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 the single vial, both the concentration and permanent stained smear can be prepared (Fig. 2.7 through 2.9). It is also possible to perform fecal immunoassay procedures from some of these vials. Make sure to ask the manufacturer about all three capabilities (concentration, permanent stained smear, fecal 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.
Figure 2.7 ECOFIX is an example of a fixative that represents the single-vial collection system. This fixative has been coupled with its own stain, the ECOSTAIN (11). Remember to inquire about the compatibility of all single-vial systems with the immunoassay procedures; not all single-vial preservatives are compatible with all fecal immunoassays. doi:10.1128/9781555819002.ch2.f2.7
Figure 2.8 ProtoFix is a single-vial collection option. It is always important to review peer-reviewed literature regarding the results of new products compared with those previously in use. doi:10.1128/9781555819002.ch2.f2.8
Figure 2.9 UNIFIX is a single-vial collection system; information on this product can be found at the company website (www.med-chem.com). This fixative works well with trichrome stain. doi:10.1128/9781555819002.ch2.f2.9
Universal Fixative (TOTAL-FIX)
The TOTAL-FIX stool collection kit is a single-vial system that provides a standardized method for untrained personnel to properly collect and preserve stool specimens for the detection of helminth larvae and eggs, protozoan trophozoites and cysts, coccidian oocysts, and microsporidian spores. Concentrations, permanent stains, most fecal immunoassays, and some molecular methods can be performed from a TOTAL-FIX preserved specimen (Fig 2.10). TOTAL-FIX is a mercury-, formalin-, and PVA-free fixative that preserves parasite morphology and helps with disposal and monitoring problems encountered by laboratories. www.med-chem.com (accessed 4/17/13). TOTAL-FIX is similar to Unifix and Zinc PVA (Z-PVA), commonly used fixatives that have been commercially available and used in many laboratories since 1992.
Figure 2.10 TOTAL-FIX is a single-vial collection system; the fixative contains no mercury, no formalin, and no PVA. It is considered a Universal Fixative and can be used for concentrations, permanent stained smears, fecal immunoassays, special stains, and some molecular methods. doi:10.1128/9781555819002.ch2.f2.10
Quality Control for Stool Fixatives
Fixatives for fecal specimens are checked for quality control by the manufacturer before sale, generally with the use of living protozoa. If you prepare your own fixatives, the following approach can be used 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, Schaudinn’s fluid containing PVA, copper- or zinc-based fixative containing PVA, SAF, MIF, and Universal Fixatives). However, the same 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 and 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.
