.
3. Prepare several fecal smears and fix immediately in Schaudinn’s fluid to be quality controlled.
4. Mix the remaining mixture of feces and buffy coat in 10 ml of PVA, modified PVA, or SAF preservative to be quality controlled.
5. Allow 30 min for fixation, and then prepare several fecal smears. Allow to dry thoroughly (60 min at room temperature or 30 to 60 min in an incubator [approximately 35°C]).
6. Stain slides by normal staining procedure.
7. After staining, if the WBCs appear well fixed and display typical morphology and color, one can assume that any intestinal protozoa placed in the same lot number of preservative would also be well fixed, provided that the fecal sample was fresh and fixed within the recommended time limits.
8. Record all quality control results. If the WBC morphology does not confirm good fixation, then describe the results and indicate what corrective action procedures were used (repeated the test, prepared new fixative).
Procedure
