a 37°C incubator or overnight at room temperature.
Note The fixative/stool/PVA mixture should be spread to the edges of the glass slide; this will cause the film to adhere to the slide during staining. It is also important to thoroughly dry the slides in order to prevent the material from washing off during staining.
4. The dry slides may then be placed into iodine-alcohol. There is no need to give them a 70% alcohol rinse before placing them in the iodine-alcohol, because the PVA smears are already dry (unlike the wet smears coming out of Schaudinn’s fixative). The iodine-alcohol is used to remove the mercuric chloride from the slides before they are stained. When reviewing the trichrome staining procedure, you will note that this step is not required if the stool specimen is preserved in a copper sulfate or zinc sulfate-based preservative.
NOTE Polyvinyl alcohol (PVA) is a plastic powder that is often added to fecal fixatives to help “glue” the stool material onto the glass slide. PVA is inert and has no fixing properties. Its main purpose is to cause the fecal material to adhere to the glass once the material is dry. HOWEVER, the Universal Fixatives do not require PVA or albumin to serve as adhesives.
California State Department of Health Modification (1) for PVA-Preserved Material
1. Thoroughly mix the fixative/stool/PVA mixture, and strain it through damp (with tap water) gauze into 15-ml centrifuge tubes.
2. After centrifugation (2 min at 500 × g), decant and discard the fluid and swab the sides of the tubes to remove any excess liquid. Although stains for Cryptosporidium spp. are not recommended from preserved material containing PVA, the centrifuge speed indicated here is probably not sufficient to recover the oocysts or microsporidian spores for special staining, fecal immunoassays using fluorescence, or other diagnostic procedures; use 500 × g for 10 min.
3. Use the remaining sediment to prepare coverslip smears (not 3- by 1-in. slides), which are immediately (without drying) stained, beginning with the iodine-alcohol step of the trichrome staining procedure (for removal of mercuric chloride).
4. The staining times can be reduced to 2 to 4 min, and dehydration steps can be reduced to 1 min each.
Note This procedure will work quite well for most stool specimens preserved in fixative containing PVA. However, for very liquid specimens or those containing large amounts of mucus, drying the smears before staining will be advantageous.
SAF-Preserved Material (31)
1. Thoroughly mix the SAF-stool mixture, and strain it through gauze into a 15-ml centrifuge tube.
2. After centrifugation (1 min at 500 × g), decant and discard the supernatant fluid. Although stains for coccidian oocysts and microsporidian spores are not recommended from PVA-preserved material, the centrifuge speed indicated here is probably not sufficient to recover the oocysts or spores. Centrifugation time and speed are recommended as 10 min at 500 × g. The final amount of sediment should be about 0.5 to 1.0 ml. If necessary, adjust by repeating step 1 or by resuspending the sediment in saline (0.85% NaCl) and removing part of the suspension.
3. Prepare a smear from the sediment for later staining by placing 1 drop of Mayer’s albumin on the slide and adding 1 drop of SAF-preserved fecal sediment. Allow the smear to air dry at room temperature for 30 min prior to staining.
4. After being dried, the smear can be placed directly into 70% alcohol (step 4) of the staining procedure (the iodine-alcohol step can be eliminated).
Merthiolate-Iodine-Formalin (MIF)-Preserved Material
1. Prepare a Mayer’s albumin-coated slide and allow it to air dry at room temperature for 30 min.
2. From the MIF vial (material allowed to settle in the vial for at least 1 h undisturbed), remove a portion of sediment and place this material onto the albumin-coated slide. Allow the slide to remain flat for 5 min; if there is still fluid left on the slide after this time, stand it up and allow the excess fluid to run off onto a paper towel.
3. After all excess fluid is removed, the slides are ready for staining in polychrome IV stain.
Universal Fixative (TOTAL-FIX)-Preserved Material
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, special stains, and some molecular testing can be performed from a TOTAL-FIX-preserved specimen. TOTAL-FIX is a mercury-, formalin-, and PVA-free fixative that preserves parasite morphology and helps with disposal and monitoring problems encountered by laboratories. The product contains zinc sulfate, acetic acid, and alcohols in water; however, the formula is proprietary and is patented (see Algorithm 3.1).
Algorithm 3.1 Method for the preparation of TOTAL-FIX-preserved fecal specimens (Universal Fixative) for the concentration, permanent stained smear, fecal immunoassays, and special stains for the coccidia and microsporidia. (a) This fixative contains NO mercury, NO formalin, and NO polyvinyl alcohol (PVA); use one-third stool and two-thirds fixative and MIX WELL. Allow 30 min prior to processing. (b) The EIA and rapid cartridge detect antigen; sample the clear fluid at the top of the vial containing the stool/fixative mixture. If the vial is shaken, make sure it stands on the counter for 5 to 10 min to allow the particulate material to settle out; work with the clear fluid only. (c) TOTAL-FIX has been tested with a number of molecular methods; however, there is no confirmation that the specimen will work with every possible available method. (d) Rinse fluids (water, saline, formalin) will PREVENT permanent stained smears from staining correctly; rinse fluids can ONLY be used for the final concentration step (after original sediment has been removed for permanent stained slide preparations—STEP 2). (e) Since the direct fluorescent antibody immunoassay (DFA) detects the actual Giardia cysts (rare trophozoites—much less intense fluorescence) and Cryptosporidium oocysts, this test sensitivity is enhanced if centrifuged sediment is used for testing; the same advantage is seen using modified acid-fast stains for Cryptosporidium (use sediment). Remember, do not use RINSED sediment, but sediment from the original centrifugation. (f) Examine entire 22-by-22-mm coverslip at low power (10× objective); examine one-third to one-half coverslip at high dry power (40× objective).
Trichrome Stain
The trichrome technique of Wheatley (22) for fecal specimens is a modification of Gomori’s original staining procedure for tissue (7). It is a rapid, simple procedure which produces uniformly well-stained smears of the intestinal protozoa, human cells, yeast cells, and artifact material in about 45 min or less.
The specimen usually consists of fresh stool smeared on a microscope slide, which is immediately fixed in liquid Schaudinn’s fixative, preserved stool (with or without PVA), the single-vial fixatives, or the Universal Fixative smeared on a slide and allowed to air dry.
Trichrome Stain
1.
